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The effect of aldosterone on the isolated frog skin epithelium (R. temporaria)
448
C. L. Voiite et al.
similarly. The cells grafted into the control mice, without cortisone conditioning, are destroyed in 4-5 days. The data obtained prove that human and animal epithelial cell lines originating from normal (FL, WISH) as well as neoplastic (Hep-2, HeLa, KB) tissues possess the common property of causing cartilage and bone induction in mice. Since primary human amnion cultures do not possess such properties [l], one may assume that established epithelial lines acquire this ability during longlasting cultivation. This can be due either to adaptation to in vitro growth or to the selection of cell lines. Cartilage and bone tissue induction after grafting of the cell lines CLV-X and CLV-4 transformed by vaccinia virus my suggest that virus(es) (latent) are responsible for this inductive properties. REFERENCES 1. Anderson, H C, Merker P C & Fogh, J, Am j path01 44 (1964) 507. 2. Wlodarski, K, Hinek, A & Ostrowski, K, J Calif, tissue. In press. 3. Koziorowska, J & Zakrzewski, K, Bull acad pol sci 14 (1966) 737. Received July 4, 1969
THE EFFECT OF ALDOSTERONE THE ISOLATED FROG SKIN EPITHELIUM (R. TEMPORARZA) A Morphological
ON
Study
C. L. VOUTE, R. DIRIX, R. NIELSEN and H. H. USSING, Institute of Biological Chemistry, University of Copenhagen, Copenhagen K, Denmark
Recently it was shown by one of the authors that aldosterone promotes a moult in the isolated frog skin [l]. The moult was accompanied by characteristic changes in potential difference and short-circuit current (fig. 1). Exptl Cell Res 57
start
lh
3h
ALDOCORT
5h
EN
7h
AWED
Fig. I. Graph illustrating the chronological bioelectric changes induced by aldosterone in the frog skin in Potential difference; x ----x , shortvitro. O----O, circuit current. Arrows indicate timing of biopsies (A-D).
The purpose of this communication is to describe the morphological changes associated with the bioelectric response. This morphological study was done according to a method previously described [2]. The timing of the biopsies is indicated in fig. I by arrows (A-D). The first changes observed after the addition of aldosterone concern the mitochondria-rich cells (MR cells). Their shape becomes pear-like; from a large base with a nucleus, the elongated apex tends to reach the subcorneal space by passing as a rather thin channel through the outermost layers of the stratum granulosum. An increasing number of these MR cells become separated from the stratum corneum by a space (“lake”) filled with some amorphous material (fig. 2B). The average number of MR cells with a lake, expressed as per cent of cells in the outermost layer of the stratum granulosum, is 9.3 in the aldosterone-treated skins, whereas it is 4.0 in the controls. The difference (5.3 %) is statistically significant (p < 0.001, N= 33).iBetween the nucleus and the top of the MR cells a varying number of granular subcellular structures are found. These organelles can be dif-
In vitro enhancement of cell clumping by surface region fractions
449
Fig. 2. Illustrations depicting the main alterations found in the chronological biopsies (A)-(D) (see also text). SC, stratum corneum; SS, subcorneal space; MR, mitochondria rich cell; L, “lake” (see text); G, granular subcellular structures. Light micrographs of toluidine blue stained 1 LI sections. x 700.
ferentiated from mitochondria by their size and density. Two to four hours after the addition of aldosterone, the cornified layer is connected to the underlying cell layer merely by a few desmosomes,becoming completely detached soon thereafter. When the lowest values of potential difference and short-circuit current are observed (fig. 2C). However, once the cornified layer is spontaneously ruptured or mechanically removed the short-circuit current rapidly raises to values above control levels. The recovery of potential difference is only partial. The biopsies in this stagedemonstrate that a new cornified layer is formed (fig. 20). These morphological observations suggest that the MR cells might be involved in the aldosterone-induced desquamation of the frog skin in vitro.
REFERENCES 1. Nielsen, R, Acta physiol Stand. In press. 2. Vofite, C L & Ussing, H H, J cell bio136 (1968) 625. Received July 14, 1969
IN VITRO ENHANCEMENT OF CELL CLUMPING BY SURFACE REGION FRACTIONS M. D. ROSENBERG, K. AUFDERHEIDE and JANE CHRISTIANSON, Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minn. 55116, USA
Summary Surface region fractions isolated from embryonic cells enhance the aggregation or clumping of cells cultured in vitro. The enhancement appears to be neither tissue nor species-specific. Fractions heattreated at lOO”C, do not enhance clumping. Exptl Cell Res 57
C. L. Voiite et al.
similarly. The cells grafted into the control mice, without cortisone conditioning, are destroyed in 4-5 days. The data obtained prove that human and animal epithelial cell lines originating from normal (FL, WISH) as well as neoplastic (Hep-2, HeLa, KB) tissues possess the common property of causing cartilage and bone induction in mice. Since primary human amnion cultures do not possess such properties [l], one may assume that established epithelial lines acquire this ability during longlasting cultivation. This can be due either to adaptation to in vitro growth or to the selection of cell lines. Cartilage and bone tissue induction after grafting of the cell lines CLV-X and CLV-4 transformed by vaccinia virus my suggest that virus(es) (latent) are responsible for this inductive properties. REFERENCES 1. Anderson, H C, Merker P C & Fogh, J, Am j path01 44 (1964) 507. 2. Wlodarski, K, Hinek, A & Ostrowski, K, J Calif, tissue. In press. 3. Koziorowska, J & Zakrzewski, K, Bull acad pol sci 14 (1966) 737. Received July 4, 1969
THE EFFECT OF ALDOSTERONE THE ISOLATED FROG SKIN EPITHELIUM (R. TEMPORARZA) A Morphological
ON
Study
C. L. VOUTE, R. DIRIX, R. NIELSEN and H. H. USSING, Institute of Biological Chemistry, University of Copenhagen, Copenhagen K, Denmark
Recently it was shown by one of the authors that aldosterone promotes a moult in the isolated frog skin [l]. The moult was accompanied by characteristic changes in potential difference and short-circuit current (fig. 1). Exptl Cell Res 57
start
lh
3h
ALDOCORT
5h
EN
7h
AWED
Fig. I. Graph illustrating the chronological bioelectric changes induced by aldosterone in the frog skin in Potential difference; x ----x , shortvitro. O----O, circuit current. Arrows indicate timing of biopsies (A-D).
The purpose of this communication is to describe the morphological changes associated with the bioelectric response. This morphological study was done according to a method previously described [2]. The timing of the biopsies is indicated in fig. I by arrows (A-D). The first changes observed after the addition of aldosterone concern the mitochondria-rich cells (MR cells). Their shape becomes pear-like; from a large base with a nucleus, the elongated apex tends to reach the subcorneal space by passing as a rather thin channel through the outermost layers of the stratum granulosum. An increasing number of these MR cells become separated from the stratum corneum by a space (“lake”) filled with some amorphous material (fig. 2B). The average number of MR cells with a lake, expressed as per cent of cells in the outermost layer of the stratum granulosum, is 9.3 in the aldosterone-treated skins, whereas it is 4.0 in the controls. The difference (5.3 %) is statistically significant (p < 0.001, N= 33).iBetween the nucleus and the top of the MR cells a varying number of granular subcellular structures are found. These organelles can be dif-
In vitro enhancement of cell clumping by surface region fractions
449
Fig. 2. Illustrations depicting the main alterations found in the chronological biopsies (A)-(D) (see also text). SC, stratum corneum; SS, subcorneal space; MR, mitochondria rich cell; L, “lake” (see text); G, granular subcellular structures. Light micrographs of toluidine blue stained 1 LI sections. x 700.
ferentiated from mitochondria by their size and density. Two to four hours after the addition of aldosterone, the cornified layer is connected to the underlying cell layer merely by a few desmosomes,becoming completely detached soon thereafter. When the lowest values of potential difference and short-circuit current are observed (fig. 2C). However, once the cornified layer is spontaneously ruptured or mechanically removed the short-circuit current rapidly raises to values above control levels. The recovery of potential difference is only partial. The biopsies in this stagedemonstrate that a new cornified layer is formed (fig. 20). These morphological observations suggest that the MR cells might be involved in the aldosterone-induced desquamation of the frog skin in vitro.
REFERENCES 1. Nielsen, R, Acta physiol Stand. In press. 2. Vofite, C L & Ussing, H H, J cell bio136 (1968) 625. Received July 14, 1969
IN VITRO ENHANCEMENT OF CELL CLUMPING BY SURFACE REGION FRACTIONS M. D. ROSENBERG, K. AUFDERHEIDE and JANE CHRISTIANSON, Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minn. 55116, USA
Summary Surface region fractions isolated from embryonic cells enhance the aggregation or clumping of cells cultured in vitro. The enhancement appears to be neither tissue nor species-specific. Fractions heattreated at lOO”C, do not enhance clumping. Exptl Cell Res 57