Metabolic Cooperation between Cells

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IhTERNATIONAL REVIEW OF CYTOLOGY VOL . 69

Metabolic Cooperation between Cells M.L. HOOPER'A N D J.H. SUBAK-SHARPE Institutes of Genetics and Virology. University of Glasgow. Glasgow. Scotland I . Introduction . . . . . . . . . . . . . . . . . . . . I1 . Detection of Metabolic Cooperation Using Variants Incapable of Incorporating Nucleotide Precursors into Their Nucleic Acid . . A . Original Autoradiographic Observations . . . . . . . . B. Conditions Necessary for Occurrence of Metabolic Cooperation C . Molecular Basis of Metabolic Cooperation . . . . . . . D. Consequences of Metabolic Cooperation: "Kiss of Death" and "Kiss of Life" . . . . . . . . . . . . . . . . . 111. Extension and Generality of the Phenomenon . . . . . . . . A . Early Observations on the Effect of Cell Type and Animal Species . . . . . . . . . . . . . . . . . . . . B. Cooperation for Other Metabolites . . . . . . . . . . C . Ionic Coupling . . . . . . . . . . . . . . . . . D. Microinjection of Tracer Molecules . . . . . . . . . . E . Introduction of Tracer Molecules via the Cut End of a Tissue F . Spontaneous Loading of Tracer Molecules . . . . . . . G . Synchronization of Cellular Behavior . . . . . . . . . IV. Quantification of Metabolic Cooperation . . . . . . . . . A . Autoradiographic Techniques . . . . . . . . . . . . B . "Kiss of Death" and "Kiss of Life" . . . . . . . . . C . Scintillation Counting . . . . . . . . . . . . . . . D. Ionic Coupling . . . . . . . . . . . . . . . . . E . Fluorescent Dye Transfer . . . . . . . . . . . . . . V . Genetics of Metabolic Cooperation . . . . . . . . . . . A . Preexisting Cells Found to Be Metabolic Cooperation-Defective B . Selected Metabolic Cooperation-Defective Variants . . . . C . Reversion to Metabolic Cooperation-Competence . . . . . D. Cell Hybrids and Heterokaryons . . . . . . . . . . . E . Permeable Junction Deficiencies in Experimental Animals and Man . . . . . . . . . . . . . . . . . . . . . VI . Properties of Permeable Junctions . . . . . . . . . . . . A . Ultrastructure . . . . . . . . . . . . . . . . . . B . Biochemical Analysis . . . . . . . . . . . . . . . C . Molecular Weight Exclusion Limit . . . . . . . . . . D. Factors Affecting Permeability . . . . . . . . . . . . VII . Incidence and Specificity of Permeable Junction Formation . . . A . Occurrence in Vivo . . . . . . . . . . . . . . . . B . Combinations of Cells from Different Species . . . . . . C . Combination of Different Cell Types . . . . . . . . .

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'Present address: Department of Pathology. University of Edinburgh Medical School. Edinburgh EH8 9AG Scotland.

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VIII. Kinetics of Permeable Junction Formation and Breakdown . . . IX. Possible Functions of Metabolic Cooperation . . . . . . . . A. Coordination of Tissue Activities . . B. Synchronization of Cellular Behavior C. Growth Control . . . . . . . . D. Differentiation and Development . . X. Conclusions . . . . . . . . . . . References . . . . . . . . . . . Note Added in Proof . . . . . . . .

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I. Introduction One of the more successful adaptations to have occurred in the course of evolution was the emergence of multicellular organisms. In these organisms, natural selection operates to ensure that each cell functions not as a fully autonomous unit but as part of an integrated system. Thus each cell must be able to receive and respond to signals that provide information about the requirements of the organism at various levels and as a whole. These signals may take the form of diffusible molecules elaborated by cells in a distant part of the organism, or alternatively, they may arise from direct contact interactions with neighboring cells. In this article, we discuss one such direct interaction, viz. the exchange of metabolites between cells via permeable intercellular junctions. Sub&-Sharpe et al. (1966, 1969) discovered that variant tissue culture cells that were genetically incapable of incorporating a nucleic acid precursor into polynucleotide could become capable of incorporation when in contact with wild-type cells (Section 11,A). To describe this phenomenon, they introduced the term “metabolic cooperation,” which they defined as “the process whereby the metabolism of cells in contact is modified (perhaps controlled) by exchange of material. It subsequently became clear that the molecular basis of this phenomenon was the transfer of small molecules (almost certainly nucleotides) from wild-type to variant cells through specialized permeable junctions formed at sites of cell contact (Section I1,C) and that these same permeable junctions were responsible for mediating the previously observed phenomena of ionic coupling and dye transfer (Sections IV,Dand E). There is now good evidence that the gap junction (Section VI) fulfils the role of permeable junction, although the possibility that other membrane specializations can also fulfill this role cannot yet be excluded. In this light, we may update the definition of metabolic cooperation as follows: Metabolic cooperation is the exchange of molecules between cells through permeable junctions formed at sites of cell contact. The phenomenon has been alternatively termed “contact feeding” by Corsaro and Migeon (1975); it must be clearly distinguished from cross feeding, where material is transferred from one cell to another via the extracellular medium. ”

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Here we will review the various techniques used to demonstrate metabolic cooperation and the evidence that these techniques share a common mechanistic basis. In doing so, we shall give particular emphasis to techniques based on the phenotypic modification of cells consequent upon cell contact and will give only brief summaries of other techniques, referring the interested reader to more detailed reviews on these topics. We will also discuss the properties of permeable junctions, the factors controlling their formation, and the possible functions of metabolic cooperation in vivo . 11. Detection of Metabolic Cooperation Using Variants Incapable of Incorporating Nucleotide Precursors into Their Nucleic Acid A. ORIGINAL AUTORADIOCRAPHIC OBSERVATIONS

The initial demonstration of metabolic cooperation made use of a thioguanine-resistantgenetic variant of the polyoma-transformed Syrian hamster cell line PyY. This variant cell line (denoted PyY/TGI) lacks HGPRT activity (Fig. I ) and is incapable of incorporating exogenously supplied hypoxanthine into nucleic acid. Therefore PyYITG I cells, after incubation in [3H]hypoxanthine, followed by extraction with trichloracetic acid (TCA), and autoradiography, remain unlabeled (cf. Fig. 2). In contrast, wild-type PyY cells are strongly labeled under these conditions. However, Subak-Sharpe et al. (1966, 1969) prepared cocultures of PyY/TG1 and PyY cells (in the ratio of 300:l) in this way for autoradiography and observed, in addition to the expected unlabeled and strongly labeled cells, a thud category of cells with an intermediate intensity of labeling. These cells were almost always in contact either directly with strongly labeled cells or with other intermediately labeled cells that in turn were in contact directly or indirectly with strongly labeled cells. The rare exceptions to this rule were in positions that suggested that they had been in such contact at some earlier time during the labeling period. As the period of coculture prior to addition of label was increased, the number of intermediately labeled cells increased too rapidly for them to have been derived from strongly labeled cells by division, suggesting that they were HGPRT- cells that had acquired the ability to incorporate hypoxanthine as a result of contact with wild-type cells. This conclusion was confirmed by Stoker ( 1967) who differentially labeled wild-type and variant cells by allowing them to ingest particles of carbon or carmine. SubakSharpe et al. (1969) postulated that the phenomenon was due to passage of some molecule from wild-type to HGPRT cells and suggested a number of possible candidates for this molecule (Section ILC). The ability to incorporate [3H]hypoxanthine could be acquired not only by cells in direct contact with wild-type cells (so-called primary cooperators) but also by cells in contact with

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M. L. HOOPER AND J . H. SUBAK-SHARPE

FIG.1. Pathways of purine nucleotide interconversion in mammalian cells (Hauschka, 1973; Kelley , 1973). (1) HGPRT (hypoxanthine guanine phosphoribosyltransferase), formerly IPP (inosinic acid pyrophosphorylase); (2) APRT (adenine phosphoribosylYansferase), formerly APP (adenylic acid pyrophosphorylase); (3) AK (adenosine kinase).

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FIG.2. Autoradiographs of cocultures of HGPRT+ and HGPRT- cells after [3Hjhypoxanthine labeling. (a) Metabolic cooperation between FC13 donor cells (arrow) and Don TG,,2 ( H G P R T ) recipient cells. (From Hooper and Slack, 1977, with permission.) (b) Adhesive, but noncommunicating contacts between heavily labeled Don cells and HGPRT Lesch-Nyhan human fibroblasts (the large, unlabeled cells). In both (a) and (b), bar represents 20 pm. (From Gaunt and Subak-Sharpe, 1979, with permission.)

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M. L. HOOPER AND J . H. SUBAK-SHARPE

FIG.2b. See legend on p. 49.

primary cooperators, and a gradient of labeling away from the genetically competent wild-type cell was often discernible. This implied that a single cell (viz. a primary cooperator) could act both as I recipient (of material from a wild-type cell) and as a donor (to a secondary cooperator). This conclusion was substan-

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tiated by Burk et al. (1968) by coculturing HGPRTAPRV cells with HGPRT+APRT- cells: the HGPRT- cells functioned as recipients when [3H]hypoxanthine was supplied, but in the presence of rH]adenine, which A P R T cells cultured by themselves cannot incorporate, the HGPRT- cells functioned as donors and conferred ability to incorporate [3H]adenineon A P R T cells in contact with them. This experiment therefore established that metabolic cooperation was a reciprocal process. Similar effects were subsequently observed using pyrimidine salvage pathway mutants (Fig. 3). Pitts (197 1) showed that cells that were defective in thymidine kinase (TK-) and therefore incapable of incorporating [3H]thymidine became

Qnovo

FIG.3. Pathways of pyrimidine nucleotide interconversion in mammalian cells (Hauschka, 1973; Kelley, 1973). (1) TK (thymidine kinase); (2) dCK (deoxycytidine kinase); (3) UK (uridine kinase).

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M, L. HOOPER AND J. H . SUBAK-SHARPE

capable of incorporation when in contact with wild-type cells. Wright et al. (1976a) used cells that were defective both in TK and deoxycytidine kinase (dCK) and were therefore incapable of deoxycytidine incorporationeither as such or after deamination (Fig. 3). These cells similarly acquired the capacity to incorporate deoxycytidine when in contact with wild-type cells.

B. CONDITIONS NECESSARY FOR OCCURRENCE OF METABOLIC COOPERATION As noted earlier, metabolic cooperation is seen only where there is cell contact, suggesting that it is due to transfer of molecules across the apposed membrane. (We define “apposed membrane” as membrane in contact-with or without permeable junctions-with other cells; “nonapposed membrane” is in contact with surrounding medium or substratum but not with other cells.) This conclusion is supported by the observations that cooperation does not occur (a) at low cell density (Dancis et al., 1969), (b) in suspension cocultures, (c) when variant cells are incubated in medium conditioned by wild-type cells, or (d) when a coverslip of variant cells and a coverslip of wild-type cells are incubated together in the same medium (Cox et al., 1970). A small amount of uptake of label is seen when variant cells are incubated in labeled medium conditioned for long periods by wild-type cells (Azamia et a1., 1972), but this is probably due to conversion of the supplied [3H]hypoxanthineto other metabolites during conditioning. An early report that the phenotype of HGPRT- cells can be corrected by incubation in a sonicate prepbred from wild-type cells (Ashkenazi and Gartler, 1971) has not been confirmed (discussed by Goldfarb et al., 1974). c.

MOLECULAR BASISOF METABOLIC COOPERATION

Five categories of molecular species were suggested by Sub&-Sharpe et al. ( 1969) as possible candidates for the material transferred between wild-type and

HGPRT- cells: (1) nucleotides formed from [3H]hypoxanthinein HGPRT+ cells; (2) labeled polynucleotides formed in HGPRV cells; (3) informational polynucleotide coding for HGPRT; (4) protein, e.g., the HGPRT enzyme; and ( 5 ) a regulator of HGPRT. In cases (3), (4), and (3, ability to incorporate hypoxanthine should persist in HGPRT- cells after separation from wild-type cells since the half-life of residual HGPRT enzyme activity (determined from the rate of decay of activity in cycloheximide-treatedwild type cells) is more than 12 hours (Cox et al., 1970); in cases (1) and (2), it should not. Cox et af. (1970) found that, when confluent 1:l cocultures of wild-type and HGPRT- cells were uypsinized and cultivated in suspension in [3H]hypoxanthine(either in the presence or absence of cycloheximide), half of the cells lost the ability to incorporate. Pitts (197 1) found a similar loss of ability to incorporate after trypsinization of cocultures and seeding at low density; and Cox et al. (1972) showed that wild-type/

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APRT- cocultures behaved in a similar way. A single report of contrary behavior (Fujimoto and Seegmiller, 1970) has not been substantiated. Uitendaal et at. (1976) have claimed evidence for transfer of HGPRT enzyme from cell to cell, based on the assay of HGPRT activities in single cells after coculturing HGPRT+ and HGPRT- cells at high density and then reseeding at low density. However, their data show only a reduction of the activity in the HGPRF cells (which could occur by a variety of mechanisms, cf. Section IX,A) and provide no critical evidence for an increase in the activity of the HGPRT cells. The available evidence therefore points to the conclusion that either labeled nucleotide or labeled nucleic acid is the material transferred. In support of this conclusion, we note that a number of techniques have failed to detect the transfer of proteins from cell to cell (Mintz and Baker, 1967; Cox et a l . , 1972; Goldfarb et a l . , 1974; Pitts and Simms, 1977). The early observation (Kanno and Loewenstein, 1966) of a spread of flourescence to adjacent cells when serum albumin coupled to a fluorescent dye was injected into arthropod salivary gland cells is now thought to have been due to the transfer of a labeled degradation product (Loewenstein, 1979). Kolodny (197 1 , 1972, 1974) has claimed that RNA can be transferred between cultured cells on the basis of experiments in which [3H]uridine-labeled donor cells, which had been allowed to ingest tantalum particles in order to increase their buoyant density, were cultured with unlabeled recipient cells and then separated from them by centrifugation in a Ficoll gradient. However, these results have not been independently confirmed and may have been due to transfer of nucleotides, to loss of tantalum from donor cells, or to incomplete separation of donor and recipient dells in the centrifugation step. Pitts and Simms (1977), in contrast, concluded that nucleotides but not RNA were transferred from cell to cell. They used donor cells prelabeled with uridine and washed so that labeled material retained by the cells consisted of nucleic acid and nucleotides. When these cells were chased with unlabeled medium for up to 24 hours, the proportion of label present in the form of nucleic acid increased slowly with time. If unlabeled recipient cells were added at various times after labeling, then following coculture and TCA-insoluble autoradiography, the extent of labeling of recipient cells correlated with the level of labeled nucleotide in the donor cells and not with the level of labeled nucleic acid. Labeling of recipient cells was predominantly nucleolar and was substantially reduced if actinomycin D was added during the coculture period. This suggested that nucleotides were transferred and incorporated into RNA in the recipient cell. Similiar experiments using cells prelabeled with thymidine indicated that DNA also cannot pass from cell to cell. The most probable hypothesis, therefore, is that metabolic cooperation between HGPRT+ and HGPRT- cells is mediated by nucleotide transfer. Since nucleotides cannot cross cell membranes without first being dephosphorylated

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M. L. HOOPER AND J . H. SUBAK-SHARPE

(Liebman and Heidelberger, 1955; Subak-Shape, 1969), this implies the existence of a “private pathway” of communication between the cytoplasms of contacting cells that is not accessible to the extracellular medium. In support of this idea, Cox et af. (1972) found that the addition of 5’-nucleotidase to the medium has no effect on the extent of metabolic cooperation. We will use the purely descriptive term “permeable junction” to refer to this private pathway: its structural basis will be discussed in Section V1.

D. CONSEQUENCES OF METABOLIC COOPERATION: “KISS “KISS OF LIFE”

OF

DEATH”A N D

Chu and Malling (1968) reported that the recovery of variant Chinese hamster cells resistant to the purine analog 8-azaguanine (Table I) was decreased at high cell density. Albertini and de Mars (1970), using mixtures of wild-type (azaguanine-sensitive) human fibroblasts with fibroblasts from a patient with Lesch-Nyhan syndrome (HGPRT-deficient and therefore azaguanine-resistant), showed that the recovery of resistant cells decreased as the density of sensitive cells was increased. Fujimoto et al. (1971) reported a similar effect with the analog 6-thioguanine and postulated that this effect was due to transfer of tGMP TABLE I ANALOGS OF PURINE A N D PYRlMlDlNE BASESA N D NUCLEOSIDES

Analog ~

Most commonly found enzyme deficiency in resistant cells“

~~

6-Thioguanine 8-Azaguanine 6-Mercaptopurine 8-Azahypoxanthine 8-Azaadenine 2.6-Diaminopurine 2-Fluoroadenine 6-Methylthiopurine riboside 7-Deazaadenosine (tubercidin) 2 -Fl uoroadenosine 6-Azauridine 5-Bromodeoxy uridine 5-Fluorodeoxyuridine Cytosine arabinoside

HGPRT HGPRT HGPRT HGPRT APRT APRT APRT AK AK AK UK TK TK dCK

“Enzyme abbreviations are defined in Figs. I and 3. For references, see Roy-Burman (1970); Clements (1975); Astrin and Caskey (1976).

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(or azaGMP in the experiment of Albertini and deMars) from wild-type to mutant cells by metabolic cooperation (Fig. 4b) and coined the term “kiss of death” to describe the process. In support of this suggestion, it has been shown that the effect does not occur with L cells (Morrow, 1972), which are defective in metabolic cooperation (SectionV,A); that it does not occur if sensitive and resistant cells are separated by a fibrin overlay (van Zeeland et al., 1972); and that it does not occur at low cell density (Albertini and de Mars, 1970; Corsaro and Migeon, 1975) or when resistant cells are incubated on coverslips suspended in medium above a monolayer of sensitive cells (Slack et af . , 1978). Similar effects have been shown for cocultures of wild-type and TK- cells in 5-bromodeoxyuridine (BUdR; Wright et al., 1976a) or 5-fluorodeoxyuridine (FUdR; Slack et al., 1976) and for cocultures of wild-type and A P R T cells in azaadenine or diaminopurine (Dickerman and Tischfield, 1978). In the case of fluoroadenine, the last-mentioned authors found an additional toxic effect, which could be transferred via the medium. In contrast to these effects, intercellular transfer of molecules can rescue a cell from the effects of an otherwise toxic environment (“kiss of life”). Thus, Fujimoto et al. (197 1) showed that HGPRT- cells could be rescued by coculture with wild-type cells from the toxic effects of the glutamine antimetabolite azaserine, which blocks de novo purine synthesis; and Pitts (1971) showed that mutual rescue of HGPRT- and TK- cells could occur in HAT medium (hypoxanthine + aminopterin + thymidine). In the latter case, aminopterin blocks de novo purine and pyrimidine synthesis so that the products of both HGPRT and TK are required for growth (Fig. 4c). Mutual rescue was not observed with L cells (Pitts, 1971), which supports the view that the “kiss of life” too is mediated by metabolic cooperation. Both the “kiss of death” and “kiss of life” have been used as selective procedures in the isolation of variants and revertants with altered metabolic cooperation properties (Section V,B and C ) . However, their importance extends somewhat more widely to the design of procedures for the isolation of a wide variety of variants: the possibility must always be considered that high cell densities will result in poor recovery of variants. In vivo, the “kiss of life” may protect a target cell from the effects of a therapeutic agent. 111. Extension and Generality of the Phenomenon

A. EARLY OBSERVATIONS ON

THE

EFFECTOF CELL TYPEAND ANIMAL SPECIES

The initial observations of metabolic cooperation in Syrian hamster kidney cells and their polyoma-transformed derivatives were soon extended to other cell types. It was shown that metabolic cooperation could occur between a variety of

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M. L. HOOPER AND J . H. SUBAK-SHARPE

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FIG. 4. (a) Postulated mechanism of metabolic cooperation for nucleotides derived from [3H)hypoxanthine. (b) “Kiss of death” between HGPRT+ and HGPRT- cells in 6-thioguanine. (c) “Kiss of life” between HGPRT+ and HGPRT cells in HAT medium. H, hypoxanthine; apt, aminopterin; tG, 6-thioguanine; NA, nucleic acid. Asterisks denote 3Hlabeled compounds.

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normal and transformed cells and in heterotypic combinations of cells derived from different tissues and different mammalian species (Stoker, 1967; Cox et a l . , 1972). Furthermore, it was not restricted to established tissue culture lines but could be demonstrated in cells from human skin biopsies (Friedmann er al., 1968) and from amniotic fluid (Fujimoto et al., 1968). However, notwithstanding the widespread occurrence of metabolic cooperation, some cell types show either a deficiency in metabolic cooperation or a selectivity in the types of cell with which they will cooperate. These types of behavior will be discussed in Sections V ,A, and VII, respectively. FOR OTHER METABOLITES B. COOPERATION

1. Alkali Metal lons

Cell cytoplasm is maintained at an ionic composition different from that of extracellular fluid or growth medium by plasma membrane Na+ ,K+-ATPase, (EC 3.6.1.3; the so-called “sodium pump”), which utilizes free energy obtained from ATP hydrolysis to pump Na+ out of the cell, exchanging it with K+, and which is inhibited by the steroid compound ouabain (reviewed by Akera, 1977). Because of this inhibition, ouabain is toxic to cultured cells although there is wide variation in the level of sensitivity between cells from different species (Baker and Ling, 1978). Somatic cell variants resistant to ouabain can be selected from sensitive cell lines; these variants possess an Na+ ,K+-ATPase with reduced sensitivity to ouabain (Baker et a l . , 1974). Fusion hybrids between resistant and sensitive CHO lines exhibit an intermediate level of sensitivity (Baker et a l . , 1974), presumably due to the presence of both wild-type and mutant ATPase in the plasma membrane (Fig. 5c). One would therefore predict that in cocultures of resistant and sensitive cells, if Na+ and K+ can be freely exchanged between cells through permeable junctions, a resistanthensitive cell pair should similarly show an intermediate level of sensitivity (Fig. 5d). Corsaro and Migeon (1977a) showed that ouabain-sensitive (human) cells did indeed show increased resistance to ouabain when cocultured with resistant (mouse 3T3) cells. This increased resistance was not seen in coculture with L cells or when sensitive and resistant cells were seeded onto separate coverslips and the coverslips incubated together in the same dish; this evidence supports the view that resistance was mediated by exchange through permeable junctions. That alkali metal ions are indeed transferred is shown by the data of Ledbetter and Lubin (1979) who, using 86Rb+as a tracer for K+ showed that cocultures of sensitive (human) and resistant (mouse) cells in ouabain showed higher intracellular 86Rb+levels than would be predicted by summing the levels in separate cultures. Rescue from ouabain toxicity thus provides a method for the detection of alkali metal ion transfer between cells, and this method can be used as an alternative to the long-

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M. L. HOOPER AND J . H. SUBAK-SHARPE

b

V

d

FIG. 5 . Postulated mechanism of rescue from ouabain toxicity of sensitive cell by coculture with resistant cell. (a) Ouabain-sensitive(ouas) cell. In the presence of ouabain the Na+,K+-ATPase( ) is inhibited and the cell dies due to an inability to pump Na+ out of the cell. (b) Ouabain-resistant (ouaR) cell. In ouabain, the Na+,K+-ATPase of this cell ( 0 ) remains active. Arrows indicate movement of Na+, which is accompanied by an equal and opposite flow of K+.(c) Fusion hybrid between ouaS and ouaRcell. Membrane contains ATPase of both parents so that in ouabain only a fraction of the ATPase is active, resulting in an intermediate level of resistance. (d) ouaR and ouas cells connected by a junction permeable to Na+. Since Na+ can pass freely from one cell to the other, the situatron is formally analogous to (c).

+

established technique of ionic coupling (Section III,C). The relative advantages of the two techniques are discussed in Section X.Ledbetter and Lubin’s data imply that the sodium pump of the resistant mouse cells was not fully extended under pure culture conditions. However, the observations by Baker et al. (1974) of

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intermediate levels of resistance in fusion hybrids suggest that the spare capacity of the sodium pump is limited. Thus we could infer that ouabain-resistant cells should become more sensitive when cocultured with sensitive cells unless their spare capacity were great and exchange through permeable junctions relatively slow. This prediction has yet to be experimentally tested. 2 . Cyclic AMP (CAMP) Metabolic cooperation, probably mediated by intercellular transfer of CAMP, was elegantly demonstrated by Lawrence et al. (1978) using the cell types diagrammatically represented in Fig. 6. Mouse myocardial cells respond to noradrenaline (NA) by an increase in beat frequency, an increase in action potential amplitude, and a decrease in action potential duration. These effects are mediated by intracellular cAMP and are enhanced in the presence of 1methyl-3-isobutylxanthine (MIX), which inhibits cAMP breakdown. They do not respond to follicle stimulating hormone (FSH). Rat ovarian granulosa cells respond to FSH by producing the enzyme plasminogen activator (PA), an effect which is also mediated by CAMP. They do not respond to NA (except at high concentration in the presence of MIX). In coculture, the existence of permeable junctions between granulosa cells and myocardial cells could be demonstrated by uridine nucleotide transfer, by ionic coupling (Section III,C), and by electron microscopy (Section V1,A). When low concentrations of NA were added to cocultures (in the presence of MIX), it was possible to demonstrate synthesis of plasminogen activator (Fig. 6c), whereas FSH, when added to cocultures, elicited the myocardial cell responses of increased beat frequency, increased action potential amplitude, and decreased action potential duration (Fig. 6d). The normal responses of granulosa cells to FSH and myocardial cells to NA were also retained. Both modes of cross stimulation (i.e.. of granulosa cells by NA and myocardial cells by FSH) were dependent on cell contact and unaffected by addition of cyclic nucleotide phosphodiesterase to the medium, thus ruling out the possibility that they were mediated by cAMP secreted into the medium. The authors concluded, therefore, that they were probably due to gap junctional transfer of a communicator of hormonal stimulation, a likely candidate being CAMP. This demonstration clearly has implications regarding the role of metabolic cooperation in vivo (Section IX). 3 . Amino Acids By using a Chinese hamster ovary cell line auxotrophic for proline, Pitts and Finbow (1977; Pitts, 1978) were able to provide evidence for metabolic cooperation involving transfer of either proline or its precursor A'-pyrroline-5-carboxylic acid. In coculture with wild-type cells capable of forming permeable junctions, these auxotropic cells would grow in proline-deficient medium, whereas in coculture with wild-type L cells, growth was poorer. However, some growth was

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Q

b

C

FIG.6 . Postulated events in metabolic cooperation between myocardial cells and ovarian granulosa cells, which probably involves intercellular transfer of cyclic AMP (CAMP). FSH.follicle stimulating hormone; NA, noradrenaline; PA, plasminogen activator. MIX ( l-methyl-3isobutylxanthine) is used with noradrenaline to block cAMP degradation (not shown for clarity). The transferred molecule is shown as cAMP although other possibilities are not excluded.

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seen in the L cell cocultures and was attributable to passage of the amino acid from cell to cell via the medium. (Unlike nucleotides and alkali metal ions, amino acids cross nonjunctional membranes readily, and this introduces a background level of transfer that complicates the demonstration of metabolic cooperation.) Hooper and Morgan (1979a), studying intercellular transfer of amino acids of the urea cycle, were able to substantially reduce this problem by including degradative enzymes in the extracellular medium (Fig. 7). They used as recipient cell type the Chinese hamster cell line Don, which lacks detectable activity of argininosuccinate synthetase (ASS), which is the enzyme responsible for converting citrulline to argininosuccinic acid (ASA; Carritt et a l . , 1977). In medium where arginine is replaced by citrulline, Don cells therefore suffer arginine starvation and consequently fail to grow and to incorporate FHIthymidine into DNA. Cells with ASS activity can grow and incorporate rH]thymidine under these conditions. In coculture, arginine and ASA released from ASS+ cells are degraded to ornithine by argininosuccinate lyase (ASL) supplied by the serum component of the medium and by arginase added to the medium and cannot therefore satisfy the growth requirement of ASS- cells. If, however, the cells form permeable junctions, a pathway of transfer inaccessible to the degradative enzymes is available, allowing incorporation of r3H]thymidine by ASS- cells. Intercellular transfer was seen when the ASS+ cells were embryonal carcinoma cells (Section IX), which form permeable junctions with Don cells, but not when the ASS+ cells were L cells, which do not. 4. Folic Acid-Derived Cofactor

Pitts and Finbow (1977; Finbow and Pitts, 1980) have claimed evidence for transfer of the cofactor tetrahydrofolate between cells. They observed that cells starved of folic acid (and therefore with much reduced ability to incorporate r3H]formateinto cellular material) showed a marked increase in formate incorporation after coculture at high density with unstarved cells, provided that both cell types were capable of permeable junction formation. In contrast, an experiment designed to investigate intercellular transfer of the polyglutamyl derivative of tetrahydrofolate gave no evidence of transfer (Finbow and Pitts, 1980). In this experiment, the recipient cell type was the AUXBI variant of the CHO cell line isolated by McBurney and Whitmore (1974), which is defective in the addition of glutamyl residues to the tetrahydrofolate molecule. This difference in ability to transfer may be a consequence of the pore size of the junction (Section V1,C).

5 . Phosphorylated Derivatives of Sugars and Choline Pitts and Finbow ( I 977) extended the prelabeling technique of Pitts and Simms (Section I1,C) to study the movement of phosphorylated derivatives of sugars (fucose and 2-deoxyglucose) and choline. They reported that in each case cells retaining labeled acid-soluble pools but not cells in which label had been chased

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Q

b

V

FIG.7. Postulated mechanism of metabolic cooperation for urea cycle amino acids. (a) In medium containing citrulline in place of arginine, ASS+ cells can produce arginine and therefore grow and incorporate rHlTTP produced from supplied rHlthymidine; ASS- cells cannot. (b) Permeable junctions between ASS' and ASS- cells allow relief of arginine starvation in the ASScell and consequent [3H]'ITP incorporation. cit, Citrulline; ASA, argininosuccinic acid; arg, arginine; om, omithine.

into macromolecules were capable of transferring label to recipient cells if permeable junctions could be formed. Peterson and Rubin (1970) had previously reported transfer of label from chick embryo fibroblasts prelabeled with [3H]cholineto unlabeled cells but interpreted their results as showing transfer of phospholipid since no quenching could be demonstrated by adding unlabeled phosphorylcholine to the medium. Their argument is however invalid since phosphorylcholine would not be expected to cross the cell membrane.

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METABOLIC COOPERATION BETWEEN CELLS

An example of metabolic cooperation possibly involving phosphoribosylpyrophosphate (PRPP) has been described by %ref et al. ( 1976). They used skin fibroblasts from a patient with a mutant superactive PRPP synthetase resistant to feedback inhibition by purine nucleotides. In medium containing hypoxanthine, uridine, and 6-methyl mercaptopurine riboside (which after phosphorylation blocks de novo purine synthesis by inhibiting PRPP amidotransferase in addition to acting as a feedback inhibitor analog for PRPP synthetase) wild-type cells were killed, whereas the mutant cells survived because of the availability of PRPP for purine nucleotide salvage pathway synthesis. In cocultures, wild-type cells were rescued by contact with mutant cells. The phenomenon could, however, be due to intercellular transfer of molecules other than PRPP, e.g., nucleotides.

C. IONICCOUPLING The statement that two adjacent cells exhibit ionic coupling means that the resistance to electrical current flow between the cytoplasms of the two cells is substantially lower than that between cytoplasm and external medium. Lowresistance junctions between cells are detected using the apparatus shown diagrammatically in Fig. 8. Two microelectrodes, A and B, are inserted into the cytoplasm of cell 1 and a third, C, into the cytoplasm of the adjacent cell 2. Current pulses are passed into cell I through microelectrode A and resulting

W m

a b FIG.8. (a) Arrangement for detection of low-resistance junctions between cells. C and D are alternative positions of a single microelectrode. To detect low-resistancejunction between cells 1 and 2, current pulses are injected into cell 1 through microelectrode A and the resulting changes in potential V , and V , recorded by microelectrodes B and C. (b) Variation of V , , V,. and V , with time in response to a rectangular pulse of current injected into cell 1, demonstrating ionic coupling between cells 1 and 2 but no ionic coupling between cells 1 and 3.

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changes in potential recorded by microelectrodes B and C. If the cells are coupled, part of the current injected into cell 1 enters cell 2 and a change in potential is recorded by microelectrode C. Ionic coupling was first demonstrated by Furshpan and Potter (1959) between the pre- and postsynapic fibers of the giant motor synapse of the crayfish. They reported that the intercellular junction here behaved not as a simple resistance but as a rectifier, i.e., electric current passed readily in one direction but not in the opposite direction. This electrical synapse thus behaved like the more commonly occumng chemical synapse in transmitting action potentials unidirectionally. However, subsequent work indicated that ionic coupling was not restricted to excitable tissues: Kuffler and Potter (1964) showed that it occurred between glial cells in the leech central nervous system, and Loewenstein and Kanno (1964) showed it between salivary gland cells in Drosophila. Unlike the giant motor synapse, the junctions in these systems were nonrectifying. Low-resistancejunctions (the overwhelming majority of which are nonrectifying) have now been demonstrated in a wide variety of tissues (Section VII). Strong correlation between the existence of ionic coupling and metabolic cooperation for nucleotides in tissue culture was reported by Gilula et al. (1972) and by Azamia et a f . (1972). This indicates that the same permeable junctions mediate both intercellular metabolite transfer and ionic coupling (see Section VI). Recent reviews by Sheridan (1978), Bennett and Goodenough (1978), and Socolar and Loewenstein (1979) discuss the various aspects of ionic coupling in more detail.

D. MICROINJECTION OF TRACER MOLECULES 1. Fluorescent Dyes

Loewenstein and Kanno (1964) demonstrated by fluorescence microscopy that, following microinjection into salivary gland cells of Drosophila, the fluorescein anion (MW 330) spread into adjacent cells in contact. The use of fluorescent dyes has since proved to be a powerful technique for investigating junctional permeability (reviewed by Bennett, 1978; Loewenstein, 1979). Azarnia et al. ( 1972) found that the capacity for transfer of fluorescein between cells of a number of tissue culture lines correlated with their ability to participate in metabolic cooperation for nucleotides and in ionic coupling. There have been many parallel studies of fluorescent dye transfer and ionic coupling (see Bennett, 1978; Loewenstein, 1979), and in general, these show good correlation except in three cases: 1. Embryonic cells of several species have been reported to show ionic coupling but not fluorescent dye transfer (Bennett, 1978;Loewenstein, 1979). Recent studies using dyes with improved properties (see later) suggest that failure to detect fluorescein transfer may have been due to leakage from the cells across the

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nonjunctional membrane, and the early studies must now be reevaluated (Bennett, 1978). A further complicating factor is introduced by the finding that electrical conductivity and dye permeability are reduced in some early amphibian embryos if an electrical potential difference is applied across the junction (Spray et al., 1979). However, neither of these factors appears to account for two recent observations of ionic coupling in the absence of dye coupling in developing systems (Goodman and Spitzer, 1979; Lo and Gilula, 1979b). The significance of these observations is discussed in Section IX,D. 2. The rectifying giant motor synapse of the crayfish has been found to be impermeable to fluorescein (Keeter et a l . , 1974). However, measurements were made under conditions where the electrical resistance of the junctionis high since no potential difference was applied across it, and further study of dye permeability as a function of potential difference is desirable (Bennett, 1978). 3. Certain somatic cell hybrids between human fibroblasts and L cells show ionic coupling but not fluorescent dye transfer (Azamia and Loewenstein, 1977). The significance of this result is discussed in Section V,D. Fluorescein permeates some nonjunctional membranes readily, and this makes it less than ideal as a probe of junctional permeability. Procion yellow M4RS (Payton et a l . , 1969), though better retained by nonjunctional membrane, has the disadvantages of low fluorescence yield, intracellular binding, and cytotoxicity (Socolar and Loewenstein, 1979). The dyes of choice now appear to be Lucifer yellow CH (Stewart, 1978) and 6-carboxyfluorescein (Socolar and Loewenstein, 1979). A further recent refinement has been the synthesis of a series of fluorescent probes with different molecular weights for investigating the molecular size limit for junctional permeation (Section VI,C); the use of fluorochromes with different emission spectra enables the spread of two different tracer molecules to be monitored simultaneously (Simpson et a l . , 1977).

2 . Colored Dyes Following the initial use of fluorescein as a probe of junctional permeability, several colored dyes with molecular weights between 300 and 1000 were used as tracers (Kanno and Loewenstein, 1966; Potter et al. , 1966). However, they have now been superseded by fluorescent dyes, which offer increased sensitivity of detection and lower levels of binding to cytoplasmic constituents. 3 . Radioactive Tracers Rieske et al. ( 1975) microinjected radioactively labeled fucose, glucosamine, glycine, leucine, orotic acid, and uridine into one of the paired, electrically coupled Retzius cells of the leech central nervous system and in each case were able to show incorporation of label into macromolecular material in the other cell

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of the pair. Other (noncoupled) cells were not substantially labeled and no transfer between the Retzius cells was seen if they were uncoupled. The material transferred after sugar or amino acid labeling was shown to be a low-molecularweight precursor of protein or glycoprotein since injection of puromycin into one cell of the pair abolished the labeling of that cell. Occasionally, incorporation of label was inhibited also in the noninjected cell, suggesting that puromycin (MW 473) also may have permeated the junction. 4. Heavy Metals Politoff et a f . (1974) demonstrated (by microinjection of CoZ+ into crayfish lateral giant axons, followed by precipitation as the sulfide prior to fixation) that Co2+ could cross the junctions between axons provided that injection was carried out slowly in order to maintain the cytoplasmic concentration at a low level; at higher concentrations, uncoupling occurred (cf. Section VI,D). Turin (1977) has reported the extension of this technique to the use of heavy metal complex ions: AuC130H- (MW 320), but not the larger PbEDTAZ- (MW 531), passed between the blastomeres of early Xenopus embryos, whereas permeability to Co2+ was intermediate. No experiments were, however, reported in the latter study to check any possible effects of the probe molecule itself on junctional permeability. 5 . Enzymes Reese er al. (1971) reported that after injection of microperoxidase into crayfish lateral giant axons followed by fixation, enzyme activity could be detected histochemically in the adjacent electrically coupled mons. However, this was subsequently shown to be an artifact caused by the fixation procedure (Bennett, 1973).

E. INTRODUCTION

OF

TRACER MOLECULES VIA

THE

CUT ENDOF

A

TISSUE

As an alternative to microinjection, tracer molecules may be introduced into a tissue by a technique developed by Imanaga (1974). Using hrkinje fibers (from sheep and calf cardiac muscle) inserted through a tight-fitting hole in a rubber membrane between two perfusion chambers, he applied Caz+-freesaline containing the tracer (in this case, Procion yellow, MW 697) to one perfusion chamber and made a cut in the tissue. Under these conditions, the damaged cells remained coupled to the rest of the tissue, allowing the tracer to enter through the cut cells. After a suitable loading period, the tracer-containing solution was replaced by Ca2+-containingsaline, thus uncoupling the damaged cells from the rest of the tissue (cf. Section VI,D). The tracer was then allowed to diffuse through the tissue and was localized by freezing the preparation, sectioning, and examining the sections for fluorescence. Similar techniques have been used to demonstrate

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67

intercellular transfer of radioactively labeled tetraethylammonium ions (MW 130; Weingart, 1974) and CAMP(Tsien and Weingart, 1976).

F. SPONTANEOUS LOADING OF TRACER MOLECULES Weidmann (1966) used the fact that K+ permeates nonjunctional membrane readily to load cardiac muscle bundles with radioactive 42K. The bundles were arranged in multicompartment perfusion chambers so that the movement of the tracer within the tissue could be followed either by monitoring the perfusion fluid or by freezing and sectioning the specimen. He argued from quantitative analysis of his data that at least a proportion of the tracer must have spread through the cells (and therefore across permeable junctions) rather than through the extracellular space. A particularly interesting example of spontaneous loading is the use of nonpolar fluorescein esters that enter cells readily and are then hydrolyzed by esterases to free fluorescein, which is more polar than its esters and leaves the cells only slowly (Rotman and Papermaster, 1966). This technique was used by Sellin et al. (1971, 1974) to load donor cells with fluorescein; after washing, they could then be cocultured with unlabeled recipients to investigate junctionforming ability. The results of these experiments are discussed in Section IX.

G . SYNCHRONIZATION OF CELLULAR BEHAVIOR In primary cultures of myocardial cells, isolated cells undergo independent spontaneous contractions. When two cells come into contact, their contractions frequently become synchronized, as in the intact tissue, where synchrony results from the transmission of action potentials from cell to cell by ionic coupling (reviewed by DeHaan and Sachs, 1972; De Mello, 1977). Myocardial cells in indirect contact via another cell type can also become synchronized; a variety of cell lines can serve as connectors in this way, but L cells cannot (Goshima, 1969), confirming that signals passing through permeable junctions are responsible for the synchrony. When the bridging function was performed by a HeLa cell, Goshima was able to observe these signals in the form of a rhythmical change in the membrane potential of the HeLa cell that had the same frequency as the observed contractions.

IV. Quantification of Metabolic Cooperation A. AUTORADIOGRAPHIC TECHNIQUES

In common with many of the other techniques we have described, autoradiographic techniques are indirect in the sense that they detect not the direct product of intercellular transfer itself but the result of its incorporation into macromolecular

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material. This must be borne in mind in quantitative studies, since factors other than junctional permeability can affect the extent of this incorporation: in particular, changes in pool sizes in the recipient cell will change the extent to which transferred labeled metabolites are diluted in specific activity and hence alter the amount of incorporation; changes in the rate of nucleic acid synthesis will also influence the result. It is clearly essential to be able to distinguish donor cells from recipients in autoradiograms. Donor cells will in general show a higher intensity of labeling than recipients, but to rely on this for identification of individual donor cells is uncritical and subjective. Differences in morphology can often be used to advantage but rarely eliminate ambiguities completely. Stoker (1967) used ingestion of carbon or carmine particles to mark cell types, but his study was complicated by transfer of particles between cells. Moreover, some particles adhere to the outside of the marked cells. Clements (1973) and Goldfarb et a / . (1974) prelabeled donor cells with [14C]- or [3H]thymidine to facilitate their identification. One frequently wishes to test whether cooperation is occurring at all in a particular cell combination. This is conveniently done by carrying out grain counts over a number of recipients in direct contact with donor cells (as observed in the light microscope) and comparing the resulting histogram with one obtained for isolated recipients (the latter representing the background level of grains). This background level may either be uniformly distributed over the film [causes of such “film background” are discussed by Rodgers (1973); q.v. for a review of autoradiographictechniques in general] or may be cell associated. The presence of cell-associated background may be due either to incomplete enzyme deficiency in a variant (e.g., HGPRT-) cell line in which case, it will be present in a control from which donor cells are omitted. Alternatively, it may be due to transfer of labeled metabolites via the medium in which case, it may be possible to reduce it by adding the corresponding unlabeled metabolite to the medium. For example, the addition of unlabeled hypoxanthine reduces the background level when the label is [3H]adenine(unpublished results). The data are conveniently presented in the form shown in Fig. 9, and the distributions can be tested to see whether they are significantly different by a nonparametric test (Siegel, 1956). The Mann-Whitney V test should be employed, although often a simple median test will be adequate to show a significant difference between the distributions. A more difficult problem arises when one wishes to compare the levels of metabolic cooperation in different cell combinations. Two approaches have been used. Slack et al. (1978) used a parameter designated the “grain count index,” which is obtained by taking the difference between the median grain count over recipients in contact with donors and the median grain count over isolated recipients in the experimental coculture and expressing the value obtained as a percentage of the corresponding value for a control coculture. This enables one to compare the results of independent experiments where absolute grain counts may

METABOLIC COOPERATION BETWEEN CELLS

3f-I

69

Number of grains

FIG.9. Distribution in a single experiment of grain numbers over recipient cells cocultured with Don donor cells in the presence of rH]hypoxanthine. (a) PC13TG8 recipients; (b) R5/3 recipients (cooperation-defective variant isolated from PC13TG8). In each panel the upper histogram gives the distribution of grain numbers over recipients in direct contact with donors, whereas the lower, inverted histogram gives the distribution over isolated recipients. Median grain counts: (a) upper, 22; lower, 2; (b) upper, 6; lower, 2. Thus in this experiment the grain count index for R5/3 = (6 - 2) f (22 - 2) x 100% = 20%. (From Slack et al., 1978,with permission.)

vary considerably-it is obviously much easier to eliminate uncontrolled variables that may influence the result within an experiment than to do so between experiments. Grain count indices are affected by parameters other than junctional permeability and are therefore suitable only for comparisons of the type AB versus AC, where one cell type A is common to both cocultures and the others, B and C, are closely related (e.g., a variant and its parental wild type). Even in these cases, independent evidence is necessary before one can conclude that differences in grain count index reflect real differences in the permeability of the junctional membrane. This can be obtained by studying metabolic coopera-

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tion for a range of metabolites not involved in the same pathway or for the same metabolite by different techniques (Slack ef a l . , 1978). The second approach to comparing different cell combinations [used by Gaunt and Subak-Sharp (1979)l is to score each combination for the percentage of donor-recipient contacts showing evidence of cooperation. This method of scoring is not affected by variation in pool size, but, since an arbitrary choice has to be made regarding the number of grains that one considers as evidence of cooperation, the method is suitable only where the background is low. It gives no information about the extent of transfer. An alternative approach to the quantification of autoradiograms has been developed by Michalke (1977). By seeding cells into wells formed by placing a plastic template in a petri dish and then removing the template after cell attachment, he was able to construct a linear border between HGPRY and HGPRT cells. After incubation in [3H]hypoxanthine and autoradiography, he photographed the border area under dark-field illumination to show the silver grains, whose density he then measured photometrically in a chromatogram scanner. The density above the HGPRT- cells formed an exponential gradient away from the border; from this gradient, a space constant (defined as the distance over which the grain density falls by a factor e-l) could be determined. This parameter, like the grain count index, is affected by parameters other than junctional permeability (e.g.. nucleotide pool size, rate of nucleic acid synthesis, and cell density).

B. “KISSOF DEATH”A N D “KISSOF LIFE” Like the autoradiographic techniques discussed in Section IV,A, the “kiss of death” and “kiss of life” techniques detect the long-term consequence of metabolite transfer rather than the transfer itself, and the final results, which can be influenced and modified by parameters other than junctional membrane permeability, must be interpreted with care. In the “kiss of death” technique using thioguanine, for instance, one measures the efficiency of colony formation of the resistant cell type in thioguanine in the presence and absence of sensitive (donor) cells. The results may be quantified either as a ratio of colony-forming efficiencies in the presence and absence of a standard density of donors (Slack et al., 1978) or as the density of donors required to reduce the colony-forming efficiency by 50% (Corsaro and Migeon, 1975); this is a parameter referred to as the MLD (mean lethal density). While these parameters are sensitive to changes in junctional membrane permeability, one cannot validly conclude [as do Corsaro and Migeon ( 1975, 1977b)I in the absence of further evidence that differences in MLD reflect differences in the extent of metabolic transfer. The “kiss of death” technique is also lacking in an inherent control measuring any transfer of toxic metabolites via the medium so that one normally includes either an L cell control

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or a control where cells are cultivated together in the same medium but not allowed to make contact, e.g., by floating a coverslip of resistant cells in the medium above a monolayer of sensitive cells. Various techniques have been used to quantify “kiss of life” experiments. In the mutual rescue technique of Pitts (1971), net growth is observed only if both rescue processes are efficient and it suffices to count the total number of cells in the coculture; but where only one cell type of the pair is subject to rescue, the problem becomes one of quantifying small numbers of rescued cells in the presence of a large background of rescuing cells. Corsaro and Migeon (1977a) used cells of different species and estimated the numbers of rescued cells by karyotyping the mixed cultures. An alternative technique makes use of the fact that cells that have been treated with mitomycin C to block cell division retain capacity for metabolic cooperation: thus, if the rescuing cell type is treated with mitomycin C prior to coculture, the rescued cells form colonies that can be scored in the presence of a background of nondividing rescuing cells (Slack et a l . , 1978; Hooper and Morgan, 1979a). Nicolas et al. (1978) prelabeled the HGPRT cell type with [3H]thymidineprior to coculture with wild-type cells in HAT, so that the death of HGPRT cells could be detected as a release of radioactivity into the medium. C. SCINTILLATION COUNTING

If it is not just a matter of sharing but one of utilization of spare potential, then one might expect to be able to estimate the incorporation of radioactivity into recipient cells as a result of metabolic cooperation by determining the difference between incorporation in the coculture and that in donors alone: in practice, with [3H]hypoxanthine,this difference is small and the method does not give useful results (Subak-Sharpe, 1969; Wood and Pinsky, 1972). With r3H]adenine, the difference is measurable but a high background is still present (Subak-Sharpe, 1969). Ledbetter and Lubin (1979) were able to quantify the transfer of 8sRb from ouabain-resistant to ouabain-sensitive cells by such a difference technique, but the method again is subject to a high background and appears less than ideal for routine use. A better experimental system would allow incorporation only as a result of metabolic cooperation. Such a system has been described by Pitts (1 978) and involves coculturing a cell type that is both TK- and ouabain-resistant (ouaR)with one that is TK+ and ouabain-sensitive in the presence of ouabain and labeling with [3H]thyrnidine. Neither cell type alone incorporates, but in the coculture, if metabolic cooperation occurs, the ouabain-resistantcell type rescues the sensitive cell type from the toxic effect of ouabain (cf. Section III,B, l), thus allowing it to incorporate the label. (Some label also crosses as nucleotide to the TK- cell type and is incorporated there.) In the absence of metabolic cooperation, little or no label is incorporated. The method also works well if the

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ouabain-resistantpartner is HGPRT and [3H]hypoxanthineis used as label (J.D. Pitts, personal communication). This technique appears well suited to the rapid screening of a wide range of cell lines for presence or absence of cooperation with a single, marked (ouaRTK- or ouaRHGPRT-) tester line. It is less suitable for quantitative work since the results are affected by such factors as pool size, cell cycle parameters, and rate of nucleic acid synthesis. Moreover, one cannot tell what proportion of the incorporation is dependent upon transfer of alkali metal ions alone and what proportion depends upon transfer of both alkali metal ions and, subsequently, nucleotides.

D. IONIC COUPLING Techniques for the quantification of ionic coupling have recently been extensively reviewed by Socolar and Loewenstein (1979). Either the coupling ratio or the junctional conductance is usually measured. The coupling ratio, which is the ratio of potentials V J V , (Fig. 8), is relatively easy to measure and provides a sensitive test for the presence of ionic coupling, but its magnitude depends both on cell geometry and the topology of interconnection and is rather insensitive to changes in junctional conductance when the latter is high. The junctional conductance is a more informative parameter, but its determination is technically more difficult since it requires several independent measurements.

E. FLUORESCENT DYETRANSFER A number of techniques are available for quantitative fluorescence microscopy (reviewed by Socolar and Loewenstein, 1979), including (a) photography and densitometry, (b) use of a TV camera and image intensifier, followed by digital processing of the resulting electronic signal, (c) measurement with an array of photodiodes, and (d) estimation with a photomultiplier. Calculation of tracer concentration from measurements of fluorescent luminance requires a knowledge of the depth of focus of the microscope (Socolar and Loewenstein, 1979). An alternative approach used by Sellin ef al. (1974) in work with suspension cells was to pass the suspension through a flow cytofluorometer, which differentiated the cells according to fluorescence intensity and provided the data in the form of a histogram.

V. Genetics of Metabolic Cooperation A. PREEXISTING CELLSFOUND TO BE METABOLIC COOPERATION-DEFECTIVE Early experiments showed that the mouse L cell and its derivatives failed to cooperate for nucleotides when tested by autoradiography in combination either with each other or with various other cell types (Pitts, 1971; Widmer-Favre,

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73

1972; Cox et a l . , 1972). L cells have subsequently been shown to have a general defect in intercellular transfer of small molecules and have been extensively used as negative controls in a variety of techniques for the demonstration of metabolic cooperation (see Sections 11, 111, and IV). However, recent work indicates that the defect is not an absolute one and that L cells form permeable junctions at low frequency with most cell types and at high frequency in certain combinations (Section V11) so that caution must be exercised in their use. A number of other cell types have been found to be defective in permeable junction formation, including A, SA-21, and A’ cells from the H-5123 rat hepatoma; XD hamster embryo cells; MCF-7 and T231 cells from human breast cancer patients; cell lines M, N-32, At, and N-18 derived from mouse neuroblastorna (21300 (see Loewenstein, 1979, for references); HTC and H-35 rat hepatoma cells (Pitts and Simms, 1977); and cells from a mast cell neoplasm (Widmer-Favre, 1972). Many of these have been tested in combination with only a small number of partner cell types and, in the light of recent studies on the cell-type specificity of metabolic cooperation (Section VII), it would seem desirable to study a wider spectrum of cell combinations before inferring the presence of an absolute defect. Cox et al. (1976) claimed that phytohemagglutinin-stimulated human lymphocyte cultures were cooperation-defective, but this conflicts with other data (Section IX) and was probably an artifact produced by their experimental conditions (90-minute incubation of agglutinated cell clumps or centrifuge pellets with [3H]hypoxanthine),which did not include positive controls to show that cooperation could be detected under these conditions. Corsaro and Migeon (1977b) claimed that certain SV40-transformed human cell lines and human fibrosarcoma cell lines showed reduced metabolic cooperation compared with normal human cells, but their results could be explained on the basis of differences in nucleotide pool size (Section IV). B . SELECTED METABOLIC COOPERATION-DEFECTIVE VARIANTS There are two reports describing the isolation of metabolic cooperationdefective variants by selection in tissue culture from a cooperating cell type. The first used as starting material a polyoma-transformed Syrian hamster cell line defective in TK, dCK, and HGPRT (PyY/HGPRT-dCK-TK-mec+; Wright and Subak-Sharpe, 1974; Wright et a l . , 1976a). Because of its TK deficiency, this cell line was resistant to growth in BUdR and treatment with blue light, but when simultaneously cocultured with TK+ cells, it became sensitive through the ‘‘kiss of death” mechanism. The authors made use of this as a selective procedure to favor the survival of cooperation-deficient cells. The survival frequency of the starting cells when exposed to this selection was high (approximately 3%) so that multiple rounds of sequential selection were necessary; 46 rounds in all were used prior to cloning the survivors. One resulting clone, mec-IA, was exten-

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sively studied. Its ability to participate in cooperation, as measured by autoradiography (Section IV,A) was reduced to undetectable levels in the case of [3H]thymidine-derivednucleotides and to low levels in the cases of r3H]adenineand [3H]hypoxanthine-derivednucleotides. In the case of [3H]thymidine-derived nucleotides, the conclusions from autoradiography experiments were confirmed by an independent technique involving the separation of donor and recipient cell DNA after density labeling. Differences between mec-IA and its parent existed also in size, morphology, growth rate, and karyotype. Subsequently, Wright et al. (1976b) showed that, if the concentration of thymidine in the medium was increased (thus increasing the pool sizes of thymidine nucleotides in donor cells), cooperation for [3H]thymidine-derived nucleotides could be detected in mec-IA, although, compared with the parental cell line, the extent of cooperation was still reduced. The differences in the measured extent of cooperation-deficiencybetween purine and pyrimidine nucleotides were therefore interpreted as a consequence of different pool sizes rather than of selectivity at the level of the permeable junction. Autoradiographic measurements are indirect (Section IV,A), and so it was necessary to eliminate the hypothesis that differences in pool size between mec-IA and mec+ cells, rather than differences at the level of the junctional membrane, were responsible for the altered incorporation. This possibility, unlikely because separate changes in both purine and pyrimidine nucleotide pool size would have to be invoked, was eliminated by the demonstration that the measured difference in cooperation for [3H]thymidine-derived nucleotides persisted in the presence of aminopterin, which inhibits de novo synthesis of thymidine nucleotides. The authors also considered the possibility that the defect in mec-IA could be at the level of a mechanism responsible for equilibration between separate nuclear and cytoplimic nucleotide pools. They eliminated this possibility by infecting the cells with a temperature-sensitive mutant of herpes simplex virus, which at 38°C led to the synthesis of a virus-coded TK activity without shutting off host cell DNA synthesis. Having thus rendered the cells phenotypically TK+, they were then able to show that [3H]thymidine could be incorporated normally into their DNA so that any pool equilibration mechanism must be intact. They thus concluded that mec -1A must have a defect in its ability to form permeable junctions. Some relationship between this defect and the altered morphology of the cells was suggested by the finding that addition of dibutyryl cAMP and theophylline to the cultures caused a substantial reduction in both of these differences between mec-IA and its parent. Wright and Marsden (1976) examined polyacrylamide gel profiles of total cell proteins from mec-IA and its parent and found a substantial number of differences. All but six of these were not abolished by dibutyryl cAMP and theophylline: this excluded their involvement in the cooperation-deficiency. Four of the remaining six polypeptide bands had increased intensity in mec-IA cells

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(MW 27,000, 15,500, 15,000, and 13,000), whereas two were decreased in intensity (MW 28,000 and 13,500). The changes at MW 28,000 and 27,000 are of particular interest in the light of recent observations on the molecular weight of the major protein component of gap junctions (Section VI,B). A second metabolic cooperation-defective variant was isolated by Slack et al. (1978) from the mouse embryonal carcinoma line PC13TG8 (Section IX). They modified the selection technique previously described by using “kiss of death” killing of an HGPRT- line by 6-thioguanine and were able to obtain more efficient killing (survival frequencies of the order of 1 OP5) so that the number of rounds of selection could be substantially reduced, thereby lowering the probability of accumulating secondary genetic changes unrelated to metabolic cooperation. After five rounds of selection, they obtained a variant R5/3, which showed a reduction in grain count index when tested by autoradiography as a recipient of [3H]hypoxanthine-derived nucleotides. The possibility that this reduction was due to a pool size difference was eliminated by comparing cooperation for adenine- and thymidine-derived nucleotides and by combining data on hypoxanthine-derived nucleotide transfer obtained by different techniques. It was therefore concluded that the reduction was due to a difference in junctional membrane properties. A small proportion of strongly interacting cells were always seen when R5/3 cells were tested for cooperation by autoradiographic methods, and this feature persisted when both donor and recipient cells were cloned, ruling out the possibility that it was due to genetic heterogeneity. This behavior was interpreted in terms of the probability model of metabolic cooperation (Section VII), the R5/3lesion having the effect of reducing the probability of forming junctions with all cell types tested. R5/3 cells were subsequently shown to be defective in intercellular transfer not only of nucleotides but also of alkali metal ions and of amino acids (Hooper and Morgan, 1979a). Aside from the metabolic cooperation defect, R5/3 and parental PC13TG8 cells show comparatively few differences compared with mec-IA and its parent; R5/3 cells have increased thioguanine resistance and a near-tetraploid karyotype (PC13TG8 is near-diploid), but otherwise the two cell lines are very similar. This makes them more suitable for investigation of the mechanism and role of metabolic cooperation. Their suitability in this regard is further improved by the existence of a revertant to cooperation-competence (Section V ,C) that makes it possible to dissociate the effects of the cooperation defect from those of secondary genetic changes. As the formation of intercellular junctions is a complex process (cf. Section VIII), it is reasonable to expect that genetic lesions affecting any of the different steps involved may have effects on metabolic cooperation. A number of variants with altered cell adhesion properties have been obtained (see Grinnell, 1978; Baker and Ling, 1978), but to date none have been examined for metabolic cooperation.

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C REVERSION TO METABOLIC COOPERATION-COMPETENCE Hooper and Morgan (1979b) used a selective system based on the rescue of HGPRT- cells from HAT toxicity by metabolic cooperation with mitomycin C-treated wild-type cells (cf. Section IV,B) to isolate a cooperation-competent revertant H2T12 from the cooperation-defective variant R5/3 (Section V,B). In H2T12, the defects in intercellular transfer of both nucleotides and alkali metal ions are repaired, indicating that they have a common genetic basis; the increased thioguanine resistance and ploidy increase also present in R5/3 remain in H2T12, indicating that their genetic basis is different. Comparison of the properties of PCl3TG8, R5/3, and H2T12 thus provides a critical method of investigating the basis of the cooperation defect and of its effects. Thus Hooper and Parry (1980) demonstrated that, compared with PC13TG8, R5/3 cells showed a decrease in gap junction area and an increased surface area of microvilli per unit cell volume and that both changes were reversed in H2T12. This provides strong evidence in favor of the view that the gap junction (Section VI) mediates metabolic cooperation and suggests that the lesion responsible for the cooperation-deficiency of R5/3 may affect the formation of both gap junctions and microvilli: one possibility being a cytoskeletal defect. Buultjens el al. (1980) have shown that a major cellular polypeptide of molecular weight 44,000 present in PC13TG8 is substantially reduced in amount in R5/3 but is again present in H2T12, and the possibility 1hat this may be a cytoskeletal protein is currently under investigation. A direct role for this protein in metabolic cooperation is, however, unlikely since it is not present in all clones of embryonal carcinoma cells capable of metabolic cooperation. Thus it may represent a protein whose synthesis is dependent upon metabolic cooperation.

D. CELLHYBRIDSA N D HETEROKARYONS Two metabolic cooperation-defective cell types have been studied in fusion hybrids and heterokaryons formed with cooperation-competent cell types, and in both cases the defect behaved as a recessive trait. Heterokaryons are here defined as the initial multinucleate products of cell fusion, whereas hybrids are the viable progeny of the incorporation of the different genomes into a single nucleus. Azarnia and Loewenstein (1973) found that both heterokaryons and hybrids formed between the cooperation-defective A cell line and either of two competent cell lines were capable both of ionic coupling and dye transfer. Hybrids between L cells and cooperation-competent cell lines show metabolic cooperation (McCargow and Pitts, 1971), ionic coupling, fluorescent dye transfer, and gap junctions (Azarnia et al., 1974) so long as appreciable chromosome loss has not occurred. In hybrids between L cells and normal human fibroblasts, however, the frequent spontaneous loss of human chromosomes from mouse-human

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hybrids (reviewed by Ringertz and Savage, 1976) generates segregants that are cooperation-deficient. These fall into two classes (Azamia and Loewenstein, 1977; Larsen et a l . , 1977): a majority, which lack the capacity for both ionic coupling and fluorescein transfer and in which no gap junctions can be detected, and a minority, which show ionic coupling but not fluorescein transfer. The latter class of hybrids do not possess normal gap junctions, but freeze-fracture of their junctional membrane shows small arrays of fibrils of a diameter similar to that of gap junction particles (Section VI). These structures are also present, together with gap junctions, in the cooperation-competent primary hybrids but are not found in either parental cell type. The authors suggest that they are deviant aggregates of gap junction particles that have either junctional channels of smaller pore size or reduced numbers of junctional channels and arise because a defective mouse gene product interferes with normal gap junction assembly by competing with the corresponding normal human gene product. According to this theory, the number of copies of the human gene present in a hybrid would determine whether fibrillar arrays only or fibrillar arrays plus normal junctions could be formed. Other interpretations are, however, not excluded. Cooperation-defective hybrids showed increased tumorigenicity and increased saturation density of growth in vitro compared with competent hybrids: this will be discussed in Section IX. A provisional assignment of a human gene that complements the L cell defect to chromosome 11 has been reported (Loewenstein, 1978). Clements and Subak-Sharpe (1975) showed that the APRT enzyme activity necessary for a cell to function as a donor in metabolic cooperation for adeninederived nucleotides could be transiently introduced into an APRT- cell by fusion with a chick erythrocyte.

E. PERMEABLE JUNCTION DEFICIENCIES I N EXPERIMENTAL ANIMALS A N D MAN To date, genetic studies of metabolic cooperation at the whole-animal level have been restricted to ultrastructural observations. These have focused on inherited conditions where cell interactions in certain tissues are visibly abnormal under the light microscope. In each case, only the affected tissue has been studied so that there is no information about the tissue specificity of the communication defect or whether it causes or is caused by the other disturbances seen or is a parallel effect of a common underlying lesion. The human syndrome, hereditary mucoepithelial dysplasia, which is inherited as an autosomal-dominant trait, involves deficiences in cell adhesion and keratinization in the epithelia of all the orificial mucosa. Ultrastructural studies of gingival biopsy material showed few desmosomes (see Section VI ,A) and the presence of cytoplasmic structures resembling gap junctions and hemidesmosomes (Witkop et a l . , 1978a,b, 1979). The authors suggested that the disease

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M. L. HOOPER A N D J . H. SUBAK-SHARPE

involved a defect in the assembly of gap junctions and desmosomes at the cell surface. Mice homozygous for the mutation t 9 , one of a series of recessive mutations at the complex T locus (Sherman and Wudl, 1977) are histologically abnormal at 9 days of gestation with an enlarged primitive streak and a deficiency of mesoderm and subsequently die in utero. Mutant mesoderm, although showing increased areas of cell apposition compared with normal mesoderm, has smaller and sparser gap junctions (Spiegelman, 1976). A similar mutation, talpid3, occurs in the chick (Ede et a l . , 1974), but no studies of its effect on gap junctions have been reported. The recessive mutation Splotch in the mouse causes a defect in closure of the neural tube:. Study of the neuroepithelium early on the ninth gestation day, just prior to the development of visible abnormalities, showed an increased incidence of gap junctional vesicles (Sections VI,A and VIII) in the homozygous mutant Sp/Sp compared with control littermates (Wilson and Finta, 1979). The control littermates., a mixture of +/+ and +/Sp embryos, showed a small number of such vesicles, but the authors reported preliminary data suggesting that these were contributed by the heterozygote only. Flint and Ede (1978) reported an increased incidence of gap junctions in the sclerotome of mouse embryos homozygous for the recessive mutation amputated; these mutant mice subsequently develop skeletal abnormalities and die at term. However, since Flint and Ede classified as gap junctions structures with an extracellular gap of 2.5 to 10 nm, this conclusion needs confirmation. Hyperplasia of the lens has been found in two strains of chick (Hy- 1 and Hy-2) that were selected for rapid growth. In these strains, the incidence of gap junctions in the lens is reduced (Odeigah et a l . , 1979).

VI. Properties of Permeable Junctions A . ULTRASTRUCTURE

Electron microscope studies have identified a number of specialized regions of contact between the plasma membranes of adjacent cells; the most widely distributed specializations are desmosomes, tight junctions, and gap junctions (Staehelin, 1974). A number of lines of evidence support the view that gap junctions are mediators of metabolic cooperation. They are widely distributed in tissues showing ionic coupling (Staehelin, 1974) and in certain cases are the only recognizable membrane specialization (Revel et a l . , 1971). Treatments that disrupt gap junctions interrupt ionic coupling (Barr et a l . , 1965; Pappas et a l . , 1971), whereas, in mammalian heart, the only other type of junction present (the desmosome) can be selectively split without effect on ionic coupling (Dreifuss et

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al., 1966). L cells, which are defective in metabolic cooperation, are also defective in gap junction formation (Gilula et al., 1972). As described earlier (Section V,D), in hybrids between L cells and human fibroblasts, ability to participate in dye-coupling segregates concordantly with ability to form gap junctions. Finally, as described in Section V,C, the incidence of gap junctions is reduced in a metabolic cooperation-defective embryonal carcinoma cell variant and restored in a cooperation-competentrevertant. At present, however, the possibility cannot be excluded that tight junctions also mediate intercellular metabolite transfer since cell types studied that form tight junctions also form gap junctions. Unfortunately, it has not yet been possible to study coupling between hair cells and supporting cells in the reticular lamina of the organ of Corti. In the chinchilla (Gulley and Reese, 1976) and the chick (Ginzberg and Gilula, 1979), although not in the alligator lizard (Nadol et al., 19761, hair cells and supporting cells are connected by tight junctions but not by gap junctions. The gap junction was first clearly distinguished from the tight junction by Revel and Karnovsky (1967) who showed by thin-section electron microscopy that the membrane bilayers of the adjacent cells were separated by a 2- to 4-nm gap that could be permeated by colloidal lanthanum. Subsequent freeze-fracture investigation showed that the bilayers contained arrays of intramembranous particles packed in a roughly hexagonal array. Purified gap junction preparations obtained by subcellular fractionation have been studied by optical diffraction analysis of electron micrographs of negatively stained material and by X-ray diffraction (reviewed by Bennett and Goodenough, 1978; see also Henderson et al., 1979). This work has led to the current view of the gap junction as an array of hydrophilic pores that run through the center of hexameric protein particles or “connexons,” the particlesof the two bilayers lying in register so as to produce a continuous aqueous channel connecting the cytoplasms of the two cells. Variations in junction shape, thin-section profile, and particle-packing geometry in gap junctions from different sources have been reported (reviewed by Larsen, 1977). In some cases, these are the result of differences in preparation techniques, and in particular, an increase in packing density and regularity of intramembranous particles is seen in preparations uncoupled by various treatments (see review by Perrachia, 1977). In isolated gap junctions, a reversible interconversion between two forms with the same lattice constant but different connexon features has been reported by Zampighi and Unwin (1979; see also Unwin and Zampighi, 1980). However, in the other cases, there do appear to be real differences between junctions from different origins: in particular, arthropod gap junctions have larger intramembranous particles than nonarthropod junctions and can also be distinguished by the membrane face to which the particles remain attached after freeze-fracture (Staehelin, 1974; Gilula, 1978). Other variants include reflexive junctions and gap junctional vesicles (Larsen, 1977; see also Section VIII), whereas in heart muscle, gap junctions and desmosomes occur

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together in an organized structure known as the intercalated disc (see review by De Mello, 1977). The only difference in architecture detected between rectifying and nonrectifying junctions is the presence of cytoplasmic vesicles in association with rectifying junctions (Hanna et al., 1978). Intramembranousparticles similar to those seen in gap junctions are also seen in developing tight junctions (Montesano et al., 1973, and this has led to a suggestion that gap junctions and tight junctions may be assembled from a common precursor (Elias and Friend, 1976). B. BIOCHEMICAL ANALYSIS Until recently, analysis of the protein and lipid composition of isolated gap junctions gave conflicting results, probably due to the use of proteolytic enzymes and lipid solvents in the purification procedures (see review by Bennett and Goodenough, 1978). More recent purifications of gap junctions from rodent liver, which avoid the use of proteolytic enzymes, give a preparation with a major protein component whose mobility in SDS-polyacrylamide gel electrophoresis corresponds to a molecular weight of approximately 26,000 (Hertzberg and Gilula, 1979; Henderson et al., 1979). The presence of a similar protein has been reported in gap junction preparations from a number of sources (Goodenough et a l . , 1978; Aka16 et a l . , 1978; Finbow et al., 1979). Additional bands (reported by several groups) at molecular weight 47,000 and 21,000 can be explained by dimerization or partial degradation of the 26,000-MW species (Hertzberg and Gilula, 1979; Hertzberg et al., 1978; Henderson et a / ., 1979). A hexamer formed from a monomer of molecular weight 26,000 would have a molecular weight in the range within which that of the connexon was deduced to lie from X-ray and optical diffraction studies (Makowski et al., 1977). C. MOLECULAR WEIGHTEXCLUSION LIMIT

The effective pore size of permeable junction channels in the salivary gland of the midge Chironomus has been investigated using a series of fluorescent probes of different molecular weights (Simpson et al., 1977; hewenstein et al., 1978a). Probes of molecular weight 1664 and below were found to pass from cell to cell, whereas those 1926 and above were found not to. As mentioned in Section VI,A however, arthropod junctions differ in ultrastructure from nonarthropod junctions and Flagg-Newton et al. (1979) have shown that the pore size of mammalian gap junctions is smaller. This agrees with the results of Pitts and Finbow (see Section 111,B ,4) who found intercellular transfer of tetrahydrofolate (MW 446) but not of its polyglutamyl derivative between hamster tissue CUlNre cells, and of Imanaga (1974) who found that Chicago blue, a dye of molecular weight about 1O00, did not pass between cells of sheep and calf heart muscle.

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Molecular weight exclusion limits in this range correspond to a pore size of the order of 15 d;, which is consistent with data from ultrastructural studies of gap junctions (Bennett and Goodenough, 1978). Weingart (1974) estimated a pore size of somewhat more than 10 d; from quantitative comparison of the permeabilities of sheep and calf heart junctions to K+, tetraethylammonium ions, and Procion yellow, and so did Brink and Dewey (1978) who extended this analysis to include a range of fluorescent probes. Sheridan et at. (1978) concluded that their quantitative measurements of gap junction area and junctional conductance in Novikoff hepatoma cells were consistent with the hypothesis that gap junctional particles contained central hydrophilic channels, wbich were about 20 d; in diameter and which had cytoplasmic resistivity. The accumulated experimental data suggest, therefore, that the gap junction functions as a molecular sieve with a pore size in'the range 10-20 d; and apparently without specificity for the permeant molecule except on the basis of molecular weight. In the salivary gland of Chironomus, the pore size can be reduced by injection of calcium ions into the cell (Section VI,D) and has been reported to change during development (Loewenstein, 1978). Such behavior has obvious implications with regard to the role of the permeable junction (Section IX). D. FACTORS AFFECTING PERMEABILITY Table I1 summarizes the effects of a number of factors on permeable junctions. It is important to distinguish between effects on junction formation and effects on established junctions: we will consider the latter here and the former in Section VIII . The effects of divalent cations, particularly Ca2+,on permeable junctions have been extensively studied. Whereas extracellular fluid typically contains about M Ca2+, the concentration in cytoplasm is more than 1000-fold lower (see discussion in Rose and bewenstein, 1971). bewenstein et at. (1967) showed that, if the cytoplasmic Caz+concentration was raised by any of three techniques in Chironomus salivary gland, the permeability of the junctional membrane fell. By using the protein aequorin, which fluoresces with an intensity that increases with Ca2+concentration, it was shown that the diffusion of Ca2+ions is restricted in cytoplasm as a consequence of energy-dependent sequestering and that only when the concentration is high at the junction itself is the permeability lowered (reviewed by Loewenstein and Rose, 1978). At cytoplasmic concentrations of Ca2+between lo-' and M, there is a progressive reduction in the molecular weight exclusion limit (Rose et ai., 1977), and it is possible to resolve changes in permeability due to the action of CaZ+ions on individual channels of the junction (see Section VIII). Difficulties have been encountered in extending these observations to mammalian tissue culture cells (Gilula and Epstein, 1973, but recent work indicates that this may be due to more efficient local calcium-buffering

82

M. L. HOOPER AND J . H. SUBAK-SHARPE TABLE I1 FACTORS AFFECTING PERMEABLE JUNCTION FORMATION AND Factor

Effect on formationaBb

PERMEABILITY

Effect on permeability of established junctions“.’

1. Ion composition and pH of

intracellular and extracellular fluid Intracellular injection of Ca 2+ Intracellular injection of Srz+ Omission of divalent cations from medium Addition of CO, to medium Intracellular injection of Na Replacement of medium Na+ by Li choline Replacement of medium C1- by propionate acetate sulfate isethionateC nitrate glycerophosphate Removal of serum from medium

I [see text]

N(1). I(2) [see text] I(3) [reversible]

+

+

I(2) [irreversible] “1, 2) I(4) [reversible] l(4) [reversible] I(4) [irreversible] I(4) [irreversible] N(4) N(4)

2. Metabolic inhibitors Cytochalasin B Colchicine Colcemid Hydroxyurea Dinitrophenol

CN Oligomycin N-Ethyl maleimide Ouabain Azide p-Chloromercuribenzene sulfonate (continued)

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TABLE I1 (continued)

Effect on formation".b

Factor

Effect on permeability of established junctions".*

~

p-Chloromercuribenzoate Reserpine Acetazolamide Fluoride Iodoacetate Cycloheximide Puromycin

"5) N(5). I(1I) [see text] N(12), I(1I) [see text]

3. Cyclic nucleotides, hormones, and vitamins Dibutyryl cyclic AMP

S(5, 13) [antagonized by

Thyroxine (ependymoglia)

S( 15) [blocked by cyclohexi-

8Br-cGMP(5)] mide] Human chorionic gonadotropin (ovarian inters(16) stitial cells) Corticosterone (Chironomus salivary gland) Aldosterone Retinoic acid (chick embryo stratified squamous epithelium) S( 17) Caerulein (analog of cholecystokinin) (pancreas acinar cells) Bombesin 1 Acetylcholine 4. Surface modification of

cells l(30) [blocked by glucocorticoids] N(1)

Trypsin Neuraminidase Phospholipase C Lipase

I(30) [blocked by glucocorticoids] N(1) l(30) [blocked by glucocorticoids]

Urea EGTA Hydrogen peroxide Fab fragment of anti-F9 (embryonal carcinoma cells)

I(1)

I(22, 23) (conrinued)

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M. L. HOPER AND J . H. SUBAK-SHARPE TABLE I1 (continued)

Factor

Effect on formation".b

Effect on permeability of established junctions",b

5. Physical parameters and treatments Temperature reduction Cell cycle phase Increased passage number Enucleation Karyoplast preparation

I( 18) 1(21) N(27, 28) I(28)

" S, Stimulation; I , inhibition; N, no effect.

bReferences: ( I ) Cox eral., 1974; (2) Rose and Loewenstein, 1971; (3) Turin and Warner, 1977; (4) Asada and Bennett, 1971; (5) Sheridan, 1978; (6) Stoker, 1975; (7) It0 et al., 1974; (8) Hulser and Webb, 1973; (9) Cox e: al., 1972; (10) Politoff e: al., 1969; ( 1 1) Griepp and Bemfield, 1978; (12)Goshima. 1971;(13) Wrightetal., 1976b; (14)Hax etal., 1974;(lS)Decker, 1976;(16)Burghardt and Anderson, 1979; (17) Elias and Friend, 1976; (IS) Kam e: al., 1978; (19) O'Lague e t a l . , 1970; (20) Merk and MacNutt, 1972; (21) Kelley e t a l . , 1979; (22) Jacob, 1978; (23) Dunia e: al., 1979; (24) DeMello, 1977; (25) Iwatsuki and Petersen, 1978; (26) Epstein etal.. 1977; (27) Bols and Ringertz, 1979; (28) Cox et al.. 1976; (29) DeMelIo, 1979; (30) Suzuki and Higashino, 1977. lsethionate = 2-hydroxyethanesulfonate.

capacities brought about by the architecture of the tissue culture cell in the region of the gap junctions, which tend to be situated in fine cell processes (FlaggNewton and bewenstein, 1979). Junctional permeability is also influenced by cytoplasmic pH (Turin and Warner, 1977) and, since pH and Ca2+concentration in the cytoplasm are interdependent, the question has been raised whether pH, rather than Ca2+concentration, is the primary controlling variable. As a result of an extensive analysis of the effect of a variety of treatments on cell coupling, pH, and Ca2+ concentration, Rose and Rick (1978) conclude that changes in junctional permeability parallel changes in Ca2+concentration rather than in pH. A similar debate has centered on the observation that a rise in cytoplasmic Ca2+ concentration leads to membrane depolarization (see Sheridan, 1978); Loewenstein and Rose (1978) argue, however, that depolarization cannot be the primary controlling variable. A direct effect of Ca2+ on the packing geometry of intramembranous particles in isolated gap junctions from calf eye lens fiber cells has been demonstrated by Peracchia (1 978): this may be the ultrastructural correlate of the change in permeability. An increase in the cytoplasmic Ca2+concentration is expected as a result of a number of the treatments (listed in Table 11) that reduce junction permeability, including, paradoxically, the omission of Ca2+ from the extracellular medium (Rose and Loewenstein, 1971). Effects of Ca2+

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omission on junctional permeability are seen in some cell types but not others (Table 11; see discussion in Loewenstein et a!., 1967). This may simply reflect differences in the rate of release of Ca2+previously bound. The drug cytochalasin B, which inhibits microfilament function, has been reported to inhibit metabolic cooperation for nucleotides (Cox et al., 1974; Stoker, 1975) but not ionic coupling (It0 et al., 1974). This may reflect differences in the sensitivity of the measurement techniques or may indicate an effect on metabolic cooperation at a step other than intercellular transfer. Dinitrophenol, which uncouples oxidative phosphorylation from electron transport, has been reported to inhibit ionic coupling in Chironomus salivary gland cells (Politoff et af ., 1969) but to have little effect on metabolic cooperation for nucleotides between mammalian tissue culture cells (Cox er al., 1972). The reason for this discrepency is unclear: no study has been reported of the effect of dinitrophenol on ionic coupling in mammalian tissue culture cells. Hulser and Webb (1973) reported a correlation between morphology and ionic coupling in established tissue culture lines, finding that all seven fibroblastic lines that they studied were capable of ionic coupling, whereas all of their seven epithelial cell lines were not. Subsequent work has shown that this is not in general true (discussed by Loewenstein, 1979).

VII. Incidence and Specificity of Permeable Junction Formation A. OCCURRENCE in Vivo

Permeable junctions are found between cells in organisms of all metazoan phyla from the porifera to the chordates (reviewed by Loewenstein, 1979; Bennett and Goodenough, 1978; Staehelin, 1974). They are formed by a wide variety of cell types, and to date, the only cell types known to be incapable of intercellular communication are ones that either do not undergo cell division or are not part of an organized tissue. In the first category, skeletal muscle myotubes do not form permeable junctions, although their dividing precursors, the myoblasts, do (Kalderon et a l . , 1977). Permeable junctions between neurons are rare, being limited to the highly cell-specific electrotonic synapses, and neurons do not form permeable junctions with glial cells although the latter form permeable junctions extensively between themselves (Kuffler and Potter, 1964). The hair cells of the organ of Corti do not form gap junctions, at least in some species (Section V1,A). In the second category, most circulating blood cells are not connected by permeable junctions, although exceptions such as stimulated lymphocytes are known (Section IX).

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M. L. HOOPER AND J . H. SUBAK-SHARPE

B. COMBINATIONS OF CELLSFROM DIFFERENT SPECIES Early experiments (Section III,A) showed that metabolic cooperation took place readily ,between cells of different mammalian species, and no species barriers to metabolic cooperation have been found within the mammals. Epstein and Gilula (1977) studied the formation of permeable junctions in heterologous combinations of tissue culture cells from mouse, chick, and different orders of arthropods, each of which formed homologous junctions readily. Permeable junctions were formed between mouse and chick cells and between lines from the same arthropod order, but little intercellular communication was found between cell lines from different arthropod orders and none between arthropod and vertebrate cell lines. This last observation is probably related to the difference in gap junction ultrastructure in the arthropods compared with other phyla (Section V1,A). A second instance of species specificity within a phylum is provided by observations on the development of ionic coupling in aggregates of sponge cells (Loewenstein, 1967).

C. COMBINATION OF DIFFERENT CELLTYPES Histiotypic preference in the formation of permeable junctions has now been described for a number of cell combinations. Pitts and Burk (1976) found that BHK cells (baby hamster kidney fibroblasts) formed permeable junctions very slowly with BRL cells (an epithelial line from rat liver), whereas each cell type formed homotypic junctions readily. A similar observation was reported for human mammary duct epithelial cells and mammary fibroblasts (Fentiman et af., 1976), and Pitts (1978) has reported that it is a common phenomenon for epithelial and fibroblastic cell lines from a variety of organs. Interestingly, this preference is not shown by certain epithelial cell lines obtained from breast tumors (Fentiman and Taylor-Papadimitriou, 1977; Fentiman et al., 1979). Lack of histiotypic preference is, however, not restricted to tumor lines: various normal cell types, including lens epithelial cells (Fentiman et af., 1976), pigmented retinal epithelial cells, and keratinocytes (Pitts, 1978), will cooperate both with fibroblastic and with epithelial cell lines. Gaunt and Subak-Sharpe (1979) extended these observations and found additional cases where heterotypic interactions were formed less readily than homotypic interactions. Furthermore, L cells differed from the other cell lines studied in forming homotypic interactions less readily than certain heterotypic interactions (e.g., with PyY cells, a line of polyoma-transformed baby hamster kidney cells). They therefore suggested that the lack of communication previously reported for L cells (Section V,A) was a consequence not of an absolute defect in ability to form junctions but of an unusual histiotypic preference. Within a given coculture, some contacts between donor and recipient cell types showed strongly positive evidence for metabolic cooperation whereas others showed none. This behavior persisted in freshly

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cloned cell populations and was therefore not due to genetic heterogeneity. It appeared, rather, that each cell pair had a certain probability of forming a permeable junction in a stated time. Gaunt and Subak-Sharpe proposed two possible explanations that could account for this: One proposal postulates that contact of two cells leads to an initial state that does not establish junctional communication, but from which, with a certain probability per unit time, they can progress to permeable junction formation. The alternative proposal postulates that cells may contact one another for limited and variable periods of time due to cell movement and that poor compatibility for junction formation may result from a requirement for a long period of contact before a junction can be formed: though, once formed, a permeable junction stabilizes the cell-to-cell contact. This raises the question of whether histiotypic preference in permeable junction formation is related to other instances of intercellular recognition such as selectivity in adhesion and “sorting out” in heterotypic cell mixtures (Marchase er af., 1976; Edwards, 1977) and density-dependent growth inhibition in mixed cultures (Weiss and Njeuma, 1971). As in the case of metabolic cooperation, cell type is more important than species (within the warm-blooded vertebrates) in determining patterns of sorting out (Marchase er a l . , 1976), and Pitts and Biirk (1976) noted that BHK and BRL cells, as well as cooperating poorly with each other, tended to sort out. Further study is needed, however, before it can be concluded that these phenomena are related. Gaunt (1979) has observed that fusion of an L cell to another cell type can modify its histiotypic preference for metabolic cooperation. Further work is needed to elucidate the laws governing permeable junction formation between different cell types.

VIII. Kinetics of Permeable Junction Formation and Breakdown The formation of permeable intercellularjunctions requires the prior formation of stable intercellular adhesions (Marchase et a l . , 1976; Edwards, 1977). This in itself involves at least two steps: an initial loose association not requiring energy is followed by an energy-dependent stabilization of binding (Umbreit and Roseman, 1975). Nevertheless, the presence of permeable junctions can be detected within a few minutes of initial cell contact in a number of systems (reviewed by Sheridan, 1978). In the early phase of junction development between Xenopus blastomeres, Loewenstein et al. (1978b) were able to resolve quanta1 increments in junctional conductance that they interpreted as the opening of individual membrane channels. The earliest detectable ultrastructural event in the formation of the gap junction is the appearence of so-called “formation plaques” (reviewed by Bennett and Goodenough, 1978), where the interacting cell membranes approach to a distance of about 10 nm and flatten. Few intramembranous particles are present, with the exception of large 10-nm-diameter particles similar to those seen in

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M. L.HOOPER AND J. H . SUBAK-SHARPE

some systems associated with gap junction edges. The large particles decrease in number as the junction develops, suggesting that they may be precursors of its intramembranous particles. Table I1 lists some of the factors affecting the rate of permeable junction formation. The different conclusions reported for the effect of puromycin are probably a consequence of the use of different concentrations of the drug, whereas, with cytochalasin B, different results have been obtained using different assays for junctional permeability. The reason for the conflict in observations on the effect of cycloheximide is, however, unclear. Where intercellular communication is stimulated, e.g., by hormones, it is not always clear whether this represents an effect on junction formation or on the permeability of existing junctions, and only studies that distinguish between these possibilities have been included in Table 11. Other studies have shown effects of gonadotropin on communication between oocytes and follicle cells (Browne et al., 1979) and of P-ecdysone on communication in mealworm epidermis (Caveney, 1978) and in Drosophila salivary gland (Haxet a l . , 1974). Yotti et al. (1979) have reported that treatment of cocultures of wild-type and thioguanine-resistant Chinese hamster V79 cells with tumor promoters interferes with “kiss of death” killing in thioguanine (Section 11,D).As yet, there is no evidence that this effect is due to a reduction in the level of intercellular communication, (cf. Section IV) but if it is, then the phenomenon of tumor promotion may find an interpretation in terms of the theory that metabolic cooperation plays a role in growth control (Section IX,C). Cooling below 30°C reduces the rate of permeable junction formation (Kam et a l . , 1978), but gap junction formation is less temperature sensitive than the formation of desmosomes so that, at 2”C, the gap junction becomes the predominant junctional specialization in aggregating fibroblasts (Lloyd et al., 1976). In general, the turnover of gap junction proteins appears to be slow (Gurd and Evans, 1973). In tissues where there is extensive gap junction breakdown, gap junctional vesicles (internalized or annular gap junctions) are found (discussed by Gilula, 1978; Ginzberg and Gilula, 1979), and it is assumed that these structures represent intermediate stages in the process of degradation. Actin-like microfilaments have been found in association with gap junctional vesicles (Larsen et a l . , 1979) and may play a role in the internalization process.

IX. Possible Functions of Metabolic Cooperation A. COORDINATION OF TISSUE ACTIVITIES The widespread occurrence of permeable junctions between cells of organized tissues suggests that such cells pool their resources of some or all lowmolecular-weight metabolites and retain their individuality principally with re-

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gard to macromolecular components. This option can be put to use in a number of ways. It allows the smoothing out of local heterogeneities in the distribution of small molecules. Pitts (1976) has estimated, on the basis of the doubling time of cells dependent upon metabolic cooperation for growth, that the rate of movement of nucleotides between cells can be at least 106 nucleotides per second per cell pair, which would allow rapid equilibration over substantial distances. This could be important where cells differ in accessibility to blood supply. An extreme example occurs in the lens, which is not vascularized: here gap junctions are extremely abundant, accounting for as much as 25% of the total cell surface (Benedetti et al., 1976). Phenotypic correction of local cellular defects arising by somatic mutation could clearly be achieved by metabolic cooperhtion. Such correction presumably occurs in females heterozygous for the X-linked recessive allele of Lesch-Nyhan syndrome, where both HGPRT+ and HGPRT- cells are present as a result not of somatic mutation but of X-chromosome inactivation (reviewed by McKusick, 1978). Another model system that may be relevant is provided by the experiments of Van Buul et al. ( 1 978). These workers showed that fibroblasts from patients with Bloom’s syndrome, which exhibit an abnormally high frequency of sister chromatid exchange, showed a reduced frequency when cocultured with normal cells. This effect is not known to involve metabolic cooperation but does depend upon cell contact. Metabolic cooperation may also play a role in the transmission of controlling signals through a tissue. Transmission of hormonally triggered stimuli from cell to cell, as demonstrated by the work of Lawrence et al. (1978; see Section 111,B,2) could be the method of achieving coordinate regulation of a whole tissue in response to the direct interaction of hormone with some of its cells. Sheridan et al. (1975, 1979) interpret their observations on the incorporation of labeled hypoxanthine and formate in cocultures of HGPRT+ and HGPRT- cells as an increase in HGPRT activity in the wild-type cells in response to a signal from the variant cells and a decrease in the activity of the de nova purine synthesis pathway of the variant cells in response to a signal from the wild-type cells. These effects did not occur with L cells, indicating a role for permeable junctions. Metabolic cooperation may also serve to coordinate the activities of different cell types in a tissue such as the pancreas (Meda et al., 1979). Specific functions of permeable junctions peculiar to individual tissues are known to exist. Permeable junctions between neurons function as synapses in the transmission of nerve impulses. Electrotonic synapses are less common than chemical synapses but are often found where speed of response is important. Ability to transmit impulses in both directions may also be an important property (Bennett, 1977). Lymphocytes activated by phytohemagglutinin or by exposure to antigen show ionic coupling, fluorescent dye transfer, and gap junctions (Hulser and Peters, 1972; Sellin etal., 1974;de Oliviera-Castro et al., 1975). AS discussed in Section V,A, the reported failure to detect metabolic cooperation for nucleotides is probably due to artifact. Coupling is minimal prior to activation

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and develops within minutes of the addition of phytohemagglutinin (Hulser and Peters, 1972). This, together with the observation that cell contact is essential (Peters, 1972), suggests a role for permeable junctions in lymphocyte activation. A role in cell-mediated cytotoxicity is suggested by the observation of Sellin et al. (197 1) that the incidence of fluorescein-permeable junctions between lymphocytes and target cells is increased by prior sensitization of the lymphocytes to the target cells. It has been suggested that in the ovary the maintenance of meiotic arrest in the oocyte is dependent upon signals transferred via gap junctions from the granulosa cell (Anderson and Albertini, 1976). The coupling of receptors in the retina via gap junctions may provide a means of regulating visual acuity in response to levels of illumination (discussed by Fain et at., 1976).

B. SYNCHRONIZATION OF CELLULAR BEHAVIOR The involvement of the gap junction in the synchronizationof muscle contraction is well established (Section 111,G). The atrioventricular node of the heart, which introduces a delay in the transmission of the contraction signal from the atrium to the ventricle, has a substantially reduced incidence of gap junctions compared with other regions of the heart (Pollack, 1976). In the myometrium of the uterus, gap junctions are absent during pregnancy, but their incidence increases markedly just prior to term. This probably plays a role in the onset of uterine contractions (Garfield et al., 1977, 1978). As discussed earlier, electrotonic synapses between neurons often occur where speed of response is essential. One such instance is in synchronized systems such as the control systems for the specialized electric organs of certain fish (Bennett, 1977). A possible third role for the gap junction in the synchronizationof cell behavior is to be found in the testis. In spermatogenesis, the germ cells develop synchronously over segments of the seminiferous tubule up to 1 mm long. Local synchrony is assured by the syncytial nature of germ cell clones, but this cannot account for synchrony over so extensive a segment. Germ cells do not form gap junctions with each other or with the fixed Sertoli cells of the tubule wall; however gap junctions do occur between adjacent Sertoli cells (Gilula et al., 1976) and this would provide a means of synchronizing the metabolism of the Sertolk cells and hence the microenvironment in which the germ cells develop.

C. GROWTHCONTROL Cells in tissue culture are subject to two distinct influences of cell density on growth rate. At low cell densities, growth rate commonly increases with cell density. A number of different mechanisms contribute to this “feeder effect” (see Weiss and Njeuma, 1971; Gaunt and Subak-Sharpe, 1977, for references).

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An influence of contact with other cells or extracellular matrix secreted by them has been demonstrated, as well as a separate effect of molecules capable of diffusion through the medium. The latter may be specific growth factors or molecules as simple as nonessential amino acids. No information is yet available concerning the question of whether metabolic cooperation plays any role in low cell density growth control. At high cell density, there is a density-dependent growth inhibition. Loss of density-dependent growth is frequently associated with the acquisition of tumorigenicity, although the correlation is not perfect (Shields, 1976). Stoker (1967) found that polyoma-transformed BHK cells, which did not show density-dependent growth inhibition when cultured alone, were subject to inhibition by normal cells. However, this is the exception rather than the rule. Although all normal cells, cultured alone, exhibit density-dependent growth inhibition, in mixed culture even two normal cell lines may fail to show reciprocal inhibition (Weiss and Njeuma, 1971). Holley (1975) has argued that density-dependent inhibition is due not to cell contact but to a quantitative increase in the requirement for macromolecular growth factors as cell density increases. However, Loewenstein has advanced evidence that favors a role for metabolic cooperation in growth control (reviewed by Loewenstein, 1979). The evidence can be summarized as follows. First, cells of all organized tissues capable of cell division possess permeable junctions. Second, some tumor-derived tissue culture lines are defective in permeable junction formation or have altered histiotypic preference. Third, all tissue culture lines defective in permeable junction formation show tumorigenicity , lack of density-dependent growth inhibition, or both. Finally, in two cases, hybrids between cooperation-defective and competent cell lines show both cooperation competence and low tumorigenicity so long as appreciable chromosome loss has not occurred. This, of course, shows only that both communication deficiency and tumorigenicity behave as recessive traits in these cells, but in the case of the L celYhuman fibroblast hybrids described in Section V,D, human chromosome loss resulted in the concordant appearanceof communication-deficiency,densityindependent in v i m growth and high tumorigenicity (Azarnia and Loewenstein, 1977). The hybrids showing ionic coupling but not fluorescent dye transfer (Section V,D) were excluded from this analysis as their chromosome constitution was too unstable. The interpretation of the data is subject to some reservations because in general the communication-incompetentsegregants had many fewer chromosomes than the competent hybrids, and in only one case could the loss of competence be associated with the loss of as few as two chromosomes. Nevertheless, while still circumstantial. these results are encouraging, and a more recent paper (Loewenstein, 1978) reports that the correlation has been substantiated by the study of a further 15 clones although the detailed analyses have not yet been published. A role for gap junctions in growth control in regenerating liver is

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suggested by the observation of Yee and Revel (1978) that, after partial hepatectomy, gap junctions between hepatocytes transiently disappear in parallel with the appearance of a peak in mitotic activity. Loewenstein (1968, 1979) has proposed a model for growth control involving the asynchronous production of bursts of a division-promotingfactor by the cells in a population and the diffusion of this factor from cell to cell via gap junctions. The concentration of the factor in a particular cell would be sensitive to the size of the coupled cell population and to whether the cell itself occupied a central or a peripheral position, allowing cell division to be regulated in response both to total and to local cell number. The model was further developed by Burton (197 1) and Burton and Canham (1973). By introducing the condition that the rate of production of the division-promotingfactor varied sinusoidally with time, they endowed the system with the further property of “superpeaking,” i.e., an increase in the amplitude of the oscillation for small cell aggregates compared with that for single cells. Such a mechanism could provide a contribution to the feeder effect. Density-dependent growth inhibition is not to be confused with contact inhibition of cell locomotion (reviewed by Harris, 1974). There is no evidence to suggest a role for metabolic cooperation in contact inhibition of cell locomotion. D. DIFFERENTIATION A N D DEVELOPMENT Embryonic development provides perhaps the most impressive examples of regulation by cell-cell interaction. The clearest instances are to be found in the phenomena of embryonic induction and pattern formation. Embryonic induction (reviewed by Saxen et a l . , 1976) is said to occur “whenever in development two or more tissues of different history and properties become intimately associated and alteration of the developmental course of the interactants results” (Grobstein, 1955). The dependence of induction on cell contact has been investigated in many inductive systems by culturing the interacting tissue types on opposite sides of a Millipore filter. In general, induction occurs under these conditions, which appeared to rule out a requirement for cell contact. However, recent work has shown that cytoplasmic processes can penetrate these filters where they may be poorly preserved on fixation unless appropriate techniques are used. On reinvestigation of the induction of kidney tubules in metanephric mesenchyme by dorsal spinal cord, it was found that where cell contact through penetrating processes did not occur, induction did not take place (Saxen et a l . , 1976). In other systems, however, such as primary induction in amphibia (SaxCn et a l . , 1976) and the production of the primary corneal stroma from the corneal epithelium under the influence of the lens (Hay, 1977), there is good evidence that cell contact is not required. The mere presence of a basement membrane between the two interacting cell types does not preclude cell contact, as cell processes can penetrate through the basement membrane (Saxen et a l . ,

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1976). Thus, at least in some cases, cell contact is required for embryonic induction, and although there is at present no evidence for or against a role for metabolic cooperation in these cases, the hypothesis that it is involved in an obvious and attractive one. Pattern formation (reviewed by Gierer, 1977) is the process of specifying the spatial pattern of cellular differentiation in an initially near-homogeneous structure. The manner in which pattern formation can be regulated by the embryo in response to various experimental manipulations implies that pattern formation occurs in two stages: a cell first acquires information about its position and then interprets this according to its own genetic information content and developmental history. Various theories have been proposed that attempt to explain the specification of positional information by “prepatterns, i.e., spatial patterns of some physical property that determine the positions at which subsequent developmental events proceed. Wolpert (1968) proposed a prepattern consisting of a linear concentration gradient of a morphogen produced by a localized group of cells (a source) and diffusing to a localized sink, where it is destroyed. Goodwin and Cohen (1969) proposed that a periodic event is propagated from a localized “pacemaker region” together with a more slowly propagated wave. The phase difference between the two waves would then provide positional information. Both these models require a preexisting pattern and therefore cannot in themselves explain pattern formation. This problem can be overcome by theories based on the work of Turing (1952). His analysis of reactions in solution predicted that if a minimum of two substances act by auto- and cross-catalysis on their own and each other’s production and if the only factor acting to remove spatial inhomogeneities in concentration is diffusion, then under certain conditions, stable spatial patterns of concentration can be spontaneously set up. The mathematical properties of such systems have since been extensively studied (reviewed by Nicolis and Prigogine, 1977) and the theory applied to prepattern formation by Gierer and Meinhardt (see Gierer, 1977). Their “lateral inhibition” theory, originally developed for hydra, proposes the existence of an activator and an inhibitor acting catalytically on their own and each other’s production, the inhibitor having a longer range than the activator due to differences in diffusion constant andor relative production and decay rates. For certain nonlinear kinetics of catalysis, a stable prepattern of concentrations can be set up and may be symmetrical, oriented at random, or oriented with respect to an initial assymmetry (termed polarity), which specifies the orientation but not the shape of the pattern. Such prepatterns are capable of explaining the observed features of pattern formation in hydra and many other systems. A similar theory was applied by Kauffman ef ul. (1978) to describe how a succession of prepatterns set up in a growing domain can provide an elegant explanation of sequential compartment formation in Drosophilu . In none of these models is there an explicit requirement for permeable intercellularjunctions, but the concept of diffusion is central to all of them and, as with embryonic induction, the theory that gap junctions may be ”

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involved is an attractive one. If gap junctions are essential components of these systems, then they would seem to place a size (molecular weight) constraint on the molecular species responsible. Wolpert (1978) has reviewed data on the distribution of gap junctions in embryos and concludes that their presence is the rule rather than the exception in developing systems. Some discrepancies in the earlier literature regarding the question of whether gap junctions occur between blastomeres of cleavage-stage embryos may be resolved by recent observations of Lo and Gilula (1979a). They found that, in mouse embryos up to the precompaction 8-cell stage, each blastomere showed ionic and dye coupling to at most one other blastomere and that this coupling was always accompanied by the ability to transfer horseradish peroxidase (MW 40,000). This transfer they therefore interpreted as due to cytoplasmic bridges left between sister blastomeres as a result of delayed completion of cytokinesis. In compacted 8-cell embryos, however, each blastomere showed ionic and dye coupling to all other blastomeres of the embryo(coup1ing no long linked to peroxidase transfer), indicating that permeable junctionmediated communication develops at the compaction stage. This correlates well with ultrastructural studies (Magnuson et ai., 1977). Wolpert (1978) lists a number of situations in later-stage embryos in which gap junctions are absent or disappear, but as he points out, in some cases uncoupling precedes morphogenetic movements and it is difficult to distinguish cause from effect. If morphogenetic molecules do pass through gap junctions, we may ask whether their movement is controlled by regulating junctional permeability. There appears to be no difference in gap junction incidence or the degree of electrical coupling at segment boundaries of the insect cuticle (Warner and Lawrence, 1973; Lawrence and Green, 1975)where sharp discontinuitiesin the gradient are expected. This would however not preclude a selective blocking of permeability to molecules of higher molecular weight such as has been reported to occur in Chironomus salivary gland during the fourth instar (Loewenstein, 1978). In mouse blastocysts forming outgrowths in v i m (Lo and Gilula, 1979b) and in certain neuroblast cells and their progeny in the developing central nervous system of the grasshopper (Goodman and Spitzer, 1979), uncoupling of fluorescent dye transfer occurs without breakdown of ionic coupling. Whether this is due to a reduction in the number of junctional channels or in their pore size or to some other cause is not clear, but it does indicate that the extent of ionic coupling may not be a reliable guide to the freedom of intercellular communication through larger molecules. The scarcity of information regarding the question of whether metabolic cooperation plays a role in embryonic development is primarily due to the unavailability of techniques for specifically inhibiting metabolic cooperation in a developing system. A genetic approach to this problem, however, is possible using teratocarcinomas. These tumors consist of a chaotic juxtaposition of various differentiated tissues, together with a rapidly dividing stem cell population of embryonal carcinoma cells. Embryonal carcinoma cells are developmentally totipotent and give rise to other cell types by differentiation within the tumor; they

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are now viewed as intrinsically normal embryonic cells endowed with malignant properties by an abnormal microenvironment (see review by Graham, 1977). Embryonal carcinoma cells participate in metabolic cooperation with each other and with a variety of other cell types (Hooper and Slack, 1977). They can be grown in tissue culture with retention of developmental capacity; the successful isolation first of a metabolic coc7eration-defective variant (Section V,B) and then a cooperation-competent revertant (Section V,C)now provides a unique opportunity for analysis of the effect of metabolic cooperation-deficiency on developmental capacity. Jacob (1978) has reported that treatment of embryonal carcinoma cells with the Fab fragment of an antiserum that was prepared against the embryonal carcinoma line F9 and that inhibits metabolic cooperation (Table 11) also inhibits differentiation. However, the antiserum causes marked morphological changes in the cells, which round up and appose less closely to each other. Since this may lead to a variety of nonspecific effects, confident interpretation of these observations is difficult. Changes in cell communication with differentiation can also be studied under controlled conditions. Thus Lo and Gilula (1978) have reported that, when embryonal carcinoma cells differentiate into endoderm cells, gap junctions are maintained and tight junctions develop. Dunia et af. (1979), however, have reported that both gap junctions and tight junctions occur between embryonal carcinoma cells. Nicolas et al. (1978) have claimed that embryonal carcinoma cells fail to undergo metabolic cooperation with their differentiated derivatives, but their detection technique (Section IV,B), being based on rescue, would probably not detect low levels of cooperation. Studies of cooperation between embryonal carcinoma cells and their differentiated derivatives by the uridine prelabeling technique have shown that some degree of cooperation occurs in most, if not all, painvise combinations, although its extent may vary (R. Morgan, personal communication). Gaunt and Papaioannou ( 1979) have studied metabolic cooperation between embryonal carcinoma cells and cells taken from several tissues of early mouse embryos. Interestingly, they find that embryonal carcinoma cells will cooperate with cells from the morula, from the inner cell mass of the blastocyst, and the endoderm, mesoderm, and embryonic ectoderm of the eighth-day egg cylinder, but not with trophectoderm and its derivatives. Since embryonal carcinoma cells are closely related to inner cell mass and embryonic ectoderm cells (Graham, 1977), this suggests the hypothesis that these cell types become uncoupled from the trophectoderm during normal development (see discussion in Gaunt and Papioannou, 1979). These observations are therefore in good agreement with the results of fluorescent dye-transfer investigations on blastocyst outgrowths (Lo and Gilula, 1979b).

X. Conclusions We have reviewed the discovery of metabolic cooperation, the various techniques used to demonstrate its occurrence, and our present knowledge of the

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properties of permeable intercellular junctions, the factors controlling their formation, and the physiological significance of metabolic cooperation. Choice of the technique used to demonstrate metabolic cooperation is governed by the purpose of the study. For precise quantitative investigations, no currently available method is entirely satisfactory. The techniques described in Sections IV,A, B, and C all provide parameters that reflect the average property of a large number of cells and have the advantage of being nonintrusive. None of them directly quantify the molecules actually transferred, and the results must therefore be interpreted with caution. These techniques provide means for investigating the intercellular transfer of physiologically significant compounds, but in many cases, the effective material transferred may not be a single compound but a class of related compounds, e.g., all hypoxanthine-derived nucleoside mono-, di-, tri-, and even higher phosphates. Perhaps their major advantage is their utility for the selection of a limited but important spectrum of genetic variants. Ionic coupling can provide a direct measure of junctional permeability but considerably fewer cells can be studied in a given experiment than with the techniques discussed above. It has the additional disadvantages of being intrusive and applicable only to the study of inorganic ions. Techniques based on microinjection of fluorescent dyes can be applied to a wide range of probes, but they again are intrusive and restricted to small numbers of cells in a given experiment. The technique that appears to hold most promise for the future is that based on spontaneous loading of a nonpolar precursor of a fluorescent probe (Section 111,F). At the moment, the technique is limited by the properties of the only presently available compounds of this type, the fluorescein esters: these release fluorescein, which, although more polar than its esters, crosses cell membranes too rapidly to be suitable for quantitative measurements. There is a need for the introduction of compounds that upon entering cells, are converted to fluorescent probes with improved retention properties. Ready availability of such substrates, coupled with analysis of the movement of the probe molecule through a population of cells by fluorescence-activated cell sorting (Herzenberg and Herzenberg, 1978) would provide a rapid and direct measure of intercellular transfer applicable to kinetic studies both of probe transfer and of gap junction formation. There is also a need for a means of investigating the permeability properties of isolated gap junctions: for instance, by incorporating them into artificial lipid bilayers. This would enable the concentration of permeant molecule at the junction to be more effectively controlled than is possible with present techniques and allow investigation of whether factors such as Ca*+ concentration that modulate junctional permeability do so by a direct action on the junction itself. It would also facilitate direct comparison of gap junctions from different sources and studies of the relation between variation in ultrastructural parameters and permeability changes. An important area of research that can be expected to produce interesting

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results in the near future is the definitive biochemical and molecular biological analysis of isolated gap junctions. The availability of purified gap junction proteins to which antibodies can be raised will be an important step in elucidating the mechanism of gap junction assembly, particularly if monoclonal antibodies become available. Identification of the polypeptides that make up gap junctions, particularly if specific antibody is also available, should make it possible to isolate the relevant messenger RNAs (for example, by immunoprecipitationor by size enrichment coupled with in vitro translation), and these could then be reverse-transcribed and cloned. Once cloned fragments were available, it would be relatively easy to identify the genomic DNA-probably from existing gene libraries-and study these gene sequences, the signals used, and the relationships between equivalent genes in different organisms. We would speculate that these genes are probably among the most ancient and stable in genetic terms and their organization is clearly of considerable interest. More work is also needed to establish the factors that govern the incidence and specificity of gap junction formation. Dyes such as Lucifer yellow CH, which has a high fluorescence yield and can be fixed in tissues for subsequent histological examination, appear ideal for investigating whether discrete domains of coupled cells can be detected in vivo. We believe that analysis of the properties of genetic variants with altered communication properties will provide a particularly powerful strategy for investigating the physiological significance of metabolic cooperation. The range of variants currently available can in principle be extended by the use of existing selective techniques to include variants with temperature-sensitive defects in metabolic cooperation and variants with alterations in any fine-control mechanism that may regulate the size or metabolite specificity of the pore. The development of selective techniques based on junction-specific transfer of molecules larger than nucleotides (e.g., folate-derived cofactors or certain antibiotics) would widen the range of possibilities. If quantification of metabolic cooperation by fluorescence-activated cell sorting, as suggested earlier, proves feasible, then this could provide a particularly powerful selective technique that would be applicable to probes of various molecular weights. Because different classes of variant may arise at different relative frequencies in cell lines from different origins, the use of a panel of starting cell lines may also widen the spectrum of isolable variants. Further investigation of genetic deficiencies such as those discussed in Section V is obviously merited, and an investigation of genetic complementation between these lesions and those present in variants selected in culture may shed fresh light on the nature of both. The experimental evidence that we have reviewed establishes that metabolic cooperation has features that make it an attractive candidate for the transmission of many kinds of intercellular signals. We are confident that application of the kinds of approaches we have suggested in this section will contribute to a more complete understanding of its mechanism and physiological role.

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Akera, T. (1977). Science 198, 569. Albertini, R. J., and DeMars, R. (1970). Science 169, 482. Alacalh, J., Bradley, R., Kuszak, J., Waggoner, P., and Maisel, H. (1978). J. Cell Biol. 79, 219a. Anderson, E., and Albertini, D. F. (1976). J. Cell Biol. 71, 680. Asada, Y., and Bennett, M. V. L. (1971). J . Cell Biol. 49, 159. Ashkenazi, Y. E.,and Gartler, S. M. (1971). Exp. Cell Res. 64, 9. Asmn, K. H., and Caskey, C. T. (1976). Arch. Biochem. Biophys. 176, 397. Azamia, R., and Loewenstein, W. R. (1973). Nature (London) 241. 455. Azamia, R., and Loewenstein, W. R. (1977). J. Membr. Biol. 34, 1. Amnia, R., Michillke, W., and Loewenstein, W. R. (1972). J. Membr. Biol. 10, 247. Azamia, R., M e n , W. J., and Loewenstein, W. R. (1974). Proc. Nurl. Acud. Sci. U.S.A. 71,880. Baker, R. M., and Ling, V. (1978). In “Methods in MembraneBiology”(E. D. Kom, ed.), Vol. 9. p. 337. Plenum, New York. Baker, R. M., Brunette, D. M., Mankovitz. R., Thompson, L. H., Whitmore, G . F., Siminovitch, L., and Till, J. E. (1974). Cell 1, 9. Barr, L., Dewey, M. M., and Berger, W. (1965). J. Gen. Physiol. 48, 797. Benedetti, E. L., Dunia, I., Bentzel, C. J., Vermorken, A. J. M., Kibbelaar, M., and Blomendal, H. (1976). Biochim. Biophys. Acta 457. 353. Bennett, M. V. L. (1973). Fed. Proc. Fed. Am. SOC. Exp. Biol. 32, 65. Bennett, M. V. L. (1977). In “Handbook of Physiology - The Nervous System I ” (E. R. Kandel, ed.), p. 357. American Physiological Society, Washington, D.C. Bennett, M. V. L. (1978). In “Intercellular Junctions and Synapses” (J. Feldman, N. B. Gilula, and J. D. Pins, eds.), p. 23. Chapman & Hall, London. Bennett, M. V. L.. and Goodenough, D. A. (1978). Neurosci. Res. Prog. Bull. 16, 377. Biggelaar, J. A. M. van den, and Domsteijn, A. W. C. (1980). Eur. J . Cell Biol. 22, 451. Blalock, J. E. (1979). Proc. SOC. Exp. Biol. Med. 162, 80. Blalock, J. E.. and Baron, S . (1977). Nurure (London) 269, 422. Blalock, J. E., and Stanton, J. D. (1980). Nature (London) 283, 406. Bols, N. C., and Ringertz, N . R. (1979). Exp. Cell Res. 120, 15. Brink, P. R., and Dewey, M. M. (1978). 1. Gen. Physiol. 72, 67. Browne, C. L., Wiley, H. S., and Dumont, J. N. (1979). Science 203, 182. Burghardt, R. C., and Anderson, E. (1979). J. Cell Biol. 81. 104. Biirk. R. R., Pitts, J. D.. and Subak-Sharpe, J. H. (1968). Exp. Cell Res. 53, 297. Burton, A. C. (1971). Persp. Biol. Med. 14, 301. Burton, A. C.. and Canham. P. B. (1973). J . Theor. Biol. 39, 555. Buul, P. P., van, Natarajan, A. T., and Verdegaal-Immerzeel, E. A. (1978). Hum.Genet. 44, 187. Buultjens, T. E. J., MacDonald, C. M., and Hooper,M. L. (1980). In preparation. Campbell, F. R. (1980). Anur. Rec. 196, 101. Carritt, B., Goldfarb, P. S. 0 . .Hooper, M. L., and Slack, C. (1977). Exp. Cell Res. 106, 71. Caveney, S. (1978). Science 199, 192. Chu, E. H. Y., and Malling, H. V. (1%8). Proc. Nutl. Acud. Sci. U.S.A. 61, 1306. Clements, G. B. (1973). Ph.D. Thesis, University of Glasgow. Clements, G. B. (1975). Adv. Cancer Res. 21, 273. Clements, G. B., and Subak-Sharpe, J. H. (1975). Exp. Cell Res. 95, 25. Corsam, C. M., and Migeon, B. R. (1975). ESP. Cell Res. 95, 39. Corsan~,C. M., and Migeon, B. R. (1977a). Nature (London) 268, 737. Corsar~,C. M., and Mineon, B. R. (1977b). Proc. Narl. Acad. Sci. U . S . A . 74. 4476. Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1970). Proc. Narl. Acad. Sci. U.S.A. 67, 1573.

METABOLIC COOPERATION BETWEEN CELLS

99

Cox, R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1972). Exp. Cell Res. 74, 251. Cox, R. P., Krauss. M. R., Balis, M. E., and Dancis, J. (1974). J . Cell. Physiol. 84. 237. Cox. R. P., Krauss, M. R., Balis, M. E., and Dancis, J. (1976). Exp. CellRes. 101, 411. Dancis, J . , Cox, R. P., Bennan, P. H., Jansen, V., and Balis, M. E. (1969). Biochem. Genet. 3, 609. Decker, R. S. (1976). J. Cell Biol. 69, 669. De Haan, R. L., and Sachs. H. G. (1972). Curr. Top. Dev. Biol. 7, 193. DeMello, W . C. (1977). In "Intercellular Communication" ( W . C. DeMello, ed.), p. 87. Plenum, New York. DeMello, W. C. (1979). Pflugers Arch. 380,267. Dickennan, L. H., and Tischfield, J. A. (1978). Murar. Res. 49, 83. Dreifuss, J. J., Girardier. L.. and Forssman, W . G . (1966). PfIugers Arch. Ges. Physiol. 292, 13. Dunia, I., Nicolas, J. F., Jakob. H.,Benedeni. E. L., and Jacob, F. (1979). Proc. Nurf.Acud. Sci. U.S.A. 76, 3387. Ede, D. A.. Bellairs, R., and Bancroft, M. (1974). J. Embryol. Exp. Morphol. 31, 761. Edwards, J. G. (1977). In "Mammalian Cell Membranes" (0.A. Jamieson and D. M. Robinson, eds.), p. 32. Buttenvorths, London. Elias. P. M., and Friend, D. S. (1976). J . Cell Biol. 68, 173. Epstein, M. L., and Gilula, N. B. (1977). J . CeIl Biol. 75, 769. Epstein, M. L., Sheridan, J. D., and Johnson. R. G. (1977). Exp. Cell Res. 104, 25. Fain, G. L., Gold, G. H., and Dowling. J. E. (1976). Cold Spring Harbor Symp. puant. B i d . 40, 547.

Fentiman, I. S., and Taylor-Papadimitriou, J. (1977). Nurure (London) 269, 156. Fentiman, 1. S.,Taylor-Papadimitriou, J., and Stoker, M. (1976). Nature (London) 264, 760. Fentiman, 1. S., Hurst, J., Ceriani, R. L., and Taylor-Papadimitriou, J. (1979). Cuncer Res. 39, 4739.

Finbow, M. E., and Pitts. J. D. (1980). Exp. Cell Res.. in press. Finbow, M. E., Yancey, B.. Johnson, R., and Revel, J. P. (1979). J . Cell Biol. 83, 85a. Flagg-Newton, J., and Loewenstein, W. R. (1979). i. Membr. Biol. 50, 65. Flagg-Newton, J. L., and Loewenstein, W. R. (1980). Science 207, 771. Flagg-Newton, J., Simpson, I., and Loewenstein, W. R. (1979). Science 205, 404. Flint, 0. P., and Ede, D. A. (1978). J . Cell Sci. 31, 275. Friedmann, T., Seegmiller, J. E., and Subak-Sharpe, J. H. (1968). Nature (London) 220, 272. Fujimoto, W. Y.,and Seegmiller, J. E. (1970). Proc. Nutl. Acud. Sci. U.S.A. 65, 577. Fujimoto, W.Y., Seegmiller. J. E., Uhlendorf, B. W., and Jacobson, C. B. (1968). Lancer 2,511. Fujimoto, W . Y., Subak-Sharpe, J. H., and Seegmiller, J. E. (1971). Proc. Nutl. Acad. Sci. U.S.A. 68, 1516. Furshpan, E. J., and Potter, D. D. (1959). J . Physiol. (London) 145, 289. Garfield, R. E., Sims. S., and Daniel, E. E. (1977). Science 198, 958. Garfield, R. E., Sims, S. M., Kannan, M. S., and Daniel, E. E. (1978). Am. J. Physiol. 235, C168.

Gaunt, S. J. (1979). Ph.D. Thesis, University of Glasgow. Gaunt, S. J., and Papaioannou, V. E. (1979). J . Embryol. Exp. Morphol. 54, 263. Gaunt, S . J., and Subak-Sharpe, J. H. (1977). Exp. Cell Res. 109, 341. Gaunt, S. J., and Subak-Sharpe, 1. H. (1979). Exp. Cell Res. 120, 307. Gierer, A. (1977). Curr. Top. Dev. Biol. 11, 16. Gilula, N. B. (1978). In "Intercellular Junctions and Synapses" (J. Feldman. N. B. Gilula, and J. D. Pitts, eds.), p. 1. Chapman & Hall, London. Gilula, N. B., and Epstein, M. L. (1975). Symp. Soc. Exp. B i d . 30,257. Gilula, N. B., Reeves, 0. R.. and Steinbach, A. (1972). Nature (London) 235, 262.

100

M. L. HOOPER AND J. H. SUBAK-SHARPE

Gilula, N. B., Fawcett, D. W., and Aoki, A. (1976). Dev. B i d . 50, 142. Ginzberg, R. D., and Gilula, N. B. (1979). Dev. B i d . 68, 110. Goldfarb, P. S . G., Slack, C., Subak-Sharpe, J. H., and Wright, E. D. (1974). Symp. Sor. Exp. Biol. 28, 463. Goodenough, D. A , , Caspar, D. L. D.. Phillips, W. C., and Makowski, L. (1978). J . Cell Biol. 79, 223a.

Goodman, C. S . . and Spitzer, N. C. (1979). Nature (London) 280, 208. Goodwin, B., and Cohen, M . H. (1969). J . Theor. Biol. 25, 49. Goshima, K. (1969). Exp. Cell Res. 58, 420. Goshima, K. (1971). Exp. Cell Res. 65, 161. Graham, C. F. (1977). In “Concepts in Mammalian Embryogenesis” (M. I. Sherman, ed.), p. 315. MIT Press, Cambridge, Massachusetts. Griepp, E. B., and Bernfield. M. R. (1978). Exp. Cell Res. 113. 263. Grinnell, F. (1978). Int. Rev. Cyrol. 53, 67. Grobstein, C. (1955). In “Aspects of Synthesis and Order in Growth” (D.Rudnick, ed.), p. 233. Princeton Univ. Press, Princeton, New Jersey. Gulley, R. L., and Reese, T. S. (1976). J . Neurocytol. 5, 479. Gurd, J. W., and Evans, W. H. (1973). Eur. J. Biochem. 36, 273. Hanna, R. B., Keeter, J. S.,and Pappas, G. D. (1978). J. Cell Biol. 79, 764. Harris, A. (1974). In “Cell Communication” (R. P. Cox, ed.), p. 147. Wiley, New York. Hauschka, P. V. (1973). i n “Methods in Cell Biology” (D. M. Prescott, ed.), Vol. 7, p. 361. Academic Press, New York. H a . W. M. A., van Verooij. G. E. P. M., and Vossenberg, J. P. J., (1974). J . Membr. B i d . 19, 253.

Hay, E. D. (1977). In “International Cell Biology 1976-1977” (B. R. Brinkley and K. R. Poner, eds.), p. 50. Rockefeller Univ. Press, New York. Henderson, D., Eibl, H., and Weber, K. (1979). J . Mol. B i d . 132, 193. Henzberg, E. L., and Gilula, N. B. ( 1979). J. Biol. Chem. 254, 2138. Henzberg, E. L., Morganstem, R. A., and Gilula, N. B. (1978). J. Cell Biol. 79, 223a. Herzenberg, L. A,, and Herzenberg, L. A. (1978). I n “Handbook of Experimental Immunology” (D.M. Weir, ed.), 3rd ed., Vol 2. p. 22.1. Blackwell, Oxford. Holley, R. W. (1975). Nature (London) 258, 487. Hooper, M. L., and Morgan, R. H. M . (1979a). Exp. Cell Res. 119, 410. Hooper, M. L., and Morgan, R. H. M. (1979b). Exp. Cell Res. 123, 392. Hooper. M. L., and Parry.J. E. (1980). Exp. Cell Res., in press. Hooper, M. L., and Slack, C. (1977). Dev. Biol. 55, 271. Hulser, D . F.. and Peters, J . H. (197:!). Exp. Cell Res. 74. 3 19. Hulser, D. F., and Webb, D. J. (1973). Exp. Cell Res. 80. 210. Imanaga, I. (1974). J . Membr. Biol. 16, 381. Ito, S., Sato, E., and Lmwenstein, W. R. (1974). J . Membr. Biol. 19, 305. Iwatsuki, N., and Petersen, 0. H. (1978). J. Cell Biol. 79, 533. Jacob, F. (1978). Proc. R. SOC.London Ser. B 201, 249. Kalderon, N., Epstein, M. L., and Gilula, N. B. (1977). J . Cell Biol. 75, 788. Kam, E., Sheridan, J., and Johnson, R. (1978). J. Cell Biol. 79, 46a. Kanno, Y., and Lmwenstein, W. R. (1966). Nature (London) 212, 629. Kauffman, S. A., Shymko, R. M., arid Tmbert, K. (1978). Science 199, 259. Keeter, J. S., Deschenes, M., Pappas, G. D., and Bennett, M. V. L. (1974). Biol. Bull. 147,485. Kelley, R. O., Vogel, K. G., Crissman, H. A., Lujan, C. T.,and Skipper, B. E. (1979). Exp. Cell Res. 119, 127. Kelley, W. N. (1973). In “Growth, Nutrition and Metabolism of Cells in Culture” (G. H. Rothblat and V. J. Cnstofalo, eds.), Vol. 1, p. 21 1. Academic Press, New York. Kolodny, G. M. (1971). Exp. CellRes. 65, 313.

METABOLIC COOPERATION BETWEEN CELLS

101

Kolodny, G . M. (1972). Fed. Proc. Fed. A m . Soc. Exp. Biol. 31, A607. Kolodny, G. M. (1974). In “Cell Communication” (R. P. Cox, ed.), p. 97. Wiley, New York. Kuffler, S. W..and Potter, D. D. (1964). J. Neurophysiol. 27, 290. h e n , W. J. (1977). Tissue Cell 9, 373. Larsen, W. J., Azamia, R., and Loewenstein, W. R. (1977). J. Membr. Biol. 34, 39. Larsen, W. J . . Tung, H.-N., Murray, S. A., and Swenson, C. A. (1979). J . Cell Biol. 83, 576. Lawrence, P. A., and Green, S. M. (1975). 1.Cell Biol. 65, 373. Lawrence, T. S . , Beers, W. H., and Gilula, N. B. (1978). Nurure (London) 272, 501. Ledbetter, M. L. S., and Lubin, M. (1979). J. Cell Biol. 80, 150. Liebman, K. C.. and Heidelberger. C. (1955). J. Biol. Chem. 216, 823. Lloyd, C. W., Rees, D. A., Smith,C. G.. and Judge, F. J. (1976). J. Cell Sci. 22, 671. Lo, C. W., and Gilula, N. 8 . (1978). J . Cell Biol. 79, 18a. Lo, C. W.. and Gilula, N. 8 . (1979a). Cell 18, 399. Lo, C. W . , and Gilula, N. 8 . (1979b). Cell 18, 441. Loewenstein, W. R. (1967). Dev. Biol. 15, 503. Loewenstein. W. R. (1968). Dev. Biol. 19 (Suppl. 2). 151. Loewenstein, W. R. (1978). I n “Differentiation and Development” (F. Ahmad, J. Schultz, T. R. Russell and R. Werner, eds.), p. 399. Academic Press, New York. Loewenstein, W. R. (1979). Biochim. Biophys. Acra 560, 1. Loewenstein. W. R., and Kanno, Y. (1964). J. Cell Biol. 22, 565. Loewenstein, W. R., and Rose, B. (1978). Ann. N . Y . Acud. Sci. 307. 285. Loewenstein, W . R., Nakas, M., and Socolar, S. J. (1967). J . Gen. Physiol. 50. 1865. Loewenstein, W. R., Kanno, Y., and Socolar, S. J. (1978a). Fed. Proc. Fed. Am. Soc. Exp. Biol. 37. 2645. Loewenstein, W. R., Kanno, Y., and Socolar,S . I . (1978b). Nurure (London) 274, 133. McBumey, M. W., and Whitmore, G. F. (1974). Cell 2, 173. McCargow, J . , and Pitts, 1. D. (1971). Biochem. J . 124, 48P. McGinley, D. M.. Posalaky. Z., Porvaznik, M., and Russell, L. (1979). Tissue Cell 11, 741. McKusick, V. A. (1978). “Mendelian Inheritance in Man,” 5th ed. Johns Hopkins Univ. Press, Baltimore, Maryland. Magnuson. T., Demsey, A,, and Stackpole, C. W . (1977). Dev. Biol. 61, 252. Makowski, L., Caspar. D. L. D., Phillips, W. C., and Goodenough, D. A. (1977). J. Cell Biol. 74, 629. Marchase, R. B., Vosbeck, K., and Roth, S. (1976). Biochim. Biophys. Acra 457, 385. Meda. P., Perrelet, A., and Orci, L. (1979). J. Cell Biol. 82, 441. Merk, F. B., and MacNutt, N. S . (1972). J . Cell Biol. 55, 51 1. Michalke, W . (1977). J . Membr. 5iol. 33, 1. Mintz, B., and Baker, W. W. (1967). Proc. Nutl. Acud. Sci. U . S. A . 58, 592. Montesano, R., Friend, D. S . , Perrelet, A., and Orci. L. (1975). J. Cell Biol. 67, 310. Morrow, J. (1972). Genetics 71, 429. Mumay, A. W., and Fitzgerald, D. J. (1980). Biochem. Biophys. Res. Commun. 91, 395. Nadol, J. 8 . . Mulroy M. J., Goodenough, D. A., and Weiss. T. F. ( I 976). A m . J . A m . 147,28 1. Nicolas, J.-F., Jakob, H., and Jacob, F. (1978). Proc. Nurl. Acod. Sci. U.S.A. 75, 3292. Nicolis, G.,and Prigogine, I. (1977). “Self-Organization in Nonequilibrium Systems. ” Wiley (Interscience), New York. Odeigah, P. G. C., Clayton, R. M., and Truman, D. E. S. (1979). Exp. Eye Res. 28, 31 I . O ’ h g u e , P., Dalen. H . , Rubin, H., and Tobias, H. (1970). Science 170, 464. Oliviera-Castro, G . M., de, Barcinski, M. A,, andGaziri, I. F. (1975). In “Conceptsof Membranes in Regulation and Excitation” (M. Rocha e Silva and G. Suarez-Kurtz, eds.), p. 135. Raven, New York. Pappas, G. D.. Asada, Y., and Bennett, M. V. L. (1971). J. Cell Biol. 49, 173. Payton, B. W., Bennett, M. V. L.. and Pappas, G. D. (1969). Science 166, 1641.

.

102

M. L. HOOPER AND J . H. SUBAK-SHARPE

Peracchia, C. (1977). Trends Biochem. Sci. 2, 26. Peracchia, C. (1978). Nurure (London) 271,669. Peters, J. H. (1972). Exp. Cell Res. 74, 179. Peterson, J . A,, and Rubin, H. (1970). Exp. Cell Res. 60,383. Pitts, J. D. (1971). in “Growth Control in Cell Cultures” (G.E. W. Wolstenholme and J. Knight, eds.), p. 89. Livingstone, London. Pitts, J. D.(1976). In “The Developmental Biology of Plants and Animals’’ (C. F. Graham and P. F. Wareing, eds.), p. 96. Blackwell, Oxford. Pitts, J. D. (1978). In “Intercellular Junctions and Synapses” (J. Feldman, N. B. Gilula, and J. D. Pitts, eds.), p. 61. Chapman & Hall, London. Pitts, J. D., and Biirk, R. R. (1976). Nature (London) 264, 762. Pitts, J. D., and Finbow, M. E. (1977). In “IntercellularCommunication”(W. C. deMello, ed.). p. 61. Plenum, New York. Pitts, J. D., and Simms, J. W. (1977). Exp. Cell Res. 104, 153. Politoff, A. C., Socolar, S . I., and Loewenstein, W. R. (1969). J. Gen. Physiol. 53, 498. Politoff, A,, Pappas, G . D., and Bennett, M. V. L. (1974). Bruin Res. 76, 343. Pollack, G. H. (1976). J. Physiol. (London) 255, 275. Potter, D. D., Furshpan, E. J., and Lennox, E. S. (1966). Proc. Nurl. Acud. Sci. U . S . A . 55, 328. Reese, T. S . , Bennett, M. V. L., and Feder, N. (1971). Anur. Rec. 169, 409. Revel, J. P.. and Karnovsky, M. J. (1967). J. Cell Biol. 33, C7. Revel, J.-P., Yee, A. G., and Hudspeth, A. J . (1971). Proc. Nurl. Acud. Sci. U.S.A. 68, 2924. Rieske, E., Schubert, P., and Kreutzberg, G. W, (1975). Bruin Res. 84, 365. Ringertz, N. R., and Savage, R. E. (1976). “Cell Hybrids.” Academic Press. New York. Rogers, A. W. (1973). “Techniques of Autoradiography.” Elsevier, Amsterdam. Rose, B., and Loewenstein, W. R. (1971). J. Membr. Biol. 5, 20. Rose, B., and Rick. R. (1978). J. Membr. Biol. 44, 377. Rose, B., Simpson, I . , and Loewenstein, W. R. (1977). Nature (London) 267, 625. Rotman, B., and Papermaster, B. W. (1966). Proc. Narl. Acud. Sci. U.S.A. 55, 134. Roy-Burman. P. (1970). “Analogues of Nucleic Acid Components. Recent Results in Cancer Research,” Vol. 25. Springer-Verlag, Berlin and New York. Saxen, L., Karkinen-Jiiskelainen, M . , Lehtonen, E., Nordling, S., and Wartiovaara, J. (1976). In “The Cell Surface in Animal Embryogenesis and Development” ( G . Poste and G. Nicholson, eds.), p. 331. Elsevier, Amsterdam. Sellin, D., Wallach. D. F. H., and Fischer, H. (1971). Eur. J. Immunol. 1, 453. Sellin, D., Wallach. D. F. H., Weltzien, H. U.,Reich, K., Springer, E., and Fischer, H. (1974). Eur. J . Immunol. 4. 189. Sheridan, J. D. (1978). In “Intercellular Junctions and Synapses” (J. Feldman. N. B. Gilula, and J. D. Pitts, eds.), p. 37. Chapman & Hall, London. Sheridan, J. D., Finbow, M. E., and Pitts, J. D. (1975). J. Cell Biol. 67, 396a. Sheridan, J. D., Hammer-Wilson, M.. Preus, D., and Johnson, R. G. (1978). J . Ceif BioI. 76, 532.

Sheridan, J. D., Finbow, M. E., and Pitts, J. D. (1979). Exp. Cell Res. 123, 11 I . Sherman, M. I., and Wudl, L. (1977) In “Concepts in Mammalian Embryogenesis” (M. I. Sherman, ed.), p. 136. MIT Press, Cambridge, Massachusetts. Shields, R. (1976). Nature (London) 262, 348. Siegel. S . (1956). “Nonparametric Slatistics for the Behavioural Sciences,” McGraw-Hill, New York. Simpson, 1.. Rose, B., and Loewenstein, W. R . (1977). Science 195, 294. Slack, C., Morgan, R. H. M., Camitt, B., Goldfarb, P. S. G., and Hooper, M. L. (1976). Exp. Cell Res. 98, 1 . Slack, C.. Morgan, R. H. M., and Htmper, M. L. (1978). Exp. Cell Res. 117, 195.

METABOLIC COOPERATION BETWEEN CELLS

103

Swolar. S. J., and bewenstein, W. R. (1979). I n “Methods in Membrane Biology” (E. D. Kom, ed.), Vol. 10, p. 123. Plenum, New York. Spiegelman, M. (1976). In “Embryogenesis in Mammals” (K. Elliott and M. O’Connor, eds.), p. 199. Elsevier, Amsterdam. Spray, D. C.. Harris, A. L., and Bennett, M. V. L. ( 1979). Science 204, 432. Staehelin, L. A. (1974). Int. Rev. Cyrol. 39, 191, Stewart, W. W. (1978). Cell 14, 741. Stoker, M. G. P. (1967). J . Cell Sri. 2, 293. Stoker. M. G. P. (1975). Cell 6, 253. Subak-Sharpe, J. H. (1969). I n “Ciba Foundation Symposium on Homeostatic Regulators” ( G . E. W. Wolstenholme and J. Knight, eds.), p. 276, Churchill, London. Subak-Sharpe, J. H., Biirk. R. R., and Pitts, J . D. (1966). Heredify 21, 342. Subak-Sharpe, J . H., Biirk, R. R.,and Pitts, J. D. (1969). J . Cell Sci. 4, 353. Suzuki. K . . and Higashino, S. (1977). Exp. Cell Res. 109, 263. Trosko, J. E., Dawson, B., Yntti, L. P., and Chang. C. C. (1980). Narure (London) 285, 109. Tsien, R. W., and Weingart, R. (1976). J . Physioi. (London) 260, 117. Turin, L. (1977). J . Physiol. (london) 269, 6P. Turin. L., and Warner. A. (1977). Nature (London) 270, 56. Turing, A. (1952). Phil. Trans. R . Soc. London Ser. B 237, 37. Uitendaal, M. P., Oei. T. L., De Bruyn. C., and Hosli, P. (1976). Biochem. Biophys. Res. Commun. 71,574. Umbreit, J., and Roseman, S. (1975). J. Biol. Chem. 250, 9360. Unwin, P. N. T., and Zampighi, G. (1980). Nature (London) 283, 545. Warner, A. E . , and Lawrence. P. A. (1973). Nature fLondonJ245, 47. Weidmann, S . (1966). J. Physiol. (London)187, 323. Weingart, R. (1974). J . Phvsiol. (London) 240. 741. Weiss, R. A., and Njeuma, D. L. (1971). In “Growth Control in Cell Cultures” (G.E. W. Wolstenholme and J. Knight, eds.), p. 169. Livingstone, London. Widmer-Favre, C. (1972). J . Cell Sci. 11, 261. Wilson, D. B . , and Finta, L. A. (1979). Terarology 19, 337. Witkop, C . J., White, J. G . , King, R. A . , Dahl, M. V., Young, W. G., and Sauk, J. J., Jr. (1978a). Arch. Dermarol. 114, 1833. Witkop, C. J., White, J. G., Sauk, 1. J.. and King, R. A. (1978b). Oral Surg. Orul Med. Oral Pathol. 46. 645. Witkop, C. J., White, J. 0.. King, R. A., Dahl, M. V.. Young, W. G., and Sauk, J. J. Jr (1979). Am. J . Hum. Genet. 31,414. Wolpert, L. (1968). I n “Towards a Theoretical Biology: I. Prolegomena” (C. H. Waddington, ed.), p. 125. Edinburgh Univ. Press. Wolpert, L. (1978). In “Intercellular Junctions and Synapses” (J. Feldman, N. B. Gilula, and J. D. Pitts. eds.), p. 8 I . Chapman & Hall, London. Wood, S . , and Pinsky, L. (1972). J . Cell. Physiol. 80, 33. Wright, E. D., and Marsden, H. (1976). Exp. Cell Res. 103. 93. Wright, E. D., and Subak-Sharpe, J . H. (1974). Heredify 32, 279. Wright, E. D.. Goldfarb. P. S . G., and Subak-Sharpe, J. H. (1976a). Exp. Cell Res. 103, 63. Wright, E. D.. Slack. C.. Goldfarb, P. S. G., and Subak-Sharpe, J. H. (3976b). Exp. CellRes. 103, 19. Yee, A. G., and Revel, J.-P. (1978). J. Cell B&l. 78, 554. Yotti, L. P., Chang, C. C.. and Trosko, J. E. (1979). Science 206, 1089. Zampighi, G.. and Unwin, P. N. T . (1979). J . Mol. Biol. 135, 451. &eland. A. A . , van. Diggelen, H. C. E., van. and Simons, J. W. 1. M. (1972). Mutat. Res. 14,355. Zoref, E., de Vries, A., and Sperling, 0. (1976). Narure (London) 260, 786.

104

M. L. HOOPER AND J. H. SUBAK-SHARPE

NOTE ADDEDIN PROOF.We here briefly summarize a number of reports which have recently appeared, classified according to the swtion of the text to which they are relevant. VI,D. Flagg-Newton and Loewenstein (1980) have evidence that asymmetrically permeable membrane junctions can be formed in (cultureby pairing two cell types whose homotypic junctions differ in exclusion limit. V111. Murray and Fitzgerald (1979) have observed an effect of tumor promoters on metabolic cooperation between mouse epidermal cells and 3T3 cells. As they studied transfer of different molecular species (uridine nucleotides) from Yotti et ai. (1979, see text) this suggests that the effects observed by both authors are due to a reduction in the level of intercellular communication, although it would still seem desirable to study a number of permeant species for each cell combination. Trosko er a / . (1980) have shown that in the assay of Yotti ef al. (1979) high concentrations of saccharin partially abolish killing by the "kiss of death," but until the effect of saccharin on metabolic cooperation for other molecular species has been studied the conclusion that saccharin behaves like a tumor promoter seems premature. 1X.A. On the basis of experiments similar in design to those of Lawrence ef al. (1978, see text), Blalock and Stanton (1980) have concluded that the antiviral action of interferon can be transmitted between cells by a mechanism similar to the one involved in transmitting hormonal responses, and that the same secondary messenger, passing through gap junctions, is responsible. This conclusion is, however, hard to reconcile with previously published data from the same laboratory showing that L cells can transmit an interferon response both to other L cells and to cells of other lines (Blalock and Baron, 1977; Blalock, 1979), and with their preliminary data indicating that the response could be transmitted by conditioned media (Blalock and Baron, 1977), and further evidence is needed to clarify the situation. 1X.B. In contrast to previous work using thin-section electron microscopy, McGinley er ai. (1979) have reported that, by using freeze-fracture techniques, small gap junctions can be detected between Sertoli cells and germ cells in the rat. A role for these junctions in synchronizing germ cell maturation must now be considered. IX,D. Campbell (1980) has documented the incidence of gap junctions between different cell types in mouse bone marrow. Of particular interest is the observation that although macrophages form gap junctions with a variety of cell types, those macrophages which form gap junctions with erythroblasts do so only with erythroblasts, and all erythroblasts forming junctions with a single macrophage are at the same developmental stage, suggesting a role for these junctions in the maintenance of developmental synchrony. van den Biggelaar and Dorresteijn (1980) have studied dye-coupling in the embryo of the limpet, in which an animal-vegetal polarity is generated by asymmetric cleavage, but left-right and dorsoventral asymmetry arise epigenetically at about the 32-cell stage. Dye-transfer is seen only from the 32-cell stage onward, and then only within discrete domains of coupled cells, suggesting a role for gap junction-mediated communication in the diversification of cell lineages.