Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+- independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.     

Uvomorulin mediates calcium-dependent aggregation of islet cells, whereas calcium-independent cell adhesion molecules distinguish between islet cell types

Rat islets of Langerhans are organized as a core of B-cells surrounded by non-B-cells. It is believed that cell type segregation during histogenesis is the result of the differential expression of cell adhesion molecules (CAMs). Since we have previously shown that in contrast to non-B-cells, homotypic adhesion of pancreatic B-cells is dependent on the presence of Ca2+, the possibility exists that Ca(2+)-dependent CAMs (cadherins) might be in part responsible for islet topography. We now demonstrate that after selective removal of Ca(2+)-independent CAMs from the surface of islet cells by mild trypsin/Ca2+ digestion (TC-treatment), there is no significant difference in homotypic adhesion between sorted B- and non-B-cells in the presence of calcium, suggesting an identical deployment of cadherins. Flow cytometric analysis reveals high levels of uvomorulin on both B- and non-B-cells, without any difference between the two populations. On a "1 to 100" scale, B-cell aggregation in the presence of Ca2+ was decreased by anti-uvomorulin Fab fragments from 67 +/- 4 to 25 +/- 3 (mean +/- SEM, n = 4, P less than 0.01). This level is not different from the degree of B-cell aggregation seen in the presence of 0.5 mM EDTA (22 +/- 2). Aggregation of non-B-cells was only slightly decreased by anti-uvomorulin Fab fragments (from 69 +/- 3 to 52 +/- 4). However, after TC-treatment, homotypic cell aggregation of both B- and non-B-cells was completely inhibited by anti-uvomorulin Fab fragments. Thus, uvomorulin appears to be the only functional cadherin on islet cells, and cell type aggregation properties diverge only by virtue of higher levels of Ca(2+)-independent CAMs on non-B-cells. Fab fragments with the property of perturbing islet cell aggregation in the absence but not in the presence of calcium also prevented pseudoislet organization in vitro, suggesting that Ca(2+)-independent CAMs play the major role in islet cell type segregation. In conclusion, the results show that uvomorulin is responsible for the Ca(2+)-dependent aggregation of islet cells and suggest that the cellular organization within islets or pseudoislets results from the different level of Ca(2+)-independent CAMs on islet cell types.     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Modification of the calcium-independent mechanism of cell adhesion in transformed BHK cells

The calcium-independent mechanism of cell adhesion was studied in normal and polyoma virus-transformed BHK cells. The degree of Ca2+-independent adhesion was greatly reduced in pyBHK cells, whereas CA2+-dependent adhesion occurred to the same degree as in BHK cells. This decrease was shown not to be caused by simple masking of the adhesion sites or by their altered sensitivity to trypsin. Adhesion-blocking antibodies were used to identify molecules responsible for Ca2+-independent adhesion. The antibodies precipitated surface molecules specific for adhesion-competent cells. These have tentatively been named CIDSBHK and CIDSpyBHK. Both were glycoproteins with respective apparent molecular weights of 120K and 125K. CIDSpyBHK incorporated 3H-glucosamine more than CIDSBHK did. Possible modification of the Ca2+-independent adhesion mechanism in pyBHK cells is discussed.     

Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells

Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.     

Role of calcium-dependent and calcium-independent mechanisms in the adhesion of retinal and pigment epithelium cells in the chick embryo

The mechanisms of adhesion of the retinal and pigment epithelium cells, as well of cell interaction within each of these tissues were studied during development. It was shown by means of separation of retina from pigment epithelium in different dissociation media that the adhesion of these tissues in 5-6 day old chick embryos is realized via a Ca2+-independent mechanism. The adhesion of these tissues decreases between days 7 and 16. Starting from day 16, both Ca2+-independent and Ca2+-dependent mechanisms are involved in the interaction of the retinal and pigment epithelium cells. By measuring the output of single cells into the suspension after the treatment of retina and pigment epithelium with different dissociating agents, it was shown that from the 5th day of incubation on the adhesion of pigment epithelium cells is mediated by Ca2+-dependent mechanism. In the retina three types of cells were found: interacting via Ca2+-dependent mechanism only, Ca2+-independent mechanism only, and both the mechanisms. In the course of differentiation, the numbers of the population of cells interacting only via Ca2+-dependent mechanism increase, while those of cells interacting via Ca2+-independent mechanism decrease. It is suggested that at each developmental stage those retinal cell possess Ca2+-dependent mechanism of adhesion which are closest to the definitive state.     

Can retinal adhesion mechanisms determine cell-sorting patterns: a test of the differential adhesion hypothesis

Embryonic chick neural retina cells possess two classes of adhesion mechanism, one Ca2+-independent, one Ca2+-dependent, responsible for short-term cell aggregation. This study investigates the role of these mechanisms in the long-term cell sorting potentially relevant to in vivo histogenesis. Retina cells are prepared either with both (E cells) or with only one mechanism (TC cells, CD; LTE cells, CI), respectively. The two types of cell preparations are differentially labelled using fluorescein or rhodamine isothiocyanate, mixed and allowed to aggregate in the presence or absence of cycloheximide at 0.5 microgram ml-1 to retard metabolic recovery of the removed adhesive mechanism. When observed by fluorescence and phase-contrast microscopy, the aggregates formed in cycloheximide show cell sorting, the cells with both mechanisms assuming a more interior position relative to those with a single adhesion mechanism. In parallel hanging-drop experiments, preformed aggregates of cells with a single adhesion mechanism are seen to spread upon aggregates of cells with both mechanisms. No sorting occurs amongst cells from a given stage prepared using any single dissociation protocol. The observed cell sorting would thus seem to derive exclusively from differential cell adhesiveness dependent upon the different dissociation conditions and maintained in the presence of cycloheximide. The experiments support the hypothesis that the dual CI and CD adhesion mechanisms in question can play a central role in governing cell-sorting behaviour during normal histogenesis.     

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm beta-D-galactoside-binding lectin and by beta-galactosidase

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.     

Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.     

Anti-CD9 monoclonal antibodies induce homotypic adhesion of pre-B cell lines by a novel mechanism

Anti-CD9 mAb are known agonists of platelet aggregation, but have not been implicated in cell-cell adhesion. We show here in an experimental system that the anti-CD9 mAb 50H.19, ALB6, and BA-2 can induce rapid, and irreversible, homotypic aggregation of the CD9-positive pre-B lymphoblastoid cell lines NALM-6 and HOON, but not of the CD9-negative B cell line Raji. The specificity of the response is indicated by the failure to effect aggregation with mAb directed to CD24, or to HLA class I Ag. The initiation of strong homotypic aggregates of lymphoid cells is a property ascribed to lymphocyte function-associated Ag-1 (LFA-1), a member of the beta 2 subfamily of leukocyte integrins. We show that CD9-induced aggregation is an active process which proceeds at 37 degrees C, but not at 4 degrees C, requires the expenditure of metabolic energy, and a functioning cytoskeleton, and is not inhibited by Arg-Gly-Asp-Ser peptide. These are properties described for LFA-1-mediated aggregation. However, because beta 2-integrins are not expressed on NALM-6 or HOON cells, they are not the mediators of CD9-induced aggregation. In contrast to LFA-1-mediated adhesion which is Mg2+ dependent, CD9-induced adhesion has an absolute requirement for Ca2+, but not Mg2+, indicating that a Ca2(+)-dependent event is sufficient for adhesion. However, Mg2+ enhances adhesion even at optimal concentrations of Ca2+, implicating an additional Mg2(+)-dependent event which requires Ca2+ to be effective. These findings suggest that CD9 Ag regulates a novel mechanism for promoting tight cell-cell adhesion which requires both Ca2+ and Mg2+ for optimal expression.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Biochemical characteristics of adhesion factors isolated from the retina of 8-day-old chick embryos

While isoelectrofocusing the extracts obtained after dissociation of the retina of eight day old chick embryos, two active protein fractions were found: AF I with pI 3 and AF II with pI below 3. The content of these fractions in the extracts depended on the method of dissociation: treatment with EDTA increased the content of AF II, whereas trypsin treatment increased the yield of AF I. After electrophoresis in the denaturating conditions both fractions were divided in two components. The effect of AF I on aggregation of retinal cells, unlike that of AF II, does not depend on the presence of bivalent cations in the medium and is realized within 1 h of interaction with the cells at 4 degrees. It is suggested that the retinal extracts of eight day old chick embryos contain substances providing two types of adhesiveness: Ca2+-independent (AF I) and Ca2+-dependent (AF II).     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Dual adhesion systems of chick myoblasts

Cultured chick myoblasts (Mb) were resuspended by incubation with 100 micrograms/ml trypsin/2.5 mM CaCl2 (to yield TC-Mb), or with 5 micrograms/ml trypsin/2.5 mM EDTA (to yield LTE-Mb). As measured in a particle counter, TC-Mb aggregation was Ca2+ dependent, whereas LTE-Mb aggregated equally well in the presence of CaCl2 or EDTA. Cells subjected to the same treatments in sequence, like cells dissociated directly with 100 micrograms/ml trypsin/2.5 mM EDTA, did not aggregate significantly in the presence or absence of Ca2+. Adhesive specificity was assessed by mixing unlabeled cells with cells labeled with a fluorescent dye and then analyzing the distribution of fluorescent and nonfluorescent cells in aggregates. No adhesive specificity was seen in controls (i.e., TC-Mb aggregated randomly with TC-Mb, or LTE-Mb with LTE-Mb), but TC-Mb and LTE-Mb did not cross-adhere. These results indicate the existence of two independent, noncomplementing, adhesion systems, and suggest that the differential treatments preserve or activate one system while destroying the other. Myoblasts dissociated with 2.5 mM EDTA in the absence of exogenous trypsin (E-Mb) have both adhesion systems active on their surfaces, as do Mb grown in Ca2+-free medium and then dissociated with 0.7 mM EDTA (Knudsen, K. A., and Horwitz, A. F., Dev. Biol. 58, 328-338, 1977). Although aggregation of E-Mb is largely Ca2+ independent and that of Knudsen/Horwitz-Mb is largely Ca2+ dependent, they adhere well to each other and to LTE-Mb while segregating from TC-Mb. Fibroblasts also have dual adhesion systems, one Ca2+ dependent and the other Ca2+ independent, but TC-Fb do not cross-adhere to TC-Mb (nor E-Fb to E-Mb). Cell type-specific adhesive selectivity may thus contribute to the selectivity of myocyte fusion.     

Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody

The molecular nature of the Ca2+-dependent cell-cell adhesion system in mouse teratocarcinoma (t-CDS) was studied using a monoclonal antibody recognizing t-CDS. We isolated a hybridoma clone producing a monoclonal antibody (ECCD-1) able to disrupt cell-cell adhesion when added to monolayer cultures of teratocarcinoma cells. This antibody bound to the cells with intact t-CDS, resulting in an inhibition of their aggregation, but did not bind to cells from which t-CDS was removed by trypsin treatment in the absence of Ca2+. The binding of ECCD-1 to cell surfaces required Ca2+ but not other ions. Western blot analysis showed that ECCD-1 recognizes multiple cell surface proteins, the major one of which is a component with a molecular weight of 124,000. The binding of ECCD-1 to these antigens was Ca2+-dependent even in cell-free systems, suggesting that the molecules involved in t-CDS undergo conformational changes by binding with Ca2+, leading to conversion of their molecular structure into an active form. ECCD-1 also reacted with 8-cell stage mouse embryos and with certain types of epithelial cells (excluding fibroblastic cells) in various differentiated tissues collected from mouse fetuses, again affecting their cell-cell adhesion. We also showed that a monoclonal antibody (DE1) raised against gp84 (F. Hyafil et al., 1981, Cell 26, 447-454) recognizes the same antigens as ECCD-1.     

Experimental manipulation of cell surface to affect cellular recognition mechanisms.

The mechanism of selective cell adhesion was studied using Chinese hamster V79 and chick embryonic neural retinal cells. Both of these cell types have been shown to have two experimentally separable mechanisms of adhesion; Ca²⁺-dependent and Ca²⁺-independent. Cells can be dispersed so that either or both of the mechanisms remain intact by use of different treatments. A method of labeling cells with FITC was devised to identify one of the two types of cells in a binary cell population. When cells with one of the two adhesion mechanisms were mixed with cells with the other mechanism, they segregated completely, forming independent aggregates, not only in the heterotypic combination of these cell types but also in the homotypic combination of each cell type. In contrast, when cells were mixed with others with the same adhesion mechanism, either Ca²⁺-dependent or -independent, they formed chimeric aggregates, even in the heterotypic cell combination. These results suggest that the specificity in each of those two mechanisms of cell adhesion plays an important role in cellular recognition processes.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.     

A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide

A functional assay has been developed to identify cell surface proteins involved in the formation of epithelial tight junctions. Transepithelial electrical resistance was used to measure the presence of intact tight junctions in monolayers of Madin-Darby canine kidney (MDCK) cells cultured on nitrocellulose filters. The strain I MDCK cells used have a transmonolayer resistance greater than 2,000 ohm . cm2. When the monolayers were incubated at 37 degrees C without Ca2+, the intercellular junctions opened and the transmonolayer resistance dropped to the value of a bare filter, i.e., less than 40 ohm . cm2. When Ca2+ was restored, the cell junctions resealed and the resistance recovered rapidly. Polyclonal antibodies raised against intact MDCK cells inhibited the Ca2+-dependent recovery of electrical resistance when applied to monolayers that had been opened by Ca2+ removal. Cross-linking of cell surface molecules was not required because monovalent Fab' fragments also inhibited. In contrast, a variety of other antibodies that recognize specific proteins on the MDCK cell surface failed to inhibit the recovery of resistance. Monoclonal antibodies have been raised and screened for their ability to inhibit resistance recovery. One such monoclonal antibody has been obtained that stained the lateral surface of MDCK cells. This antibody, rr1, recognized a 118-kD polypeptide in MDCK cell extracts and an 81-kD fragment released from the cell surface by trypsinization in the presence of Ca2+. Sequential immunoprecipitation with antibody rr1 and a monoclonal antibody to uvomorulin showed that this polypeptide is related to uvomorulin. The role of uvomorulin-like and liver cell adhesion molecule (L-CAM)-like polypeptides in the establishment of the epithelial occluding barrier is discussed.     

Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.     

Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion

Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg2+ and/or Ca2+ ions. Although Mg2+ is directly involved in ligand binding, the role of Ca2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca2+, but not Mg2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca(2+)-dependent epitope on LFA-1, we found that Ca2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non- responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca2+ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA- 1. Mg(2+)-dependent ligand binding does not affect binding of Ca2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr2+ ions can replace Ca2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface. We conclude that Ca2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg2+ is important in regulation of the affinity of LFA-1 for its ligands.