- Email: [email protected]
Historical Background
C H A P T E R
1 Historical Background Our understanding of the way multicellular organisms operate most often has developed from the awareness of different biological activities long before the mechanisms and molecular components involved begun to be identified. In contrast, knowledge that the neighboring cells of most tissues directly communicate with each other developed more or less in an inverted order. In fact, before the mid-1960s, no one considered the possibility that neighboring cells of most tissues might exchange small cytosolic molecules. What for? Why would hepatocytes, for instance, need to exchange small molecules if they all perform the same function? Actually, knowledge of direct cell-to-cell communication via membrane channels permeable to small, charged, and neutral molecules came as a surprise. Indeed, this important function was discovered by accident, and its meaning in (electrically) nonexcitable cells puzzled the scientists who discovered it.
1.1 MEMBRANE CHANNELS The “cell theory,” independently developed in the late 1830s by Matthias Jacob Schleiden (1804e1881) and Theodor Schwann (1810e1882), stated that tissues of plants and animals are made of independent units (cells)1,2 (Schleiden, 1839; Schwann, 1839); rev. in (Turner, 1890). The concept of “independent units” obviously implied the existence of a wall-like structure (now known as the plasma membrane) functioning as a protective barrier to prevent the exchange of molecules with adjacent cells and the extracellular space.3 However, while the cell theory did not consider the possible existence of membrane transport, toward the end of the 19th century, it became clear that certain molecules freely crossed the plasma membrane. The earliest studies on membrane transport did not consider the existence of channels but rather gave importance to the degree of lipid solubility of molecules (their oilewater partition coefficient) in determining their ability to diffuse in and out of cells. The “units” were actually discovered two centuries earlier by Robert Hooke (1665) in cork, who called them “pores or cells” - the name “cells” stuck. 1
2
Rudolf Virchow also contributed to the theory, but sadly he is not credited as much for it.
The “cell membrane concept” was actually pioneered by Charles Ernest Overton, who presented it in a series of lectures (1895e99); rev. in (Kleinzeller, 1997). 3
Gap Junction Structure and Chemical Regulation https://doi.org/10.1016/B978-0-12-816150-0.00001-0
1
Copyright © 2019 Elsevier Inc. All rights reserved.
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1. HISTORICAL BACKGROUND
Plasma membranes were believed by the British physiologist Charles Ernest Overton (1865e1933) and others to be entirely made of lipids; rev. in (Kleinzeller, 1997). In the early 20th century, however, it became clear that this idea contradicted evidence for membrane transport of some lipid-insoluble molecules such as ions. This prompted the formulation of hypotheses suggesting the existence of hydrophilic pores or channels. Thereafter, membranes started being described as mosaics of “lipoid and sieve-like” structures (Collander, 1937; Höber, 1936; Jacobs, 1924, 1935). Significantly, based on frictional properties and selectivity characteristics, in the mid-1930s membrane channels were already envisioned as narrow conduits with diameters similar to those of the permeant molecules (Jacobs, 1935) and composed of “some fibrous protein” (Höber, 1936). The earliest, definitive evidence of the existence of membrane channels came in the late 1940s and early 1950s through the work of Alan Lloyd Hodgkin (1914e1998) and coworkers (Hodgkin & Huxley, 1952a,b; Hodgkin, Huxley, & Katz, 1949; Hodgkin, Huxley, & Katz, 1952; Hodgkin & Katz, 1949). This major discovery was made possible by the earlier discovery of the giant squid axon (Young, 1938) and the inventions of microelectrodes by Ida Henrietta Hyde and the voltage-clamp technique (Cole, 1949); rev. in (Hille, 1992). Several years later, evidence for the existence of channels capable of mediating direct cellto-cell communications started emerging as well; rev. in (Peracchia, 1980; Loewenstein, 1975). These channels, now known as gap junction channels, are rather unconventional membrane channels not only because they span two membranes but also because, unlike other membrane channels, they are poorly selective, being permeable to both positively and negatively charged molecules, as well as to neutral molecules. Nonetheless, over the years, they have provided an excellent model system for studying the structure and function of membrane channels due to the fact that they are accessible to a large variety of technical approaches, spanning from crystallography to molecular biology, biochemistry, electrophysiology, biophysics, electron microscopy, and so on. For a review of 20th-century’s history of membrane channels see (Hille, 1992; Peracchia, 1994).
1.2 DIRECT CELL-TO-CELL COMMUNICATION FIRST REPORTED IN INVERTEBRATE NERVOUS SYSTEM Evidence for the existence of membrane channels that mediate direct cell-to-cell communication in (electrically) nonexcitable cells emerged serendipitously in the mid-1960s (Loewenstein & Kanno, 1964; Kuffler & Potter, 1964; Kanno & Loewenstein, 1964). But, knowledge of direct ionic communication in some (electrically) excitable cells surfaced in the early 20th century from studies on invertebrate nervous systems (Stough, 1926, 1930).
1.2.1 Giant Axons In the second half of the 19th century, zoologists discovered that certain annelids (earthworms), crustaceans (crayfish), and cephalopods (squid) display relatively large tube-like structures that extend from the rostral to the caudal end of the body. Most notable among them was the discovery of this type of structure in the squid (Williams, 1909)da structure
1.2 DIRECT CELL-TO-CELL COMMUNICATION
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destined to become one of the most relevant research tools for studying the mechanism of action potential generation and the behavior of ion channels in general. Although most scientists of the time attributed different functions to these unusual structures, Franz von Leydig (1821e1908) was the first to suggest their nervous function (Leydig, 1864). His interpretation, however, was by and large ignored until the first half of the 20th century when John Zachary Young4 (1907e97) unequivocally proved it right (Young, 1938). 1.2.1.1 Septa in Giant AxonsdEvidence for Direct Axo-Axonal Electrical Communication In the early 20th century, it became apparent that some of these giant tube-like structures (axons) were discontinuous. The first to realize it was George Edwin Johnson, who made this pivotal discovery in giant axons of crustaceans (Cambarus and Palaemonetes) (Johnson, 1924). Johnson’s report clarified earlier data of Louis Boulé, who had shown by silver-impregnation neurofibril discontinuities that repeated at regular intervals along the length of the Lumbricus terrestris’ giant axon (Boule, 1908)da finding confirmed a couple of decades later (Smallwood & Holmes, 1927). Two years after Johnson’s report, similar structures segmenting earthworms’ axons were named “septa” (Stough, 1926). In earthworms, there are three giant axons: a median and two lateral, all of them segmented. Howard B. Stough (1887e1976) described the septa as follows5: “.. there is a structure in the interior of the giant fiber which is most striking .. this is the cross-partition which completely divides the giant fibers segmentally” (Stough, 1930). In his 1930s study, Stough found that when the median axon is cut, only the anterior portion of the worm contracts, extending posteriorly up to the cut area; in contrast, when the lateral axons are cut, leaving the median intact, posterior stimulation causes the posterior portion of the worm to contract up to the cut area. These data led Stough to conclude that: “. the median giant fiber conducts antero-posteriorly and the lateral giant fibers conduct postero-anteriorly.” The apparent polarization of these axons, however, was proven wrong 2 years later by John Carew Eccles (1903e97) and coworkers who recorded with an oscillograph impulses in the isolated nerve cord elicited by stroking with a feather either the head or the tail of the earthworm (Eccles, Granit, & Young, 1932). In the concluding remarks of their brief article, they stated that “. the transverse membranes have no influence on the conduction of impulses, although the separation of the segments by means of these transverse membranes appears to be as complete as that existing at vertebrate synaptic junctions.” A few years later, their findings were confirmed by several studies (Bullock, 1945; Rushton, 1945; Kao & Grundfest, 1957; Wilson, 1961). Nonetheless, Stough deserves credit for being the first to demonstrate direct cell-to-cell electrical communication across morphologically definable septal barriers (Stough, 1926, 1930). In the absence of high-resolution images, however, it was not known then whether the current spread occurred via low-resistance membrane pathways or protoplasmic bridges. 4
During a 1969 reception at J. David Robertson’s home in Durham, NC, the Author remembers asking Professor Young how he discovered the neurological function of the squid axon. He answered: “we were all puzzled by this structure - some of us thought it was a vein, others that it was an axon. I thought that if it were an axon, by dropping on it citrate it would fire an action potential. Indeed, it fired!”
Note that in early studies, giant “axons” were called giant “fibers.” Since the term nerve “fibers” refer to myelinated axons, and invertebrate axons are not myelinated, this book will refer to them as “axons.” 5
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1. HISTORICAL BACKGROUND
Three decades later, with the advent of electron microscopy, the detailed ultrastructure of the earthworms’ septa was described as a very close apposition of plasma membranes with a total thickness of w200 Å (Hama, 1959) (see in the following). Incidentally, by observing the apparent polarization of earthworms’ median and lateral giant axons, Stough might have also inadvertently discovered the ability of injured cells to become electrically independent from each other (Stough, 1930). This property, now known as cell-to-cell uncoupling, is mediated by the gating mechanism of gap junction channelsd most likely, in Stough’s experiments the chemical gates of gap junction channels closer to the cut became activated by extracellular Ca2þ diffusing into the axoplasm. In 1947 the nonpolarized transmission of electrical impulses across anatomically welldefined septa (Fig. 1) was also demonstrated in lateral giant axons of crayfish (Wiersma, 1947). This finding was later confirmed by studies that used microelectrodes for a more precise intracellular recording of electrical signals (Watanabe & Grundfest, 1961; Kao, 1960). By the late 50s and early 60s, evidence of electrical cell-to-cell coupling was also reported in several other invertebrates, such as the lobster’s cardiac ganglion (Hagiwara & Bullock, 1957; Hagiwara, Watanabe, & Saito, 1959; Watanabe, 1958; Watanabe & Bullock, 1960) and
Cross-sectioned Crayfish Ventral Nerve Cord Septum
L
M
Gap Junctions at Septum
M 50 µm
a
Septum
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FIGURE 1 Crayfish ventral nerve cord. (A) Phase contrast image of cross-sectioned nerve cord showing median (M) and lateral (L) giant axons; the lateral axons are sectioned at the region (septum) where two axonal segments meet (see inset, white circle). (B) Electron micrograph showing a process of the caudal segment of a lateral giant axon penetrating through the septum to form gap junctions with the cranial segment; this junctional area is also seen in a phase contrast micrograph (inset a). At higher magnification (inset b), the cross-sectioned gap junction shows beaded profiles. The beads are junctional channels that link to each other across the extracellular gap. The channels’ center-tocenter spacing is w200 Å. The junction is flanked on both sides by rows of 500e800 Å vesicles.
1.2 DIRECT CELL-TO-CELL COMMUNICATION
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muscle fibers (Reuben, 1960) and the segmental ganglia of the leech (Hagiwara & Morita, 1962; Eckert, 1963). 1.2.1.2 Fenestrated Septa in Crayfish Axons Incidentally, in addition to the septa, crayfish axons display other unusual structures that, although likely to be present also in vertebrate axons, are more easily recognizable in crayfish (Figs. 2 and 3). Phase-contrast images of longitudinally sectioned crayfish nerves, fixed with glutaraldehyde-H2O2 (Peracchia & Mittler, 1972), display regularly spaced striations crossing the axoplasm every w2 mM (Peracchia, 1970; Fig. 2A). Electron microscope images revealed that each striation is made of two cross-sectioned membranes separated by 150e400 Å spacing (Fig. 2BeE). The two membranes are the profiles of “fenestrated septa” (Peracchia,
Fenestrated Septa in Crayfish axons
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(C)
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FIGURE 2 Longitudinal sections of crayfish axons, displaying membranous partitions that repeat every w2 mM. The partitions, “fenestrated septa” (Peracchia, 1970), are seen in a phase-contrast image of longitudinal sections (A) as well as in the electron micrograph (B). Each partition is composed of two cross-sectioned membranes, separated by 150e400 Å spacing (BeD), which frequently join forming 0.1e0.2 mM pores (BeE), each occupied by a single microtubule (C). Neighboring septa are joined by longitudinal membranous tubules (B and D). The diagram E is a schematic representation of the architecture of the fenestrated septa. Originally published in the Journal of Cell Biology Peracchia, C. (1970). A system of parallel septa in crayfish nerve fibers. Journal of Cell Biology, 44, 125e133.
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1. HISTORICAL BACKGROUND
1 µm 500 Å
a
500 Å
b (B)
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g 400 Å
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FIGURE 3 Cross-sections of crayfish axons, displaying face views of the fenestrated septa (A, arrows). Each of the pores (fenestrae) contains at its center a cross-sectioned microtubule (AeC). The gap between pore and microtubule measures 80e100 Å (B). Occasionally, filamentous structures are seen bridging the microtubule to the pore’s edge (C). A (inset a) shows a specialized region of axon-glia contact where the two plasma membranes project into the axoplasm, separated by a gap of 130e140 Å (Peracchia, 1974). In freeze-fracture replicas, the P-face of the axonal projections (A, inset b) appears as an elongated indentation 0.5e1.2 mm long, 0.12e0.15 mm wide, containing parallel chains of particles that repeat every 120e125 Å. The particles repeat along the chain every 80e85 Å. This structure has a rhomboidal unit cell of 80e85 120e125 Å (A, inset b). A (inset c) shows a region where axonal (a) and glial (g) membranes fuse, creating an axo-glial pore. Originally published in the Journal of Cell Biology Peracchia, C. (1970). A system of parallel septa in crayfish nerve fibers. Journal of Cell Biology, 44, 125e133.
1970) or transverse cisternae (Fig. 2CeE). The membranes of the fenestrated septa frequently join, forming 0.1e0.2 mM pores each occupied by a single microtubule (Fig. 2C). Neighboring fenestrated septa are joined by longitudinal membranous tubules (Fig. 2B, D and E). In crosssectioned axons the septal membranes, now seen in face view, show clear images of the pores each containing a cross-sectioned microtubule (Fig. 3AeC). Occasionally, thin filaments bridge the microtubule to the edge of the pore (Fig. 3C). These structures (Fig. 2E) are present in different size axons and may be a general feature of axon structure because we observed them, although in a less organized fashion, also in mouse sciatic and vagus nerves (Fig. 4).6 Their function is unclear, but their connection to microtubules suggests a potential role in the axoplasmic flow mechanism; rev. in (Ochs, 1982). If so, the filaments seen bridging the microtubule to the edge of the pore could be composed of dynein or kinesin (Gennerich & Vale, 2009).
These data were reported as “C. Peracchia, unpublished observation” in (Peracchia, 1970).
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1.2 DIRECT CELL-TO-CELL COMMUNICATION
Fenestrated Septa in Mammalian Axons
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FIGURE 4 Cross-section views of mouse sciatic and vagus nerves. Fenestrated septa similar to those seen in crayfish axons (Figs. 2 and 3) are occasionally seen (arrows), although in a less organized fashion. Also in these mammalian axons, cross-sectioned profiles of microtubules are seen at the center of the pores (fenestrae; arrows). These mouse structures were reported as “C. Peracchia, unpublished observation” in Peracchia, 1970.
1.2.1.3 Particle Arrays in Crayfish Axo-Glial Membrane Projections Crayfish axons also display regions in which axonal and glial plasma membranes are regularly curved and project into the axoplasm, separated by a gap of 130e140 Å (Peracchia, 1974) (Fig. 3A, a, arrow). In freeze-fracture replicas (Fig. 3A, b) the P-face of the axonal projection appears as an elongated indentation 0.5e1.2 mm long, 0.12e0.15 mm wide, containing parallel chains of particles repeating every 120e125 Å and oriented obliquely such that the axis of the chains is skewed with respect to the long axis of the indentations by an angle of 55e 60 degrees. The particles repeat along the chain every 80e85 Å. This structure can be defined as a particle array with a rhomboidal unit cell of 80e85 120e125 Å (Fig. 3A, b). On the axonal E-face the complementary image of these structures is seen. The projections of the satellite’s membrane also contain particle aggregates, but these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. The functional meaning of these structures is unknown, but they could be areas of cell-to-cell adhesion (Peracchia, 1974). 1.2.1.4 Are There Axo-Glial Pores in Crayfish Axons? In crayfish axons, we also observed regions where axonal and glial plasma membranes appear to fuse (Fig. 3A, c), creating images of intercellular pores (Peracchia, 1981). If present, these pores would be expected to allow direct communication between the axoplasm and the glial cytoplasm. The pores may actually form transiently but are unlikely to be a preparation artifact because the radius of membrane fusion at the pores is consistent with the minimum curvature radius of biological membranes. Recently, similar pores have also been seen in crayfish stretch receptors (Fedorenko, Neginskaya, Fedorenko, & Uzdensky, 2015).
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1. HISTORICAL BACKGROUND
1.2.2 Evidence for Rectifying Gap Junctions in Invertebrate Nervous System By the mid-40s, it was generally believed that electrical communication between adjacent cells was nonpolarized (bidirectional). Therefore, it came as a surprise when in 1947 Cornelis A. G. Wiersma (1905e79) demonstrated that in crayfish the transmission of the electrical impulse between giant and motor fibers is polarized, such that current only spreads from giant to motor fibers (rectifying junctions) (Wiersma, 1947). Twelve years later, Wiersma’s data were confirmed by a careful study using intracellular recording (Furshpan & Potter, 1959).
1.3 DIRECT ELECTRICAL COMMUNICATION BETWEEN MAMMALIAN CARDIAC FIBERS In the early 50s the Swiss physiologist Silvio Weidmann (1921e2005) made a major discovery while working on single Purkinje fibers of kid’s heart (Weidmann, 1952), although at first, he did not recognize its importance. Weidmann measured the electrical resistance of the myoplasm by passing polarizing pulses through a microelectrode inserted at one end of the fiber and recording the electrotonic potential with microelectrodes placed at various distances from the current-passing microelectrode. He reported that: “. The relatively low value of the specific d.c. resistance of myoplasm (twice that of Tyrode solution) suggests (i) that the smaller units making up the Purkinje fibre, the Purkinje cells, are not surrounded by ionic barriers of any importance, (ii) that Purkinje fibres are not subdivided by transverse membranes . and (iii) that most of the intracellular ions must be free to move under the influence of an electric field.” Weidmann’s study not only demonstrated that Purkinje cells are electrically coupled, which is the earliest example of direct cell-to-cell communication in vertebrates (indeed, mammals), but also further confirmed the capacity of these cells to “heal-over.” The “healing-over” phenomenon was first described by Theodor Wilhelm Engelmann (1843e1909), who wrote: “. cardiac cells live together and die alone .” (Engelmann, 1877). This phenomenon, later confirmed by K.E. Rothschuh (1951), is now known as cell-to-cell uncoupling, a function mediated by the gating properties of gap junction channels (see in the following). Weidmann is rightly credited for having provided the first demonstration of direct electrical communication between adjacent cells in vertebrates (indeed, in mammals). However, while we know now that this is true, in 1952, Weidmann actually believed that the cells making the Purkinje fibers were not independent of each other, but rather interconnected via cytoplasmic bridges. He thought that the heart, the uterus, or other organs made of smooth muscle fibers (intestine, arteries, etc.) known to contract globally or sequentially were in fact “functional syncytia” rather than aggregates of individual cells bordered by biological membranes. Curiously, the cellular (rather than syncytial) organization of Purkinje fibers was actually described by Sunao Tawara as early as in 1906, a finding that was confirmed by J. M. Tufts (1921) through maceration experiments. In spite of Tawara’s and Tufts’ findings, however, in the first half of the 20th century most scientists believed in the syncytial nature of Purkinje fibers (Blair & Davies, 1935; DeWitt, 1909; Field, 1951; Jordan & Banks, 1917). Actually, Jordan and Banks reported that ox’s Purkinje fibers are cellular at the embryo stage of
1.4 DIRECT CELL-TO-CELL COMMUNICATION
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development, but they thought that later in development they would fuse into a syncytium (Jordan & Banks, 1917). Eventually, the accuracy of Tawara’s original observation was vindicated by evidence from electron microscope studies, which proved that Purkinje cells are indeed independent units of tissue bordered by plasma membranes (Van Breeman, 1953; Sjostrand & Andersson, 1954; Price, Weiss, Hata, & Smith, 1955; Muir, 1957; Muir, 1965; Rhodin, Del, & Reid, 1961; Hayashi, 1962). When evidence for the cellular organization of Purkinje fibers eventually surfaced, Weidmann was actually disappointed, not realizing at first that he had made an important discovery. Indeed, in a 1976 review article,7 Weidmann wrote: “Up to 1954 there was little doubt that cardiac muscle could be considered as a syncytium. This view was based on functional properties such as all-or-nothing excitability and conduction without decrement. A short communication by Sjostrand and Andersson (1954) came as a rather unpleasant surprise. Using high resolution electron microscopy these authors clearly demonstrated the existence of morphologically distinct cells. There were two major possibilities to reconcile morphology and function: either to postulate cell-to-cell transmission by a chemical mediator (see for instance Sperelakis, 1963) or to assume that adjoining cells may have specialized regions of low resistance. The evidence, which has accumulated over the past 20 years, seems to favor the second alternative” (Weidmann, 1976).
1.4 DIRECT CELL-TO-CELL COMMUNICATION IN THE VERTEBRATE NERVOUS SYSTEM Aside from the Weidmann’s (1952) study on kid’s heart, up to the late 1950s most of the data for direct electrical communication between cells were only reported in invertebrate (electrically) excitable cells. It made news, therefore, a 1959 publication reporting electrical communication between supramedullary neurons of a vertebrate: the puffer fish Spheroides maculatus (Bennett, Crain, & Grundfest, 1959). This important finding proved that electrical coupling is not a function only restricted to invertebrate excitable cells. This study also explained some puzzling phenomena that were observed in frog spinal motor neurons. In experiments on isolated spinal cords the stimulation of one motor neuron resulted in prolonged depolarization of several adjacent motor neurons (Washizu, 1960; Kubota & Brookhart, 1962; Katz & Miledi, 1963). This phenomenon was puzzling because it was not believed to result from the activity of chemical synapses. The report on fish neurons (Bennett et al., 1959) provided an explanation for this phenomenon, as it demonstrated that it is based on electrical cell-to-cell coupling. Indeed, Yoshiaki Washizu had proposed this explanation for the behavior of frog motor neurons, but none of these authors (Washizu, 1960; Kubota & Brookhart, 1962; Katz & Miledi, 1963) seems to have been aware of the earlier study of Bennett and coworkers (Bennett et al., 1959). Four years after Bennett and coworkers’ publication, electrical coupling was also reported in two other vertebrate systems: the large caliciform synapses of the chick’s ciliary ganglion (Martin & Pilar, 1963a,b) and the Mauthner neurons of goldfish (Furukawa & Furshpan, 1963).
7
I felt very honored when Silvio Weidmann sent me this review article with his signature and a nice note!
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1. HISTORICAL BACKGROUND
1.5 DIRECT CELL-TO-CELL COMMUNICATION IN NONEXCITABLE CELLS A major discovery in the history of direct cell-to-cell communication was made in 1964 by two independent studies: one on insect salivary glands (Kanno & Loewenstein, 1964; Loewenstein & Kanno, 1964) and the other on leech glia (Kuffler & Potter, 1964). Interestingly, these discoveries were serendipitous, as no one conceived then that nonexcitable cells might directly communicate with each other. Yoshinobu Kanno and Werner R. Loewenstein (1926e2014) were actually studying the electrical properties of the nuclear envelope in the gland cells of Drosophila flavorepleta larvae and had no idea that neighboring gland cells might be able to directly communicate with each other. To their surprise, upon current injection into the nucleus of one of these giant cells, a change in membrane potential was recorded not only in the injected cell but also in the adjacent cells (Loewenstein & Kanno, 1964; Kanno & Loewenstein, 1964). Stephen W. Kuffler (1913e1980) and David D. Potter also discovered by accident the electrical coupling of glial cells, as their study was actually aimed at determining whether neurons interact with glial cells (Kuffler & Potter, 1964). However, the electrical interaction of glial cells might not have been that surprising because Alan Peters reported, 2 years earlier, tight membrane appositions at glial cell contacts (Peters, 1962). In any event, it is undeniable that these two studies opened a new exciting chapter in the history of cell biology because thereafter it became apparent that the vast majority of cells would no longer be though as independent units of tissue, but rather as members of intimately communicating cell communities.
1.5.1 Why Nonexcitable Cells Communicate Directly With Each Other The major question then was: why do (electrically) nonexcitable cells need to communicate electrically with each other? The answer came from the first study of Lowenstein and Kanno, which demonstrated that cells are also capable of exchanging molecules larger than small ions, such as fluorescein (MW ¼ 332) (Loewenstein & Kanno, 1964). This suggested for the first time the possibility of direct metabolic cooperation among cells, a function that was proven by John D. Pitts and coworkers a few years later (Pitts & Simms, 1977; SubakSharpe, Burk, & Pitts, 1966; Subak-Sharpe, Burk, & Pitts, 1969). Soon it became obvious that cell-to-cell communication is not only a property of (electrically) nonexcitable “invertebrate” cells but is also a property of (electrically) nonexcitable “vertebrate” cells such as those of toad urinary bladder (Loewenstein, Socolar, Higashino, Kanno, & Davidson, 1965) and mouse liver (Penn, 1966). Later on, most neighboring cells were found to exchange metabolites such as amino acids, nucleotides, second messengers, and so no, confirming that most tissues are more than just aggregates of independent cells but are rather metabolically interlinked cell communities harmoniously cooperating for maintaining homeostasis, amplifying hormonal signals, distributing signal transduction intermediates, and so on.
1 Historical Background Our understanding of the way multicellular organisms operate most often has developed from the awareness of different biological activities long before the mechanisms and molecular components involved begun to be identified. In contrast, knowledge that the neighboring cells of most tissues directly communicate with each other developed more or less in an inverted order. In fact, before the mid-1960s, no one considered the possibility that neighboring cells of most tissues might exchange small cytosolic molecules. What for? Why would hepatocytes, for instance, need to exchange small molecules if they all perform the same function? Actually, knowledge of direct cell-to-cell communication via membrane channels permeable to small, charged, and neutral molecules came as a surprise. Indeed, this important function was discovered by accident, and its meaning in (electrically) nonexcitable cells puzzled the scientists who discovered it.
1.1 MEMBRANE CHANNELS The “cell theory,” independently developed in the late 1830s by Matthias Jacob Schleiden (1804e1881) and Theodor Schwann (1810e1882), stated that tissues of plants and animals are made of independent units (cells)1,2 (Schleiden, 1839; Schwann, 1839); rev. in (Turner, 1890). The concept of “independent units” obviously implied the existence of a wall-like structure (now known as the plasma membrane) functioning as a protective barrier to prevent the exchange of molecules with adjacent cells and the extracellular space.3 However, while the cell theory did not consider the possible existence of membrane transport, toward the end of the 19th century, it became clear that certain molecules freely crossed the plasma membrane. The earliest studies on membrane transport did not consider the existence of channels but rather gave importance to the degree of lipid solubility of molecules (their oilewater partition coefficient) in determining their ability to diffuse in and out of cells. The “units” were actually discovered two centuries earlier by Robert Hooke (1665) in cork, who called them “pores or cells” - the name “cells” stuck. 1
2
Rudolf Virchow also contributed to the theory, but sadly he is not credited as much for it.
The “cell membrane concept” was actually pioneered by Charles Ernest Overton, who presented it in a series of lectures (1895e99); rev. in (Kleinzeller, 1997). 3
Gap Junction Structure and Chemical Regulation https://doi.org/10.1016/B978-0-12-816150-0.00001-0
1
Copyright © 2019 Elsevier Inc. All rights reserved.
2
1. HISTORICAL BACKGROUND
Plasma membranes were believed by the British physiologist Charles Ernest Overton (1865e1933) and others to be entirely made of lipids; rev. in (Kleinzeller, 1997). In the early 20th century, however, it became clear that this idea contradicted evidence for membrane transport of some lipid-insoluble molecules such as ions. This prompted the formulation of hypotheses suggesting the existence of hydrophilic pores or channels. Thereafter, membranes started being described as mosaics of “lipoid and sieve-like” structures (Collander, 1937; Höber, 1936; Jacobs, 1924, 1935). Significantly, based on frictional properties and selectivity characteristics, in the mid-1930s membrane channels were already envisioned as narrow conduits with diameters similar to those of the permeant molecules (Jacobs, 1935) and composed of “some fibrous protein” (Höber, 1936). The earliest, definitive evidence of the existence of membrane channels came in the late 1940s and early 1950s through the work of Alan Lloyd Hodgkin (1914e1998) and coworkers (Hodgkin & Huxley, 1952a,b; Hodgkin, Huxley, & Katz, 1949; Hodgkin, Huxley, & Katz, 1952; Hodgkin & Katz, 1949). This major discovery was made possible by the earlier discovery of the giant squid axon (Young, 1938) and the inventions of microelectrodes by Ida Henrietta Hyde and the voltage-clamp technique (Cole, 1949); rev. in (Hille, 1992). Several years later, evidence for the existence of channels capable of mediating direct cellto-cell communications started emerging as well; rev. in (Peracchia, 1980; Loewenstein, 1975). These channels, now known as gap junction channels, are rather unconventional membrane channels not only because they span two membranes but also because, unlike other membrane channels, they are poorly selective, being permeable to both positively and negatively charged molecules, as well as to neutral molecules. Nonetheless, over the years, they have provided an excellent model system for studying the structure and function of membrane channels due to the fact that they are accessible to a large variety of technical approaches, spanning from crystallography to molecular biology, biochemistry, electrophysiology, biophysics, electron microscopy, and so on. For a review of 20th-century’s history of membrane channels see (Hille, 1992; Peracchia, 1994).
1.2 DIRECT CELL-TO-CELL COMMUNICATION FIRST REPORTED IN INVERTEBRATE NERVOUS SYSTEM Evidence for the existence of membrane channels that mediate direct cell-to-cell communication in (electrically) nonexcitable cells emerged serendipitously in the mid-1960s (Loewenstein & Kanno, 1964; Kuffler & Potter, 1964; Kanno & Loewenstein, 1964). But, knowledge of direct ionic communication in some (electrically) excitable cells surfaced in the early 20th century from studies on invertebrate nervous systems (Stough, 1926, 1930).
1.2.1 Giant Axons In the second half of the 19th century, zoologists discovered that certain annelids (earthworms), crustaceans (crayfish), and cephalopods (squid) display relatively large tube-like structures that extend from the rostral to the caudal end of the body. Most notable among them was the discovery of this type of structure in the squid (Williams, 1909)da structure
1.2 DIRECT CELL-TO-CELL COMMUNICATION
3
destined to become one of the most relevant research tools for studying the mechanism of action potential generation and the behavior of ion channels in general. Although most scientists of the time attributed different functions to these unusual structures, Franz von Leydig (1821e1908) was the first to suggest their nervous function (Leydig, 1864). His interpretation, however, was by and large ignored until the first half of the 20th century when John Zachary Young4 (1907e97) unequivocally proved it right (Young, 1938). 1.2.1.1 Septa in Giant AxonsdEvidence for Direct Axo-Axonal Electrical Communication In the early 20th century, it became apparent that some of these giant tube-like structures (axons) were discontinuous. The first to realize it was George Edwin Johnson, who made this pivotal discovery in giant axons of crustaceans (Cambarus and Palaemonetes) (Johnson, 1924). Johnson’s report clarified earlier data of Louis Boulé, who had shown by silver-impregnation neurofibril discontinuities that repeated at regular intervals along the length of the Lumbricus terrestris’ giant axon (Boule, 1908)da finding confirmed a couple of decades later (Smallwood & Holmes, 1927). Two years after Johnson’s report, similar structures segmenting earthworms’ axons were named “septa” (Stough, 1926). In earthworms, there are three giant axons: a median and two lateral, all of them segmented. Howard B. Stough (1887e1976) described the septa as follows5: “.. there is a structure in the interior of the giant fiber which is most striking .. this is the cross-partition which completely divides the giant fibers segmentally” (Stough, 1930). In his 1930s study, Stough found that when the median axon is cut, only the anterior portion of the worm contracts, extending posteriorly up to the cut area; in contrast, when the lateral axons are cut, leaving the median intact, posterior stimulation causes the posterior portion of the worm to contract up to the cut area. These data led Stough to conclude that: “. the median giant fiber conducts antero-posteriorly and the lateral giant fibers conduct postero-anteriorly.” The apparent polarization of these axons, however, was proven wrong 2 years later by John Carew Eccles (1903e97) and coworkers who recorded with an oscillograph impulses in the isolated nerve cord elicited by stroking with a feather either the head or the tail of the earthworm (Eccles, Granit, & Young, 1932). In the concluding remarks of their brief article, they stated that “. the transverse membranes have no influence on the conduction of impulses, although the separation of the segments by means of these transverse membranes appears to be as complete as that existing at vertebrate synaptic junctions.” A few years later, their findings were confirmed by several studies (Bullock, 1945; Rushton, 1945; Kao & Grundfest, 1957; Wilson, 1961). Nonetheless, Stough deserves credit for being the first to demonstrate direct cell-to-cell electrical communication across morphologically definable septal barriers (Stough, 1926, 1930). In the absence of high-resolution images, however, it was not known then whether the current spread occurred via low-resistance membrane pathways or protoplasmic bridges. 4
During a 1969 reception at J. David Robertson’s home in Durham, NC, the Author remembers asking Professor Young how he discovered the neurological function of the squid axon. He answered: “we were all puzzled by this structure - some of us thought it was a vein, others that it was an axon. I thought that if it were an axon, by dropping on it citrate it would fire an action potential. Indeed, it fired!”
Note that in early studies, giant “axons” were called giant “fibers.” Since the term nerve “fibers” refer to myelinated axons, and invertebrate axons are not myelinated, this book will refer to them as “axons.” 5
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1. HISTORICAL BACKGROUND
Three decades later, with the advent of electron microscopy, the detailed ultrastructure of the earthworms’ septa was described as a very close apposition of plasma membranes with a total thickness of w200 Å (Hama, 1959) (see in the following). Incidentally, by observing the apparent polarization of earthworms’ median and lateral giant axons, Stough might have also inadvertently discovered the ability of injured cells to become electrically independent from each other (Stough, 1930). This property, now known as cell-to-cell uncoupling, is mediated by the gating mechanism of gap junction channelsd most likely, in Stough’s experiments the chemical gates of gap junction channels closer to the cut became activated by extracellular Ca2þ diffusing into the axoplasm. In 1947 the nonpolarized transmission of electrical impulses across anatomically welldefined septa (Fig. 1) was also demonstrated in lateral giant axons of crayfish (Wiersma, 1947). This finding was later confirmed by studies that used microelectrodes for a more precise intracellular recording of electrical signals (Watanabe & Grundfest, 1961; Kao, 1960). By the late 50s and early 60s, evidence of electrical cell-to-cell coupling was also reported in several other invertebrates, such as the lobster’s cardiac ganglion (Hagiwara & Bullock, 1957; Hagiwara, Watanabe, & Saito, 1959; Watanabe, 1958; Watanabe & Bullock, 1960) and
Cross-sectioned Crayfish Ventral Nerve Cord Septum
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Gap Junctions at Septum
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FIGURE 1 Crayfish ventral nerve cord. (A) Phase contrast image of cross-sectioned nerve cord showing median (M) and lateral (L) giant axons; the lateral axons are sectioned at the region (septum) where two axonal segments meet (see inset, white circle). (B) Electron micrograph showing a process of the caudal segment of a lateral giant axon penetrating through the septum to form gap junctions with the cranial segment; this junctional area is also seen in a phase contrast micrograph (inset a). At higher magnification (inset b), the cross-sectioned gap junction shows beaded profiles. The beads are junctional channels that link to each other across the extracellular gap. The channels’ center-tocenter spacing is w200 Å. The junction is flanked on both sides by rows of 500e800 Å vesicles.
1.2 DIRECT CELL-TO-CELL COMMUNICATION
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muscle fibers (Reuben, 1960) and the segmental ganglia of the leech (Hagiwara & Morita, 1962; Eckert, 1963). 1.2.1.2 Fenestrated Septa in Crayfish Axons Incidentally, in addition to the septa, crayfish axons display other unusual structures that, although likely to be present also in vertebrate axons, are more easily recognizable in crayfish (Figs. 2 and 3). Phase-contrast images of longitudinally sectioned crayfish nerves, fixed with glutaraldehyde-H2O2 (Peracchia & Mittler, 1972), display regularly spaced striations crossing the axoplasm every w2 mM (Peracchia, 1970; Fig. 2A). Electron microscope images revealed that each striation is made of two cross-sectioned membranes separated by 150e400 Å spacing (Fig. 2BeE). The two membranes are the profiles of “fenestrated septa” (Peracchia,
Fenestrated Septa in Crayfish axons
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FIGURE 2 Longitudinal sections of crayfish axons, displaying membranous partitions that repeat every w2 mM. The partitions, “fenestrated septa” (Peracchia, 1970), are seen in a phase-contrast image of longitudinal sections (A) as well as in the electron micrograph (B). Each partition is composed of two cross-sectioned membranes, separated by 150e400 Å spacing (BeD), which frequently join forming 0.1e0.2 mM pores (BeE), each occupied by a single microtubule (C). Neighboring septa are joined by longitudinal membranous tubules (B and D). The diagram E is a schematic representation of the architecture of the fenestrated septa. Originally published in the Journal of Cell Biology Peracchia, C. (1970). A system of parallel septa in crayfish nerve fibers. Journal of Cell Biology, 44, 125e133.
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1. HISTORICAL BACKGROUND
1 µm 500 Å
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FIGURE 3 Cross-sections of crayfish axons, displaying face views of the fenestrated septa (A, arrows). Each of the pores (fenestrae) contains at its center a cross-sectioned microtubule (AeC). The gap between pore and microtubule measures 80e100 Å (B). Occasionally, filamentous structures are seen bridging the microtubule to the pore’s edge (C). A (inset a) shows a specialized region of axon-glia contact where the two plasma membranes project into the axoplasm, separated by a gap of 130e140 Å (Peracchia, 1974). In freeze-fracture replicas, the P-face of the axonal projections (A, inset b) appears as an elongated indentation 0.5e1.2 mm long, 0.12e0.15 mm wide, containing parallel chains of particles that repeat every 120e125 Å. The particles repeat along the chain every 80e85 Å. This structure has a rhomboidal unit cell of 80e85 120e125 Å (A, inset b). A (inset c) shows a region where axonal (a) and glial (g) membranes fuse, creating an axo-glial pore. Originally published in the Journal of Cell Biology Peracchia, C. (1970). A system of parallel septa in crayfish nerve fibers. Journal of Cell Biology, 44, 125e133.
1970) or transverse cisternae (Fig. 2CeE). The membranes of the fenestrated septa frequently join, forming 0.1e0.2 mM pores each occupied by a single microtubule (Fig. 2C). Neighboring fenestrated septa are joined by longitudinal membranous tubules (Fig. 2B, D and E). In crosssectioned axons the septal membranes, now seen in face view, show clear images of the pores each containing a cross-sectioned microtubule (Fig. 3AeC). Occasionally, thin filaments bridge the microtubule to the edge of the pore (Fig. 3C). These structures (Fig. 2E) are present in different size axons and may be a general feature of axon structure because we observed them, although in a less organized fashion, also in mouse sciatic and vagus nerves (Fig. 4).6 Their function is unclear, but their connection to microtubules suggests a potential role in the axoplasmic flow mechanism; rev. in (Ochs, 1982). If so, the filaments seen bridging the microtubule to the edge of the pore could be composed of dynein or kinesin (Gennerich & Vale, 2009).
These data were reported as “C. Peracchia, unpublished observation” in (Peracchia, 1970).
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1.2 DIRECT CELL-TO-CELL COMMUNICATION
Fenestrated Septa in Mammalian Axons
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FIGURE 4 Cross-section views of mouse sciatic and vagus nerves. Fenestrated septa similar to those seen in crayfish axons (Figs. 2 and 3) are occasionally seen (arrows), although in a less organized fashion. Also in these mammalian axons, cross-sectioned profiles of microtubules are seen at the center of the pores (fenestrae; arrows). These mouse structures were reported as “C. Peracchia, unpublished observation” in Peracchia, 1970.
1.2.1.3 Particle Arrays in Crayfish Axo-Glial Membrane Projections Crayfish axons also display regions in which axonal and glial plasma membranes are regularly curved and project into the axoplasm, separated by a gap of 130e140 Å (Peracchia, 1974) (Fig. 3A, a, arrow). In freeze-fracture replicas (Fig. 3A, b) the P-face of the axonal projection appears as an elongated indentation 0.5e1.2 mm long, 0.12e0.15 mm wide, containing parallel chains of particles repeating every 120e125 Å and oriented obliquely such that the axis of the chains is skewed with respect to the long axis of the indentations by an angle of 55e 60 degrees. The particles repeat along the chain every 80e85 Å. This structure can be defined as a particle array with a rhomboidal unit cell of 80e85 120e125 Å (Fig. 3A, b). On the axonal E-face the complementary image of these structures is seen. The projections of the satellite’s membrane also contain particle aggregates, but these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. The functional meaning of these structures is unknown, but they could be areas of cell-to-cell adhesion (Peracchia, 1974). 1.2.1.4 Are There Axo-Glial Pores in Crayfish Axons? In crayfish axons, we also observed regions where axonal and glial plasma membranes appear to fuse (Fig. 3A, c), creating images of intercellular pores (Peracchia, 1981). If present, these pores would be expected to allow direct communication between the axoplasm and the glial cytoplasm. The pores may actually form transiently but are unlikely to be a preparation artifact because the radius of membrane fusion at the pores is consistent with the minimum curvature radius of biological membranes. Recently, similar pores have also been seen in crayfish stretch receptors (Fedorenko, Neginskaya, Fedorenko, & Uzdensky, 2015).
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1. HISTORICAL BACKGROUND
1.2.2 Evidence for Rectifying Gap Junctions in Invertebrate Nervous System By the mid-40s, it was generally believed that electrical communication between adjacent cells was nonpolarized (bidirectional). Therefore, it came as a surprise when in 1947 Cornelis A. G. Wiersma (1905e79) demonstrated that in crayfish the transmission of the electrical impulse between giant and motor fibers is polarized, such that current only spreads from giant to motor fibers (rectifying junctions) (Wiersma, 1947). Twelve years later, Wiersma’s data were confirmed by a careful study using intracellular recording (Furshpan & Potter, 1959).
1.3 DIRECT ELECTRICAL COMMUNICATION BETWEEN MAMMALIAN CARDIAC FIBERS In the early 50s the Swiss physiologist Silvio Weidmann (1921e2005) made a major discovery while working on single Purkinje fibers of kid’s heart (Weidmann, 1952), although at first, he did not recognize its importance. Weidmann measured the electrical resistance of the myoplasm by passing polarizing pulses through a microelectrode inserted at one end of the fiber and recording the electrotonic potential with microelectrodes placed at various distances from the current-passing microelectrode. He reported that: “. The relatively low value of the specific d.c. resistance of myoplasm (twice that of Tyrode solution) suggests (i) that the smaller units making up the Purkinje fibre, the Purkinje cells, are not surrounded by ionic barriers of any importance, (ii) that Purkinje fibres are not subdivided by transverse membranes . and (iii) that most of the intracellular ions must be free to move under the influence of an electric field.” Weidmann’s study not only demonstrated that Purkinje cells are electrically coupled, which is the earliest example of direct cell-to-cell communication in vertebrates (indeed, mammals), but also further confirmed the capacity of these cells to “heal-over.” The “healing-over” phenomenon was first described by Theodor Wilhelm Engelmann (1843e1909), who wrote: “. cardiac cells live together and die alone .” (Engelmann, 1877). This phenomenon, later confirmed by K.E. Rothschuh (1951), is now known as cell-to-cell uncoupling, a function mediated by the gating properties of gap junction channels (see in the following). Weidmann is rightly credited for having provided the first demonstration of direct electrical communication between adjacent cells in vertebrates (indeed, in mammals). However, while we know now that this is true, in 1952, Weidmann actually believed that the cells making the Purkinje fibers were not independent of each other, but rather interconnected via cytoplasmic bridges. He thought that the heart, the uterus, or other organs made of smooth muscle fibers (intestine, arteries, etc.) known to contract globally or sequentially were in fact “functional syncytia” rather than aggregates of individual cells bordered by biological membranes. Curiously, the cellular (rather than syncytial) organization of Purkinje fibers was actually described by Sunao Tawara as early as in 1906, a finding that was confirmed by J. M. Tufts (1921) through maceration experiments. In spite of Tawara’s and Tufts’ findings, however, in the first half of the 20th century most scientists believed in the syncytial nature of Purkinje fibers (Blair & Davies, 1935; DeWitt, 1909; Field, 1951; Jordan & Banks, 1917). Actually, Jordan and Banks reported that ox’s Purkinje fibers are cellular at the embryo stage of
1.4 DIRECT CELL-TO-CELL COMMUNICATION
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development, but they thought that later in development they would fuse into a syncytium (Jordan & Banks, 1917). Eventually, the accuracy of Tawara’s original observation was vindicated by evidence from electron microscope studies, which proved that Purkinje cells are indeed independent units of tissue bordered by plasma membranes (Van Breeman, 1953; Sjostrand & Andersson, 1954; Price, Weiss, Hata, & Smith, 1955; Muir, 1957; Muir, 1965; Rhodin, Del, & Reid, 1961; Hayashi, 1962). When evidence for the cellular organization of Purkinje fibers eventually surfaced, Weidmann was actually disappointed, not realizing at first that he had made an important discovery. Indeed, in a 1976 review article,7 Weidmann wrote: “Up to 1954 there was little doubt that cardiac muscle could be considered as a syncytium. This view was based on functional properties such as all-or-nothing excitability and conduction without decrement. A short communication by Sjostrand and Andersson (1954) came as a rather unpleasant surprise. Using high resolution electron microscopy these authors clearly demonstrated the existence of morphologically distinct cells. There were two major possibilities to reconcile morphology and function: either to postulate cell-to-cell transmission by a chemical mediator (see for instance Sperelakis, 1963) or to assume that adjoining cells may have specialized regions of low resistance. The evidence, which has accumulated over the past 20 years, seems to favor the second alternative” (Weidmann, 1976).
1.4 DIRECT CELL-TO-CELL COMMUNICATION IN THE VERTEBRATE NERVOUS SYSTEM Aside from the Weidmann’s (1952) study on kid’s heart, up to the late 1950s most of the data for direct electrical communication between cells were only reported in invertebrate (electrically) excitable cells. It made news, therefore, a 1959 publication reporting electrical communication between supramedullary neurons of a vertebrate: the puffer fish Spheroides maculatus (Bennett, Crain, & Grundfest, 1959). This important finding proved that electrical coupling is not a function only restricted to invertebrate excitable cells. This study also explained some puzzling phenomena that were observed in frog spinal motor neurons. In experiments on isolated spinal cords the stimulation of one motor neuron resulted in prolonged depolarization of several adjacent motor neurons (Washizu, 1960; Kubota & Brookhart, 1962; Katz & Miledi, 1963). This phenomenon was puzzling because it was not believed to result from the activity of chemical synapses. The report on fish neurons (Bennett et al., 1959) provided an explanation for this phenomenon, as it demonstrated that it is based on electrical cell-to-cell coupling. Indeed, Yoshiaki Washizu had proposed this explanation for the behavior of frog motor neurons, but none of these authors (Washizu, 1960; Kubota & Brookhart, 1962; Katz & Miledi, 1963) seems to have been aware of the earlier study of Bennett and coworkers (Bennett et al., 1959). Four years after Bennett and coworkers’ publication, electrical coupling was also reported in two other vertebrate systems: the large caliciform synapses of the chick’s ciliary ganglion (Martin & Pilar, 1963a,b) and the Mauthner neurons of goldfish (Furukawa & Furshpan, 1963).
7
I felt very honored when Silvio Weidmann sent me this review article with his signature and a nice note!
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1. HISTORICAL BACKGROUND
1.5 DIRECT CELL-TO-CELL COMMUNICATION IN NONEXCITABLE CELLS A major discovery in the history of direct cell-to-cell communication was made in 1964 by two independent studies: one on insect salivary glands (Kanno & Loewenstein, 1964; Loewenstein & Kanno, 1964) and the other on leech glia (Kuffler & Potter, 1964). Interestingly, these discoveries were serendipitous, as no one conceived then that nonexcitable cells might directly communicate with each other. Yoshinobu Kanno and Werner R. Loewenstein (1926e2014) were actually studying the electrical properties of the nuclear envelope in the gland cells of Drosophila flavorepleta larvae and had no idea that neighboring gland cells might be able to directly communicate with each other. To their surprise, upon current injection into the nucleus of one of these giant cells, a change in membrane potential was recorded not only in the injected cell but also in the adjacent cells (Loewenstein & Kanno, 1964; Kanno & Loewenstein, 1964). Stephen W. Kuffler (1913e1980) and David D. Potter also discovered by accident the electrical coupling of glial cells, as their study was actually aimed at determining whether neurons interact with glial cells (Kuffler & Potter, 1964). However, the electrical interaction of glial cells might not have been that surprising because Alan Peters reported, 2 years earlier, tight membrane appositions at glial cell contacts (Peters, 1962). In any event, it is undeniable that these two studies opened a new exciting chapter in the history of cell biology because thereafter it became apparent that the vast majority of cells would no longer be though as independent units of tissue, but rather as members of intimately communicating cell communities.
1.5.1 Why Nonexcitable Cells Communicate Directly With Each Other The major question then was: why do (electrically) nonexcitable cells need to communicate electrically with each other? The answer came from the first study of Lowenstein and Kanno, which demonstrated that cells are also capable of exchanging molecules larger than small ions, such as fluorescein (MW ¼ 332) (Loewenstein & Kanno, 1964). This suggested for the first time the possibility of direct metabolic cooperation among cells, a function that was proven by John D. Pitts and coworkers a few years later (Pitts & Simms, 1977; SubakSharpe, Burk, & Pitts, 1966; Subak-Sharpe, Burk, & Pitts, 1969). Soon it became obvious that cell-to-cell communication is not only a property of (electrically) nonexcitable “invertebrate” cells but is also a property of (electrically) nonexcitable “vertebrate” cells such as those of toad urinary bladder (Loewenstein, Socolar, Higashino, Kanno, & Davidson, 1965) and mouse liver (Penn, 1966). Later on, most neighboring cells were found to exchange metabolites such as amino acids, nucleotides, second messengers, and so no, confirming that most tissues are more than just aggregates of independent cells but are rather metabolically interlinked cell communities harmoniously cooperating for maintaining homeostasis, amplifying hormonal signals, distributing signal transduction intermediates, and so on.