Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific epidermal protein markers are modulated during calcium-induced terminal differentiation

Extracellular calcium concentration has been shown to be an important determinant of proliferation rate in a number of cell culture models. Recently, the role of calcium as a regulator of cellular differentiation has also become apparent. This effect of calcium was exemplified by the discovery that keratinocytes of mouse or human origin grew as a proliferating monolayer in medium with a calcium concentration of 0.02-0.09 mM but that proliferation ceased and cells stratified and cornified when calcium was increased greater than 0.1 mM. While the morphological and biological effects of changes in calcium concentration are dramatic in keratinocyte cultures, it has been difficult to identify specific protein changes associated with the modulation of maturation. In vivo, however, several proteins that are markers for stratified squamous epithelia have been identified by specific autoimmune sera. Pemphigoid antigen is a 220-kdalton protein found in the basement membrane and closely associated with the plasma membrane of the basal cell. Pemphigus antigen is a 130-kdalton glycoprotein found on the cell surface of stratifying epithelial cells. Immunofluorescence staining of cells cultured in low Ca2+ or cells switched to high Ca2+ for 48 h before staining demonstrated that pemphigoid antigen was detected in low Ca2+ cultures but was diminished or absent in high Ca2+ cultures and that pemphigus antigen was seen only in high Ca2+ cultures. The synthesis of each antigen was studied in immunoprecipitates of cell lysates radiolabeled with 14C-amino acids or D-[1-14C]glucosamine. Pemphigoid antigen was synthesized mainly by proliferating cells in low Ca2+ medium and its synthesis was decreased by greater than 90% in cells switched to high Ca2+ medium. In contrast, synthesis of pemphigus antigen was detected only in stratifying cells cultured in high Ca2+ medium. These studies indicate that extracellular calcium concentrations which modulate the transition between proliferating and stratifying epidermal cells also modulate, in parallel, the synthesis of specific marker proteins for these cell types.     

Aberrant in vitro expression of keratin K13 induced by Ca2+ and vitamin A acid in mouse epidermal cell lines.

Normally the expression of the murine type I keratin K13 is restricted to differentiating cells of internal squamous epithelia which line the oral cavity and the upper digestive tract. Recently, however, we were able to show that K13 is aberrantly but constitutively expressed without its normal type II partner K4 also in differentiating parts of 7,12-dimethylbenz(a)anthracene (DMBA/TPA) 12-O-tetradecanoylphorbol-13-acetate-induced squamous cell carcinomas of mouse back skin, whereas its likewise suprabasal expression in papillomas is variable (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988). In an attempt to reproduce the aberrant expression of K13 in a mouse in vitro system, we have investigated eight established murine epidermal cell lines for their putative ability to express K13. The cell lines differed distinctly in their derivation and comprised cell lines originating from DMBA/TPA induced papillomas (line SP1) or DMBA-treated adult mouse epidermis (line 308) as well as cell lines derived from DMBA or DMBA/TPA-treated primary epidermal keratinocytes (lines PDV and MCA 3D) and cell lines which arose spontaneously by long-term culture of normal epidermal keratinocytes (lines HEL 30 degrees HEL 37 degrees, HELP I and HELP III). We show that, independent of their derivation, all cell lines possess the intrinsic property to aberrantly express K13. Invariably the K13 gene is not expressed when the lines are cultured under low Ca2+ conditions (0.05 mM) and thus prevented from differentiation. Its expression can, however, be induced either by increasing the extracellular Ca2+ concentration or by the addition of physiological concentrations of vitamin A acid to low Ca2+ medium. Whereas in the latter case, K13 expression occurs without concomitant induction of morphological differentiation of the cells, Ca2+ elevation in the culture medium induces squamous differentiation and K13 expression occurs only in differentiating cells, thus reflecting the situation observed in in vivo tumors. All cell lines exhibit a concentration optimum for the stimulatory agents; however, the degree of maximal K13 expression varies considerably among the individual cell lines and shows a striking correlation with the reported tumorigenicity of the lines after transplantation to animals. In contrast, a tentatively suggested correlation between the activation of the Ha-ras gene and the aberrant expression of K13 (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988) could not definitely be confirmed since we observed K13 expression also in three cell lines which did not carry a mutation in codon 61 of the Ha-ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level

Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.     

Effects of hypoxia on the contractions and lactate release induced by high-K+, ouabain and epinephrine in guinea-pig aorta.

1. The 60 mM K+, 152 mM K+, Na-deficient medium and oubain-induced contractions of aorta were not so affected by severe hypoxia. 2. The 60 mM K+, 152 mM K+, Na(+)-deficient medium-induced responses were greatly reduced by deprivation of external Ca2+ in normoxia. 3. As the concentration of epinephrine increased, the remaining tensions which were expressed as a percentage of the original tensions became progressively greater in hypoxic condition. 4. The percentage of resistant components of the norepinephrine-induced contraction by the lower concentration was further reduced in Ca(2+)-free medium by severe hypoxic condition. 5. The tensions under normoxia and lactate release under severe hypoxia induced by 60 mM K+ or 2.5 x 10(-6) M epinephrine were of the same extent. 6. In conclusion, the inhibition of aortic response to epinephrine with severe hypoxia could not solely be explained by depression of the oxygen supply into the oxidative metabolism. Severe hypoxia did not affect Ca2+ influx through voltage-operated Ca2+ channels, but reduced both receptor-operated Ca2+ influx and intracellular Ca2+ release in the aorta.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines

The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically-induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mM extracellular Ca2+, i.e. low Ca2+ conditions) or differentiation (0.1 mM to 1.4 mM Ca2+), as previously shown using primary cultures of mouse keratinocytes. Two independently-derived normal keratinocyte lines demonstrated Ca2(+)-dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca2+ and time-dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12-dimethylbenz[alpha]anthracene). Papilloma cells cultured under conditions of low extracellular Ca2+ were comprised of three subpopulations: cells reactive only with pemphigoid anti-serum, cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca2+. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation-inducing agents in carcinoma therapy.     

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E- cadherin

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.     

Effects of lanthanum on calcium-dependent phenomena in human red cells.

Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or ATP + 2,3-diphosphoglycerate-depleted cells, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak +/ S.D. amounts to 0.28 +/ 0.08 mumol/1 of cells per min, whereas in KC1 medium to 0.15 +/ 0.04 mumol/1 of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1. Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol. Lanthanum at 0.2--0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations.     

Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro

The role of intracellular Ca²⁺ in the regulation of Ca²⁺-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca²⁺ chelator 1,2-bis(o-aminophenoxy)-ethane-N, N, N′, N′-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca²⁺ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca²⁺. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca²⁺, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca²⁺, the concentration of intracellular free Ca²⁺ (Ca_i) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca²⁺-sensitive fluorescent probe fura-2, and Ca²⁺ influx was measured by ⁴⁵Ca²⁺ uptake studies. Increase in extracellular Ca²⁺ (Ca_o) in the culture medium of keratinocytes caused a sustained increase in both Ca_i and Ca²⁺ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Ca_i concentration and prevented the Ca_i increase. After 12 hours of BAPTA treatment, the basal level of Ca_i returned to the control value, but the Ca²⁺ localized in intracellular stores was substantially decreased. ⁴⁵Ca²⁺ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total ⁴⁵Ca²⁺ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Ca_i and total cellular Ca²⁺ content in the presence of increased amount of intracellular Ca²⁺ buffer (e.g., BAPTA) by depleting intracellular Ca²⁺ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca²⁺-stores since this is the major change in intracellular Ca²⁺ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Ca_i change. © 1995 Wiley-Liss, Inc.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts.

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ or K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate. Addition of 1.8 mEGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

Multiple calcium pathways induce the expression of SNAP-25 protein in chromaffin cells

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.     

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.