Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.     

4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes.

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.     

Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+

The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action.     

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.     

Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools

Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.     

Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis.

The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.     

Cyclic AMP raises cytosolic Ca2+ and promotes Ca2+ influx in a clonal pancreatic beta-cell line (HIT T-15)

The effect on cytosolic Ca2+ concentration ([Ca2+]i) of cAMP analogues and the adenylate cyclase-stimulating agents forskolin, isoproterenol and glucagon has been examined in an insulin-secreting beta-cell line (HIT T-15) using fura 2. All these manipulations of the cAMP messenger system promoted a rise in [Ca2+]i which was blocked by the Ca2+ channel antagonists verapamil and nifedipine or by removal of extracellular Ca2+. The action of the adenylate cyclase activator forskolin was glucose-dependent. The results suggest that cAMP elevates [Ca2+]i in HIT cells by promoting Ca2+ entry through voltage-sensitive Ca2+ channels, not through mobilization of stored Ca2+. Activation of Ca2+ influx may be an important component of the mechanisms by which cAMP potentiates fuel-induced insulin release.     

A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).     

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.     

Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium

Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2- [methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha- phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.     

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.     

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than of cAMP

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine 3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+]i. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs. Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+]i resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+]i and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+]i rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP.     

Role of Ca2+ in H+ transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca2+ chelator

The role of Ca2+ in stimulation of H+ gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca2+ ionophore, A23187, and an intracellular Ca2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca2+ concentration, [Ca2+]i, measured in gastric glands loaded with Ca2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca2+-ionophore A23187 also induced an increase in [Ca2+]i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca2+]i. BAPTA-AM (5-25 microM) induced a transient decrease of resting [Ca2+]i which returned to basal level due to extracellular Ca2+ entry. Increases in [Ca2+]i produced by ACh and TG were abolished by BAPTA and those produced by Ca2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H+ secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca2+]i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca2+ and cAMP.     

Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C

CD43 is a constitutively phosphorylated 115-kDa sialoglycoprotein expressed on a variety of blood cells including lymphocytes and monocytes. L10, a mAb directed against CD43, triggers T cell activation and enhances hydrogen peroxide production in monocytes. Activation of mononuclear cells by L10 initiates phosphoinositides hydrolysis, C2+ mobilization, and protein kinase C (PKC) activation. In turn, activated PKC hyperphosphorylates CD43, suggesting a potential role for PKC in the regulation of signaling via CD43. To address this issue, we have analyzed the effect of PKC activation by the tumor promoter PMA on L10-triggered rise in intracellular free Ca2+ concentrations ([Ca2+]i). Treatment of mononuclear cells with PMA profoundly inhibited the increase in [Ca2+]i induced by L10. The inhibition of CD43-mediated signaling by PMA was due, in part, to uncoupling of CD43 from the signal-transducing G protein. This was evidenced by the comparatively modest inhibition by PMA of the increase in [Ca2+]i induced by the direct G protein activator AlF4-. PMA treatment did not affect the surface expression of CD43. However, it induced the hyperphosphorylation of CD43, the extent of which correlated with the inhibition of CD43-mediated increase in [Ca2+]i. Staurosporine, a potent inhibitor of PKC, abrogated the hyperphosphorylation of CD43 and normalized CD43-mediated signaling in PMA-treated cells. Significantly, in the absence of PMA, staurosporine enhanced the rise in [Ca2+]i triggered by L10, suggesting that engagement of CD43 by activating ligands results in feedback inhibition by PKC. It is concluded that activation of PKC inhibits signaling via CD43 by mechanisms involving phosphorylation and uncoupling of CD43 from the signal-transducing apparatus and by distal, post-receptor events.     

Calcium stimulates luteinizing-hormone (lutropin) exocytosis by a mechanism independent of protein kinase C.

Using permeabilized gonadotropes, we examined whether Ca2(+)-stimulated luteinizing-hormone (LH) exocytosis is mediated by the Ca2(+)-activated phospholipid-dependent protein kinase (protein kinase C). In the presence of high [Ca2+]free (pCa 5), alpha-toxin-permeabilized sheep gonadotropes secrete a burst of LH and then become refractory to maintained high [Ca2+]free. The protein kinase C activator phorbol myristate acetate (PMA) is able to stimulate further LH release from cells made refractory to high [Ca2+]free, suggesting that Ca2+ does not stimulate LH release by activating protein kinase C. Staurosporine, a protein kinase C inhibitor, inhibited PMA-stimulated (50% inhibition at 20 nM), but not Ca2(+)-stimulated, LH exocytosis. In cells desensitized to PMA by prolonged exposure to a high PMA concentration, Ca2(+)-stimulated LH exocytosis (when corrected for depletion of total cellular LH) was not inhibited. Ba2+ was able to stimulate LH exocytosis to a maximal extent similar to Ca2+, although higher Ba2+ concentrations were necessary. Ba2+ and Ca2+ stimulated LH exocytosis with a similar time course, and both were inhibitory at high concentrations. Furthermore, cells made refractory to Ca2+ were also refractory to Ba2+. These data strongly suggest that Ba2+ and Ca2+ act through the same mechanism. Since Ba2+ is a poor activator of protein kinase C, these findings are additional evidence against a major role for protein kinase C in mediating Ca2(+)-stimulated LH exocytosis.     

Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell

Effects of adrenocorticotropin (ACTH) on cytoplasmic free calcium concentration, [Ca2+]c, have been measured in adrenal glomerulosa cells using a calcium-sensitive photoprotein, aequorin. ACTH causes a rapid and transient increase in [Ca2+]c. Dose response study demonstrates that 1 pM ACTH induces an elevation of [Ca2+]c and that effect of ACTH appears to be saturated at 100 pM. ACTH action is greatly inhibited but not abolished by removal of extracellular calcium and is completely blocked in medium containing no added calcium and 1 mM EGTA. Under similar conditions, angiotensin II induces a remarkable rise in [Ca2+]c. ACTH action is not affected by pretreatment with dantrolene, which considerably decreases angiotensin II action on [Ca2+]c. One micromolar forskolin, which mimics 1 nM ACTH-mediated elevation of intracellular cAMP, does not increase [Ca2+]c nor modulates changes in [Ca2+] induced by a low dose of ACTH. One hundred micromolar forskolin or 1 mM 8-bromo-cAMP, however, increases [Ca2+]c even in calcium-free medium containing 1 mM EGTA. When glomerulosa cells are co-loaded with aequorin and quin2, angiotensin II-induced change in aequorin signal is greatly reduced, and ACTH-induced change is abolished. Quin2 loading results in accumulation of calcium in the cell under both unstimulated and stimulated conditions. These results indicate that ACTH increases [Ca2+]c by cAMP-independent mechanism, that ACTH action on [Ca2+]c is exclusively dependent on extracellular calcium, and that quin2 is unable to detect the rapid change in [Ca2+]c because of its calcium chelating activity.     

ACh对人垂体腺瘤细胞内蛋白激酶C、胞内游离钙及cAMP/cGMP的影响

我们曾发现ACh可明显地抑制垂体腺瘤细胞的增殖代谢,为深入探讨ACh抑制垂体腺瘤细胞增殖作用的机制,观察了ACh作用后垂体腺瘤细胞内蛋白激酶C(PKC)、[Ca^2 ]i及cAMP／cGMP的变化。结果发现：(1)与空白处理组相比,使用PKC的激动剂PMA处理培养的人垂体腺瘤细胞时可使胞浆、胞膜和细胞总PKC活性浓度均升高,但ACh(10μmol/L)作用15min后,胞浆、胞膜和细胞总PKC活性均下降,且此作用可被阿托品阻断;(2)ACh(10μmol/L)作用于单个人垂体腺瘤细胞后,立即使垂体腺瘤细胞[Ca^2 ]i相对水平降低,但此作用可被阿托品阻断;(3)ACh作用于人垂体腺瘤细胞15min后,胞内cAMP水平均明显升高,而cGMP没有改变。该结果为探讨ACh抑制垂体腺瘤细胞增殖的分子机制提供了重要线索,同时提示,ACh对垂体瘤细胞增殖分化的调控作用是细胞内多信息系统相互整合的结果。     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.