Ca2+-dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis

Incubating T51B rat liver cells in Ca2+-deficient, serum-rich medium containing only 0.02 mM Ca2+ strikingly decreased the phosphorylation of several trypsin-removable cell surface proteins and arrested the cells in late G1 phase. Raising the Ca2+ concentration in the Ca2+-deficient medium from 0.02 mM to 0.5 mM or adding 80 nM TPA (12-O-tetradecanoyl-phorbol-13-acetate), a protein kinase C activator, stimulated the phosphorylation of a certain set of surface proteins within 5 min and the initiation of DNA replication within the next 2 hr. By contrast, incubation in the same Ca2+-deficient medium, which does not affect the proliferation of neoplastic T51B-261B cells, did not reduce the phosphorylation of cell surface proteins. These observations suggest that the stimulation of a Ca2+-dependent protein kinase (possibly protein kinase C) directly or indirectly phosphorylates certain cell surface proteins that might be part of the mechanism that triggers the Ca2+-dependent G1----S transition of normal cells. They also suggest that an alteration of this Ca2+-dependent protein kinase might be the reason for neoplastic cells being able to proliferate in the face of an external Ca2+ shortage that would stop the proliferation of normal cells.     

Involvement of the Ca2+/phospholipid-dependent protein kinase in the G1 transit of T51B rat liver epithelial cells

The G0----G1 and G1----S transitions (but not the intervening events) in the G1 phase of T51B rat liver epithelial cells in serum-stimulated confluent cultures required a high concentration of extracellular Ca2+ and were accompanied or immediately preceded by increases in the amount of EDTA-extractable protein kinase C, a Ca2+/phospholipid-dependent enzyme. Involvement of this Ca2+-dependent enzyme in the two Ca2+-dependent transitions was further indicated by the facts that 12-O-tetradecanoyl phorbol-13-acetate (TPA), a compound that stimulated protein kinase C from T51B cells even in the absence of Ca2+, enabled these cells to transit G1 in Ca2+-deficient medium, while a TPA analogue (4 alpha-phorbol-12, 13-didecanoate (4 alpha-PDD) that did not stimulate the enzyme in cell-free preparations did not promote G0----G1 or G1----S transit in Ca2+-deficient medium.     

Inositol 1,3,4,5-tetrakisphosphate stimulates the initiation of DNA synthesis in Ca2+-deprived rat liver cells

The G1-S boundary of non-neoplastic cells requires extracellular Ca2+ for successful transition. Inositol 1,3,4,5-tetrakisphosphate but not inositol 1,4,5-trisphosphate can partially replace Ca2+ and stimulate the initiation of DNA synthesis of Ca2+-deprived T51B rat liver cells but only if sufficient extracellular Ca2+ (i.e., 0.075 mM) is present. The potent tumor promoter and protein kinase C activator 12-O-tetradecanoylphorbol acetate is also capable of replacing extracellular Ca2+ and partially stimulating the initiation of DNA synthesis. In addition, both inositol-1,3,4,5-tetrakisphosphate and 12-O-tetradecanoylphorbol acetate added together elicit a full DNA synthetic response.     

The glucocorticoid dexamethasone and the tumor-promoting artificial sweetener saccharin stimulate protein kinase C from T51B rat liver cells

Diacylglycerols, such as 1,2-diolein, and tumor-promoting phorbol compounds, such as TPA (12-0-tetradecanoyl phorbol-13-acetate), stimulate the Ca2+/phospholipid-dependent protein kinase C from T51B rat liver cells, probably by sensitizing it to activation by Ca2+, and they reduce the liver cells' content of EDTA-extractable (i.e., soluble) protein kinase C activity. Evidence is presented that indicates that the glucocorticoid, dexamethasone, and the tumor-promoting artificial sweetener, saccharin, also trigger a Ca2+-dependent increase in the activity of the protein kinase C from T51B liver cells and reduce the cells' content of EDTA-extractable protein kinase C activity. However, these novel stimulators do not activate the enzyme by binding to the same site as diacylglycerols and TPA, although they do alter this site as indicated by an increase in the binding of the TPA analogue PDBu (phorbol 12,13-dibutyrate).     

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.     

The 38 kDa Ca2+/membrane-binding protein of pig granulocytes needs a high Ca2+ concentration to be phosphorylated by protein kinase C

The 38 kDa Ca2+/membrane-binding protein reported to be the dominant substrate of protein kinase C in the extracts of pig neutrophil granulocytes was purified partially and its phosphorylation was investigated. In pig granulocytes type II protein kinase C was the major isoform, while type III isoenzyme was present only as a minor activity. Phosphorylation of the 38 kDa protein was performed with rat brain protein kinase C. Each of the three isoenzymes purified from rat brain was able to phosphorylate this protein, though on the conditions used in our experiments it was phosphorylated most intensively by type II protein kinase C. A phospholipid-dependent, but Ca2(+)-independent, form of protein kinase C was demonstrated with the aid of a synthetic oligopeptide substrate. Phosphorylation of the 38 kDa protein by the Ca2(+)-independent enzyme proceeded exclusively in the presence of Ca2+. The Ca2+ concentration necessary for the phosphorylation of the 38 kDa by either form of protein kinase C was by orders of magnitude higher than that required for the activation of protein kinase C.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Effect of cellular Ca2+ loading on alpha 1-agonist or protein kinase C activators-mediated stimulation of phosphorylase "a" in liver cells

Long chain unsaturated fatty acids stimulate phosphorylase "a" activity in liver cells. Similar degree of activation was achieved by increasing cellular Ca2+ content or by treatment with agents other than oleate, like 1,2-diolein or phorbol esters, sharing in common their ability to activate protein kinase C. In Ca2+-loaded liver cells only phenylephrine was capable of inducing a further stimulation of phosphorylase "a" activity. It is concluded that: 1) The state of activation of protein kinase C may play a role in the hormonal control of liver glycogen metabolism; 2) alpha 1-agonist-mediated activation of phosphorylase "a" can occur by a mechanism which is not related to a Ca2+-dependent activation of protein kinase C.     

Identification of isoenzymic forms of hepatic Ca2+-activated-phospholipid-dependent protein kinase in various animal models

We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75-85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH4)2SO4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80-88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca2+ (approximately 25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg2+ (5-10 mM) and lysine rich histone (H1). Both diolein and TPA decreased the Ca2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.     

Protein kinase C binding to isolated nuclei and its activation by a Ca2+/phospholipid-independent mechanism

The direct interaction of protein kinase C with the nucleus was examined utilizing endogenous protein phosphorylation and [3H]PDBu binding to detect the enzyme. Rat brain nuclei were relatively rich in phorbol ester receptors whereas liver nuclei contained less than 10% of their brain counterpart. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4 degrees C or at 24 degrees C reaching apparent equilibrium by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca2+ with an EC50 of 0.5 microM. Chelation of Ca2+ with EGTA resulted in rapid loss of phorbol ester receptors from nuclei. Differential extraction experiments with Triton X-100 and NaCl suggested that about 50% of the acquired phorbol ester receptors were bound to chromatin and 25% were associated with the nuclear matrix. Protein Kinase C bound to nuclei was also able to phosphorylate several endogenous nuclear substrates in a Ca2+/phospholipid-independent reaction. These data suggest that protein kinase C can associate with nuclear components leading to the phosphorylation of nuclear substrates.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.     

Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism

The Ca2+(calmodulin (CaM))-dependent protein kinase II, purified from either rabbit liver or rat brain, was preincubated under conditions that are known to promote its autophosphorylation. When kinase activity was assayed after this preincubation, it was observed that excess EGTA could block no more than 40-60% of the total Ca2+- and CaM-dependent activity compared to 95% inhibition by EGTA prior to preincubation. In the EGTA assay, free Ca2+ was calculated to be less than 1 nM; therefore, this activity was designated Ca2+-independent activity. Formation of this Ca2+-independent form of the kinase was shown to be associated with autophosphorylation based on the following observations: (a) it required the presence of Ca2+, CaM, and ATP; (b) the ATP analogs adenylyl imidodiphosphate and adenylyl methylenediphosphate could not substitute for ATP; (c) generation of the independent form was associated with incorporation of phosphate into the kinase; and (d) addition of protein phosphatase partially dephosphorylated the kinase and restored its Ca2+ dependence. This phenomenon may be of physiological importance because it would prolong the effects of extracellular signals that only transiently increase the intracellular Ca2+ level.     

Protein kinase C phosphorylation of protamine is Ca2+ independent, but the addition of DNA renders it Ca2+ dependent

Protamine is a unique substrate of protein kinase C for its Ca2+-independent phosphorylation. The interaction between protein kinase C and protamine and the effect of DNA on the interaction was studied. Protein kinase C was retained in a protamine-immobilized Sepharose 4B column, even in the absence of Ca2+ and was eluted with ammonium sulfate or L-arginine. The eluted enzyme was fully activated by phosphatidylserine alone, when protamine was used as substrate. When DNA was included in the assay system, the activity elicited by phosphatidylserine alone was inhibited. The DNA effect on the activity in the presence of both Ca2+ and phosphatidylserine was much lower than on the activity elicited by phosphatidylserine alone, thereby demonstrating the Ca2+ sensitivity of protamine phosphorylation.     

Phosphorylation of troponin in the heart and skeletal muscle by Ca2+-phospholipid-dependent protein kinase

The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Ca(2+)-induced reversible translocation of phospholipase A2 between the cytosol and the membrane fraction of rat liver macrophages

In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be rapidly associated with the particulate fraction in a Ca(2+)-dependent manner at Ca2+ concentrations of 0.1-1.0 microM. This is also the range of the levels of intracellular Ca2+ reported for basal and various stimulated conditions. After translocation, phospholipase A2 could be released from the membranes in the presence of Ca2+ chelators, increasing the specific activity of phospholipase A2 in the supernatant fraction. These findings support the view that translocation is a regulatory mechanism of phospholipase A2 by bringing the enzyme to its substrate. Unlike the situation with protein kinase C, Mg2+ exerted little effect on phospholipase A2 translocation, indicating that this process is regulated in vivo mainly by fluctuations of the intracellular Ca2+ content.     

Characterization of a mitogen-activated, Ca2+-sensitive microtubule-associated protein-2 kinase

We have previously found that treatment of quiescent mammalian fibroblast cells with several mitogenic factors activates in common a Ca2+-sensitive serine/threonine-specific protein kinase activity toward microtubule-associated protein 2 (MAP2) [Hoshi, M., Nishida, E. and Sakai, H. (1988) J. Biol. Chem. 263, 5396-5401]. Here, we characterized the mitogen-activated MAP2 kinase activity in rat 3Y1 cells. The activated kinase activity was detected in the cytosolic fraction but not in the membrane fraction. The inhibitory effect of Ca2+ on the kinase activity was reversible. Kinetic analyses revealed that the apparent Km values of the kinase activity for MAP2 and ATP were 1.6 microM and 30 microM, respectively. Free Ca2+ at 4 microM decreased apparent Vmax values for MAP2 and ATP without changing the apparent Km values. The MAP2 kinase had an apparent molecular mass of about 40 kDa as determined by gel filtration and by sucrose density gradient centrifugation. Myelin basic protein as well as MAP2 could serve as good substrates for this kinase, but 40S ribosomal protein S6, casein, histone, phosphorylase b, protamine, tubulin, actin and tau could not. These properties of the enzyme indicate that the Ca2+-sensitive MAP2 kinase may be a previously unidentified enzyme. Down-regulation of protein kinase C by prolonged phorbol ester treatment abolished the MAP2 kinase activation by phorbol ester, but did not prevent the MAP2 kinase activation by epidermal growth factor (EGF) or fresh serum. This suggests that the Ca2+-sensitive MAP2 kinase could be activated through protein-kinase-C-dependent and -independent pathways. Activation of the MAP2 kinase occurred shortly after the addition of EGF or phorbol ester even in the presence of protein synthesis inhibitors (cycloheximide, puromycin and emetin). Moreover, treatment of the EGF- or phorbol-ester-activated MAP2 kinase with acid phosphatase inactivated the kinase activity. Thus, the MAP2 kinase may be activated through phosphorylation.     

Glucocorticoids increase Ca2+ influx through dihydropyridine-sensitive channels linked to activation of protein kinase C in vascular smooth muscle cells.

To clarify whether protein kinase is associated with glucocorticoid-induced Ca2+ influx into vascular smooth muscle cells, we investigated the effects of protein kinase inhibitors on dexamethasone-induced 45Ca2+ uptake and dihydropyridine binding in A7r5 cells. Protein kinase C inhibitors (staurosporine and UCN-01) abolished the dexamethasone-induced 45Ca2+ uptake and [methyl-3H]PN 200-110 binding. In contrast, KT5720 and KT5823, which are more specific inhibitors of cAMP-dependent protein kinase and cGMP-dependent protein kinase, respectively, did not affect the effects of dexamethasone. Treatment with 100 nM dexamethasone for 48 hours increased protein kinase C activity in A7r5 cells. These results suggest that glucocorticoids increase Ca2+ influx through dihydropyridine-sensitive channels, linked to activation of protein kinase C in vascular smooth muscle cells.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.