Protein kinase C activates store-operated Ca(2+) channels in human glomerular mesangial cells

Store-operated Ca(2+) channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin. The measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](i). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca(2+)](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N',N'tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP(O) of SOC, whereas calphostin C did not affect base-line channel activity. In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 microm ATP. Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC. Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.     

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Uncoupling of ATP-induced inositol phosphate formation and Ca(2+) mobilization by phorbol ester in canine cultured tracheal epithelial cells

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca(2+) mobilization. The concentrations of PMA that gave half-maximal (EC(50)) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca(2+)](i) were 5-10 and 4-12 nM, respectively. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -theta, and -zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, and -theta from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-alpha, -betaI, -betaII, -delta, -epsilon, -gamma, and -theta induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca(2+) mobilization in TECs.     

Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca2+ signaling in single resistance arterial smooth muscle cells

Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.     

Vascular regulation of type II cell exocytosis

To determine whether lung capillary pressure regulates surfactant secretion, we viewed alveoli of the constantly inflated, isolated blood-perfused rat lung by fluorescence microscopy. By alveolar micropuncture we infused fura 2 and lamellar body (LB)-localizing dyes for fluorescence detection of, respectively, the alveolar cytosolic Ca(2+) concentration ([Ca(2+)](i)) and type II cell exocytosis. Increasing left atrial pressure (Pla) from 5 to 10 cmH(2)O increased septal capillary diameter by 26% and induced marked alveolar [Ca(2+)](i) oscillations that abated on relief of pressure elevation. The rate of loss of LB fluorescence that reflects the LB exocytosis rate increased fourfold after the pressure elevation and continued at the same rate even after pressure and [Ca(2+)](i) oscillations had returned to baseline. In alveoli pretreated with either 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, the intracellular Ca(2+) chelator, or heptanol, the gap junctional blocker, the pressure-induced exocytosis was completely inhibited. We conclude that capillary pressure and surfactant secretion are mechanically coupled. The secretion initiates in a Ca(2+)-dependent manner but is sustained by Ca(2+)-independent mechanisms.     

Stimulation of fibroblast proliferation by neokyotorphin requires Ca influx and activation of PKA, CaMK II and MAPK/ERK

Neokyotorphin [TSKYR, hemoglobin alpha-chain fragment (137-141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4beta-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+ L-type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin.     

Protein kinase C-dependent and -independent inhibition of Ca(2+) influx by phorbol ester in rat pancreatic beta-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic beta-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca(2+) concentration ([Ca(2+)](i)) elevated by the glucose stimulation when tested in isolated rat beta-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized beta-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca(2+) currents through L-type Ca(2+) channels. BIM prevented the PMA inhibition of the Ca(2+) currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic beta-cells, defeating its own inhibition of the Ca(2+) influx through L-type Ca(2+) channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.     

Protein kinase activation increases insulin secretion by sensitizing the secretory machinery to Ca2+

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).     

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used G?6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

Differential regulation of the apical plasma membrane Ca(2+) -ATPase by protein kinase A in parotid acinar cells

Cross-talk between intracellular calcium ([Ca(2+)](i)) signaling and cAMP defines the specificity of stimulus-response coupling in a variety of cells. Previous studies showed that protein kinase A (PKA) potentiates and phosphorylates the plasma membrane Ca(2+)-ATPase (PMCA) in a Ca(2+)-dependent manner in parotid acinar cells (Bruce, J. I. E., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172-48181). The aim of this study was to further investigate the spatial regulation of [Ca(2+)](i) clearance in parotid acinar cells. Par-C10 cells were used to functionally isolate the apical and basolateral PMCA activity by applying La(3+) to the opposite side to inhibit the PMCA. Activation of PKA (using forskolin) differentially potentiated apical [Ca(2+)](i) clearance in mouse parotid acinar cells and apical PMCA activity in Par-C10 cells. Immunofluorescence of parotid tissue slices revealed that PMCA1 was distributed throughout the plasma membrane, PMCA2 was localized to the basolateral membrane, and PMCA4 was localized to the apical membrane of parotid acinar cells. However, in situ phosphorylation assays demonstrated that PMCA1 was the only isoform phosphorylated by PKA following stimulation. Similarly, immunofluorescence of acutely isolated parotid acinar cells showed that the regulatory subunit of PKA (RIIbeta) translocated to the apical region following stimulation. These data suggest that PKA-mediated phosphorylation of PMCA1 differentially regulates [Ca(2+)](i) clearance in the apical region of parotid acinar cells because of a dynamic translocation of PKA. Such tight spatial regulation of Ca(2+) efflux is likely important for the fine-tuning of Ca(2+)-dependent effectors close to the apical membrane important for the regulation of fluid secretion and exocytosis.     

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 μM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 μM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 μM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Novel action of pituitary adenylate cyclase-activating polypeptide. Stimulation of extracellular acidification in rat pituitary GH4C1 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide/secretin family. Using microphysiometry, we have found that PACAP acutely (1 min) increased the extracellular acidification rate (ECAR) in GH4C1 cells approximately 40% above basal in a concentration-dependent manner. ECAR, maximally induced by PACAP, can be increased further by thyrotropin-releasing hormone (TRH), indicating that the signalling pathways for these two neuropeptides are not identical. In studies on the mechanism of PACAP-enhanced ECAR, we found that maximum stimulation of the cAMP/PKA pathway by treatment with FSK, or the PKC pathway with PMA, did not inhibit the ECAR response to PACAP. The PKC inhibitor calphostin C and the MAP kinase inhibitor PD98059 had no effect on the ECAR response to PACAP. Furthermore, PACAP induced little or no change in cytosolic Ca(2+) ([Ca(2+)](i)), while TRH induced a large increase in [Ca(2+)](i). However, the tyrosine kinase inhibitor genistein completely blocked PACAP-induced ECAR, suggesting involvement of tyrosine kinase(s). We conclude that PACAP causes an increase in ECAR in GH4C1 rat pituitary cells, which is not dependent on the PKA, PKC, MAP kinase or Ca(2+) signalling pathways, but does require tyrosine kinase activity.     

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular Ca(2+) regulates the cellular iron uptake in K562 cells

Fluorescence quenching was used to study the kinetics of the transferrin receptor (TfR)-mediated iron uptake in the calcein-loaded K562 cells. It was found that elevation of intracellular free Ca(2+) ([Ca(2+)](i)) by thapsigargin (TG) speeds up the initial rate of iron uptake and increases the overall capacity of the cells in taking up iron. Depletion of intracellular Ca(2+) or complete chelation of extracellular Ca(2+) results in complete inhibition of the iron uptake in cells. To gain insight into molecular mechanism, IANBD-labeled transferrin (Tf) and microscopic fluorescence imaging were used to observe the endocytosis and recycling of the Tf-TfR complex in single live cells. The study showed that the preincubation of cells with TG or phorbol myristate acetate (PMA), the direct activator of protein kinase C (PKC), accelerated the endocytosis and recycling of the complex in a dose-dependent manner. W-7, the calmodulin antagonist, and GF109203X, a selected cell-permeant inhibitor of PKC, can reverse the acceleration. Analysis of actin polymerization in controlled, [Ca(2+)](i)-elevated and W-7-treated cells revealed that the actin polymerization is enhanced as [Ca(2+)](i) is raised, but reduced by W-7. The results suggest that the regulation of actin polymerization by intracellular Ca(2+) may play a central role in Ca(2+)-dependent iron uptake.