Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Okadaic acid gives concentration-dependent reciprocal effects on the fluid phase endocytosis activated by Ca2+ and phorbol 12-myristate 13-acetate

Incubation of a human fibrosarcoma cell line HT-1080 in increasing concentration of Ca²⁺ was found to enhance endocytic internalization of a fluid phase marker, horseradish peroxidase. At 16.8 mM Ca²⁺, generation of the effect required incubation for more than 45 min. The effect was reversed by removal of the excess ion for 30 min. Monitoring the intracellular concentration showed that the incubation induced a transient large Ca²⁺ influx followed by a recovery to 230 ± 50 nM instead of the normal level of 83 ± 5 nM. The activation was not inhibited by inhibitors of protein kinases nor a cAMP antagonist. In contrast, the effect was prevented by okadaic acid (OKA) at 100 nM without detectable effect on the basal activity. Fluid phase uptake by HT-1080 cells was also enhanced by phorbol 12-myristate 13-acetate (PMA). In contrast to the case with Ca²⁺, OKA at 100 nM did not prevent the PMA effect but further enhanced the endocytosis. The effect of OKA was concentration-dependent, as the reagent at 1 μM inhibited not only both the activation but also the basal activity. In Ca²⁺-or PMA-stimulated cells, FITC-dextran was delivered to endosomes that had been labeled with TRITC-transferrin. In contrast, following treatment with a combination of PMA and 100 nM OKA, fluid phase was internalized in vesicular compartments devoid of transferrin labeling. These results suggest that, through differential modifications of protein phosphorylation, endocytosis can be enhanced distinctively either by employing conventional receptor-bearing compartments or generating a new endosomal population. © 1996 Wiley-Liss, Inc.     

Effect of Modulators of Protein Kinase C Activity on Ca2+ Transport in Retinal Rod Microsomes

The effect of modulators of protein kinase C (PKC) activity on Ca²⁺ translocation in retinal rod microsomes was studied. It is shown that PKC activators (phorbol 12-myristate-13-acetate (PMA) and diacylglycerol (DAG)) and inhibitors (chelerythrine chloride, polymyxin B, and phloretin) stimulate and inhibit ATP-dependent Ca²⁺ uptake in retinal rod microsomes, respectively. This effect is apparently due to an influence of PKC on Ca-ATPase contained in these vesicular structures. It was found that PKC inhibitors (chelerythrine chloride, polymyxin B, and phloretin) and activators (PMA and DAG) potentiate Ca²⁺ release from Ca²⁺ -loaded retinal rod microsomes. Specific and nonspecific mechanisms of Ca-release stimulation by the modulators of PKC activity are discussed.     

Mechanism of Catecholamine Synthesis Inhibition by Neuropeptide Y: Role of Ca2+ Channels and Protein Kinases

Abstract: We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca²⁺ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca²⁺ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by ～90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca²⁺/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.     

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²⁺ signalling. In HEK293 and Jurkat cells, the Ca²⁺ release and Ca²⁺ uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²⁺ responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²⁺ concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²⁺ flux.     

Modulation of Ca-activated K channels of human erythrocytes by endogenous protein kinase C

Single IK_Ca channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC_19-31, a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC_19-31 or chelerythrine. Channel activity was not inhibited by the PMA analog 4α-phorbol 12,13-didecanoate (4αPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK_Ca channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK_Ca channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK_Ca channels.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

A calcium-dependent protein kinase is present in tetrahymena

A Ca²⁺-dependent protein kinase of Tetrahymena thermophila has been partially purified and characterized. The molecular mass of the enzyme is less than that of similar enzymes (for example protein kinase C), being about 55 kDa. After purification and in the presence of Ca²⁺ the enzyme activity increased. The promoter of protein kinase C (PKC) activity, phorbol myristate acetate (PMA), increased the activity while the protein kinase inhibitor H-7 decreased the activity of the enzyme. The experiments demonstrate the presence, activity and similarity to vertebrate enzymes of a protein kinase at a low level of phylogeny.     

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253

The expression of protein kinase C (PKC) isoforms and the modulation of Ca²⁺ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP₃ formation, Ca²⁺ release and Ca²⁺ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP₃ formation but inhibited the Ca²⁺ release and the Ca²⁺ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca²⁺ release induced by TG, but reduced the influx of Ca²⁺ seen in the presence of this Ca²⁺-ATPase inhibitor. These results suggest that PKC modulates elements of the IP₃/Ca²⁺ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca²⁺ channels to IP₃, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca²⁺ ATPase, and (3) modulating Ca²⁺ entry pathways.     

ATP7B activity is stimulated by PKCɛ in porcine liver

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)–ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10⁻⁸ M), a PKC activator, increased Cu(I)–ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca²⁺, whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced V_max without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.     

Calcium Channels: Unanswered Questions

Despite decades of intensive research, many questions remain unresolved regarding the structure and function of voltage-dependent calcium channels. This review considers some of those questions: Where is the activation gate? Where are the inactivation gates? How are the voltage sensors coupled to the gates? Are bacterial K⁺ channels good models for the Ca²⁺ channel pore? Are protein–protein interactions fundamental to Ca²⁺ channel function? Do voltage-dependent Ca²⁺ channels regulate basal intracellular Ca²⁺? Are N and P/Q channels specialized for fast neurotransmitter release?     

Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries

LocalCa²⁺ transients("Ca²⁺ sparks") caused bythe opening of one or the coordinated opening of a number of tightlyclustered ryanodine-sensitiveCa²⁺-release (RyR) channels in thesarcoplasmic reticulum (SR) activate nearbyCa²⁺-dependentK⁺(K_Ca) channels to cause anoutward current [referred to as a "spontaneous transientoutward current" (STOC)]. TheseK_Ca currents cause membranepotential hyperpolarization of arterial myocytes, which would lead tovasodilation through decreasingCa²⁺ entry throughvoltage-dependent Ca²⁺ channels.Therefore, modulation of Ca²⁺spark frequency should be a means to regulation ofK_Ca channel currents and hencemembrane potential. We examined the frequency modulation ofCa²⁺ sparks and STOCs byactivation of protein kinase C (PKC). The PKC activators, phorbol12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),decreased Ca²⁺ spark frequency by72% and 60%, respectively, and PMA reduced STOC frequency by 83%.PMA also decreased STOC amplitude by 22%, which could be explained byan observed reduction (29%) inK_Ca channel open probability inthe absence of Ca²⁺ sparks. Thereduction in STOC frequency occurred in the presence of an inorganicblocker (Cd²⁺) ofvoltage-dependent Ca²⁺ channels.The reduction in Ca²⁺ sparkfrequency did not result from SRCa²⁺ depletion, sincecaffeine-induced Ca²⁺ transientsdid not decrease in the presence of PMA. These results suggest thatactivators of PKC can modulate the frequency ofCa²⁺ sparks, through an effect onthe RyR channel, which would decrease STOC frequency (i.e.,K_Ca channel activity).

     

Cysteine strings,calcium channels and synaptic transmission

Multidisciplinary studies have led to the discovery and characterization of cysteine string proteins (csps) in both Drosophila and Torpedo. Phenotypic analysis of csp mutants in Drosophila demonstrates a crucial role for csp in synaptic transmission. Expression studies of Torpedo csp (Tcsp) in Xenopus oocytes suggests that the protein has some role in the function of presynaptic Ca²⁺ channels. However, biochemical purification of Tcsp indicates that is associated with synaptic vesicles rather than with the plasma membrane of presynaptic terminals where Ca²⁺ channels reside. These results suggest a model in which csps serve as a link by which docked synaptic vesicles could modulate the activity of presynaptic Ca²⁺ channels.     

Inhibitory effect of glutamate release from rat cerebrocortical synaptosomes by dextromethorphan and its metabolite 3-hydroxymorphinan

Dextromethorphan (DM), a widely used antitussive, has demonstrated an effective neuroprotective effect. Excessive release of glutamate is considered to be an underlying cause of neuronal damage in several neurological diseases. In the present study, we investigated whether DM or its metabolite 3-hydroxymorphinan (3-HM) could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). DM or 3-HM inhibited the Ca²⁺-dependent release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this presynaptic inhibition was concentration-dependent. Inhibition of glutamate release by DM or 3-HM was resulted from a reduction of vesicular exocytosis, because the vesicular transporter inhibitor bafilomycin A1 completely blocked DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release. DM or 3-HM did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release was blocked by ω-conotoxin MVIIC, an antagonist of N- and P/Q-type Ca²⁺ channel, not by dantrolene, an intracellular Ca²⁺ release inhibitor. DM or 3-HM modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitors GF109203X or Ro318220 all effectively occluded the inhibitory effect of DM or 3-HM. Furthermore, 4-AP-induced phosphorylation of PKC was reduced by DM or 3-HM. These results suggest that DM or 3-HM inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca²⁺ entry and PKC activity. This may explain the neuroprotective effects of DM against neurotoxicity.     

An Early Loss in Membrane Protein Kinase C Activity Precedes the Excitatory Amino Acid-Induced Death of Primary Cortical Neurons

Abstract: Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14–18 cortical neurons to 100 µM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30–50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca²⁺, but less so on Na⁺, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca²⁺ entry through specific routes because the bulk increase in intracellular free [Ca²⁺] effected by the Ca²⁺ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral ischemia in vivo.     

Ca<Superscript>2+</Superscript>-mediated Potentiation of the Swelling-induced Taurine Efflux from HeLa Cells: On the Role of Calmodulin and Novel Protein Kinase C Isoforms

The present work sets out to investigate how Ca²⁺ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca²⁺-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca²⁺-free or high-K⁺ media. Addition of Ca²⁺-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca²⁺-activated K⁺ channels. The taurine release from control cells and cells exposed to Ca²⁺ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca²⁺-augmented taurine release. Exposure to Ca²⁺-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca²⁺-concentration has returned to the unstimulated level. The Ca²⁺-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca²⁺-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca²⁺ regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).This revised version was published online in June 2005 with a corrected cover date.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.