Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

Organization of cytokeratin bundles by desmosomes in rat mammary cells

In a rat mammary epithelial cell line, LA-7, cytokeratin bundles recognized in immunofluorescence by a monoclonal antibody (24B42) disappear after trypsinization of cultures and are gradually reformed after replating. We have followed the time course of cytokeratin filament reappearance by growing cells in low calcium medium (0.1 mM) which prevents desmosome formation, and then shifting to high calcium (1.8 mM) to start the process. By fixing the cells at various intervals and staining them in immunofluorescence for 24B42 cytokeratin and for desmosomal proteins, we found that cell to cell contact and desmosome formation are prerequisites for keratin filament formation in these cells. EGTA treatment, by disassembling desmosomes, causes the cytokeratin filaments to disappear and the 24B42 protein to pass into a soluble form in this cell line, as ascertained by 100,000 g fractionation and immunoenzymatic assay. Cycloheximide treatment also causes cytokeratin filaments to disappear, indicating that protein synthesis is needed for normal filament maintenance. In another related cell line (106A-10a) and in HeLa cells, trypsinization and EGTA exposure do not cause a complete loss of 24B42 immunofluorescence, although distinct filaments disappear, indicating the presence in these cells of different organizing centers, besides desmosomes, for cytokeratin bundle formation. LA7 cells therefore seem to have a cytokeratin system strictly dependent on the presence of desmosomes, which act as an organizing center for filament assembly. 106A-10a cells (also rich in desmosomes) and HeLa cells (showing instead a reduced number of desmosomes) have a cytokeratin system partially or totally independent from that of desmosomes, with different organizing centers.     

A role for plakophilin-1 in the initiation of desmosome assembly

Plakophilins (pkp-1, -2, and -3) comprise a family of armadillo-repeat containing proteins that are found in the desmosomal plaque and in the nucleus. Plakophilin-1 is most highly expressed in the suprabasal layers of the epidermis and loss of plakophilin-1 expression results in skin fragility-ectodermal dysplasia syndrome, which is characterized by a reduction in the number and size of desmosomes in the epithelia of affected individuals. To investigate the role of plakophilin-1 during desmosome formation, we fused plakophilin-1 to the hormone-binding domain of the estrogen receptor to create a fusion protein (plakophilin-1/ER) that can be activated in cell culture by the addition of 4-hydroxytamoxifen. When plakophilin-1/ER was expressed in A431 cells it was incorporated into endogenous desmosomes and did not disrupt desmosome formation. A derivative of A431 cells (A431D) do not form desmosomes, even though they express all the components believed to be necessary for desmosome assembly. Expression and activation of plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining on the cell surface. Co-expression of a classical cadherin (N-cadherin) and plakophilin-1/ER in A431D cells resulted in punctate desmoplakin staining at cell-cell borders. These data suggest that plakophilin-1 can induce assembly of desmosomal components in A431D cells in the absence of a classical cadherin; however a classical cadherin (N-cadherin) is required to direct assembly of desmosomes between adjacent cells. The activatable plakophilin-1/ER system provides a unique culture system to study the assembly of the desmosomal plaque in culture.     

Structure and assembly of desmosome junctions: biosynthesis and turnover of the major desmosome components of Madin-Darby canine kidney cells in low calcium medium

Neither stratifying (primary keratinocytes) nor simple (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney [MDBK]) epithelial cell types from desmosomes in low calcium medium (LCM; less than 0.1 mM), but they can be induced to do so by raising the calcium level to physiological concentrations (standard calcium medium [SCM], 2 mM). We have used polyclonal antisera to the major bovine epidermal desmosome components (greater than 100 kD) in a sensitive assay involving immunoprecipitation of the components from metabolically labeled MDCK cell monolayers to investigate the mechanism of calcium-induced desmosome formation. MDCK cells, whether cultured in LCM or SCM, were found to synthesize the desmosome protein, DPI and desmosome glycoproteins DGI and DGII/III with identical electrophoretic mobility, and also, where relevant, with similar carbohydrate addition/processing and proteolytic processing. The timings of these events and of transport of DGI to the cell surface were similar in low and high calcium. Although the rates of synthesis of the various desmosome components were also similar under both conditions, the glycoprotein turnover rates increased dramatically in cells cultured in LCM. The half-lives decreased by a factor of about 7 for DGI and 12 for DGII/III and, consistent with this, MDCK cells labeled for 48 h in SCM had three and six times the amount of DGI and DGII/III, respectively, as cells labeled for 48 h in LCM. The rate of turnover and the levels of DPI were changed in the same direction, but to much lesser extents. Possible mechanisms for the Ca2+-dependent control of desmosome formation are discussed in the light of this new evidence.     

Procaine effects on the sodium transport in frog skin

A study on the influence of procaine on the sodium transport properties in frog skin was carried out. The application of procaine hydrochloride on either the mucosal or the serosal sides of the isolated frog skin has opposite effects. When added to the mucosal compartment, the procaine (as well as two procaine based drugs: Gerovital H3 and Aslavital) biphasically increase the short-circuit current (Isc) with a noticeable "recline" phenomenon, and decrease the slope resistance, as given by the I-V curves. When applied in the serosal compartment, Isc is decreased and the slope resistance of the epithelium is increased. The procaine effect on the apical membranes shows a pronounced dependence on the external sodium concentration. The shift of the E2 inflection point (which indicates the critical intensity of the electric field at which the epithelial conductance changes), with respect to the transepithelial open-circuit potential, shows a rapid and quasi-exponential increase following the application of 25 mM procaine in addition to the different mucosal Na concentrations.     

EXPERIMENTAL MANIPULATION OF DESMOSOME FORMATION

In the corneal epithelium of the embryonic chick there is a 3- to 4-fold increase in desmosomes between the 15th and 16th days of incubation which has not been noted in earlier studies of this tissue. This finding has made it feasible to study the effects of the local cell environment on desmosome formation. Cells of 15-day corneas which were forming desmosomes rapidly, were dispersed and combined in culture with cells from 10-day corneas which were forming few desmosomes. Surfaces of the same 15-day cell which were confronted with either another 15-day cell or a 10-day cell were compared. Desmosomes formed preferentially on the surface adjacent to a like cell. When 15-day cells were confronted with pigment cells, desmosomes formed almost exclusively on the surface adjacent to a like cell. Evidence for such localized differences on the same cell surface emphasize the importance of the immediate cell environment in desmosome formation. The observation that single desmosome plaques form occasionally on lateral cell surfaces has been noted previously. This finding was confirmed.     

Carboxyl terminus of Plakophilin-1 recruits it to plasma membrane, whereas amino terminus recruits desmoplakin and promotes desmosome assembly

Plakophilins are armadillo repeat-containing proteins, initially identified as desmosomal plaque proteins that have subsequently been shown to also localize to the nucleus. Loss of plakophilin-1 is the underlying cause of ectodermal dysplasia/skin fragility syndrome, and skin from these patients exhibits desmosomes that are reduced in size and number. Thus, it has been suggested that plakophilin-1 plays an important role in desmosome stability and/or assembly. In this study, we used a cell culture system (A431DE cells) that expresses all of the proteins necessary to assemble a desmosome, except plakophilin-1. Using this cell line, we sought to determine the role of plakophilin-1 in de novo desmosome assembly. When exogenous plakophilin-1 was expressed in these cells, desmosomes were assembled, as assessed by electron microscopy and immunofluorescence localization of desmoplakin, into punctate structures. Deletion mutagenesis experiments revealed that amino acids 686-726 in the carboxyl terminus of plakophilin-1 are required for its localization to the plasma membrane. In addition, we showed that amino acids 1-34 in the amino terminus were necessary for subsequent recruitment of desmoplakin to the membrane and desmosome assembly.     

Intermediate filaments and the initiation of desmosome assembly

The desmosome junction is an important component in the cohesion of epithelial cells, especially epidermal keratinocytes. To gain insight into the structure and function of desmosomes, their morphogenesis has been studied in a primary mouse epidermal (PME) cell culture system. When these cells are grown in approximately 0.1 mM Ca2+, they contain no desmosomes. They are induced to form desmosomes when the Ca2+ level in the culture medium is raised to approximately 1.2 mM Ca2+. PME cells in medium containing low levels of Ca2+, and then processed for indirect immunofluorescence using antibodies directed against desmoplakins (desmosomal plaque proteins), display a pattern of discrete fluorescent spots concentrated mainly in the perinuclear region. Double label immunofluorescence using keratin and desmoplakin antibodies reveals that the desmoplakin-containing spots and the cytoplasmic network of tonofibrils (bundles of intermediate filaments [IFB]) are in the same juxtanuclear region. Within 1 h after the switch to higher levels of Ca2+, the spots move toward the cell surface, primarily to areas of cell-cell contact and not to free cell surfaces. This reorganization occurs at the same time that tonofibrils also move toward cell surfaces in contact with neighboring cells. Once the desmoplakin spots have reached the cell surface, they appear to aggregate to form desmosomes. These immunofluorescence observations have been confirmed by immunogold ultrastructural localization. Preliminary biochemical and immunological studies indicate that desmoplakin appears in whole cell protein extracts and in Triton high salt insoluble residues (i.e., cytoskeletal preparations consisting primarily of IFB) prepared from PME cells maintained in medium containing both low and normal Ca2+ levels. These findings show that certain desmosome components are preformed in the cytoplasm of PME cells. These components undergo a dramatic reorganization, which parallels the changes in IFB redistribution, upon induction of desmosome formation. The reorganization depends upon both the extracellular Ca2+ level and the establishment of cell-to-cell contacts. Furthermore, the data suggests that desmosomes do not act as organizing centers for the elaboration of IFB. Indeed, we postulate that the movement of IFB and preformed desmosomal components to the cell surface is an important initiating event in desmosome morphogenesis.     

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.     

Control of desmosome formation in aggregating embryonic chick cells

Corneal epithelial cells have been used to study cell surface changes during cell aggregation. Tissue was taken from developmental stages in which desmosomes were forming rapidly. When corneal cells are dispersed, adjacent desmosome plaques are separated and single plaques are left on the cell surface. As cells aggregate, changes in the frequency of single plaques or of full desmosomes (double plaques) per micrometer of cell surface cross section can be followed. Single plaques are lost from the surface by endocytosis. Quantitative studies show a loss of single plaques beginning in the first hour of culture and formation of double plaques at 2 to 3 hr. In cells treated with cytochalasin B or D, single plaques are not lost during the first 2 hr and double plaques form with a higher frequency. Formation of double plaques is suppressed by actinomycin D, cycloheximide, and dinitrophenol. Thus desmosome formation requires de novo protein synthesis. In addition, inhibition of cell surface turnover by drugs which modify the cytoskeleton will enhance the rate at which desmosomes form.     

Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells

In cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron-dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell-cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506-517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron-dense bodies associated with intermediate filaments at sites of cell-cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a "string" of intermediate filaments at areas of cell-cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron-dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of "cycling" of desmoplakin through these bodies in proliferative cells.     

Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation

Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.     

The Role of Potassium in Active Transport of Sodium by the Toad Bladder

Studies were carried out on the isolated urinary bladder of the toad, Bufo marinus, in order to explain the dependence of active sodium transport on the presence of potassium, in the serosal medium. Attempts to obtain evidence for coupled sodium-potassium transport by the serosal pump were unsuccessful; no relation between sodium transport and uptake of K⁴² from the serosal medium was demonstrable. Rather, the predominant effect of serosal potassium appeared to be operative at the mucosal permeability barrier, influencing the permeability of this surface to sodium. The mucosal effects of serosal potassium were correlated with effects on cellular cation content. When sodium Ringer's solution was used as serosal medium, removal of potassium resulted in significant decrease in tissue potassium content, commensurate increase in tissue sodium content, and marked depression of mucosal permeability and sodium transport. When choline replaced sodium in the serosal medium, removal of potassium resulted in only slight alterations of tissue electrolyte content, and effects on mucosal permeability and sodium transport were minimal.     

Pemphigus vulgaris antigen, a desmoglein type of cadherin, is localized within keratinocyte desmosomes

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of cadherin cell adhesion molecules. Because autoantibodies in this disease cause blisters due to loss of epidermal cell adhesion, and because desmoglein is found in the desmosome cell adhesion junction, we wanted to determine if PVA is also found in desmosomes. By immunofluorescence, PV IgG bound, in a dotted pattern, to the cell surface of cultured human keratinocytes induced to differentiate with calcium, suggesting junctional staining. However, by preembedding, immunogold electron microscopic studies only slight labeling could be detected in desmosomes, presumably because of difficulty in gold penetration of intact desmosomes. We therefore treated the keratinocytes with 0.01% trypsin in 1 mM calcium, conditions known to preserve cadherin antigenicity but that caused slight separation of desmosomes, before immunogold staining. In this case there was extensive labeling of the extracellular part of desmosomes but not of the interdesmosomal cell membrane which was stained with anti-beta 2- microglobulin antibodies. To confirm the specificity of this binding we showed that antibodies raised in rabbits against the extracellular portions of PVA also bound desmosomes in these cultures. In intact mouse epidermis we could also show slight, but specific, immunogold desmosomal labeling with PV IgG. Furthermore, neonatal mice injected with PV IgG affinity purified on PVA showed desmosomal separation with the IgG localized to desmosomal cores. These results indicate that PVA is organized and concentrated within the desmosome where it presumably functions to maintain the integrity of stratifying epithelia.     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.     

Ultrastructural observations on effects of different concentrations of calcium and thyroxine in vitro on larval epidermal cells of Rana catesbeiana tadpoles

Summary During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5mM) calcium (Ca²⁺) in the absence or presence of thyroxine (10⁻⁷ M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca²⁺ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca²⁺ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca²⁺ concentrations (0.5mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation inRana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

Benzodiazepines stimulate sodium ion transport in frog skin epithelium

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.