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.     

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 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.     

A role of protein kinase C in signal reactions in outer segments of the bovine retinal rods

The effect of modulators of protein kinase C activity on Ca2+ translocation in dark-adapted and bleached retinal rod outer segments (ROS) was studied. The activators (1,2-diacyl glycerol and phorbol-12-myristate-13-acetate) and the inhibitor (chelerythrine chloride) of protein kinase C were shown to stimulate and inhibit the ATP-dependent Ca(2+)-uptake in dark-adapted retinal ROS, correspondingly. Apparently, this action is due to the influence of protein kinase C on Ca(2+)-ATPase activity in these vesicular structures. No involvement of modulators of protein kinase C activity on ATP-dependent Ca(2+)-uptake in bleached retinal ROS was found. The influence of protein kinase C on Ca(2+)-release from retinal ROS was observed. It was shown that the activators and inhibitors of protein kinase C increased the efficiency of this process both in dark-adapted and bleached retinal ROS. The mechanisms of action of the protein kinase C activity modulators on the Ca(2+)-uptake and Ca(2+)-release in retinal ROS are discussed.     

Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin

Summary Activators of protein kinase C (PKC) stimulate Na^– transport (J _Na) across frog skin. We have examined the effect of Ca²⁺ on PKC stimulation ofJ _Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC₈) were used as PKC activators. Blocking Ca²⁺ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na⁺ channels, since the stimulations produced by blocking Ca²⁺ entry and adding cyclic AMP were simply additive.The Ca²⁺ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca²⁺ on PKC or an indirect action on the final receptor site (the Na⁺ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca²⁺, and in fact was inhibited at millimolar concentrations of Ca²⁺.We conclude that the effects of Ca²⁺ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca²⁺ may have stimulated Na⁺ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na⁺ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca²⁺ independent and seems related to thenPKC or PKC observed in other systems.     

The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K⁺ channel inhibition increases [Ca²⁺]_i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K⁺ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K⁺ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K⁺-channel (Kv1.5 and Kv2.1) and large-conductance Ca²⁺-activated K⁺-channel (BK_Ca) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K⁺-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non-specific attenuation of receptor-dependent cell activation?

Receptor-mediated elevations of intracellular Ca²⁺ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI₂) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol-12-myristate-13-acetate (PMA) (3-300 nM) or 1-oleyl-2-acetyl-glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI₂ from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol-12, 13-didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN-01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin-stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN-01) elevation of intracellular Ca²⁺ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca²⁺ or basal endothelial autacoid release. Several isoforms of PKC (namely PKC_α, PKC_δ, PKC_?, and PKC_ζ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype-specific anti-PKC antibodies, which, except PKC_?, were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane-bound PKC_α immunoreactivity, whereas there was no translocation of PKC_α from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor-dependent agonist. © 1993 Wiley-Liss, Inc.     

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.     

Responsiveness of a human parotid epithelial cell line (HSY) to autonomic stimulation: Muscarinic control of K+ transport

Summary Salivary electrolyte secretion is under the control of the autonomic nervous system. In this paper we report that HSY, an epithelial cell line derived from the acinar-intercalated duct region of the human parotid gland, responds to muscarinic-cholinergic (generation of Ca²⁺ signal) andβ-adrenergic (generation of cAMP signal), but not toα-adrenergic (lack of Ca²⁺ signal), receptor stimulation. The muscarinic response was studied in detail. Carbachol (10⁻⁴ M, muscarinic agonist) or A23187 (5 μM, calcium ionophore) stimulation of HSY cells increases both⁸⁶Rb (K⁺) influx and efflux, resulting in no change in net equilibrium⁸⁶Rb content. Atropine (10⁻⁵ M, muscarinic antagonist) blocks both the carbachol-generated Ca²⁺ signal and carbachol-stimulated⁸⁶Rb fluxes, but has no effect on either the A23187-generated Ca²⁺ signal or A23187-stimulated⁸⁶Rb fluxes. Carbachol- and A23187-stimulated⁸⁶Rb fluxes are substantially inhibited by two K⁺ channel blockers, quinine (0.3 mM) and scorpion venom containing charybdotoxin (33 μg/ml). The inhibition of these stimulated fluxes by another K⁺ channel blocker, tetraethylammonium chloride (5 mM), is less pronounced. Protein kinase C (PKC) seems to be involved in the regulation of the⁸⁶Rb fluxes as 10⁻⁷ M PMA (phorbol ester, phorbol-12-myristate-13-acetate) substantially inhibits the muscarinic-stimulated⁸⁶Rb efflux and influx. Because this concentration of PMA totally inhibits the carbachol-generated Ca²⁺ signal and only 80% of the muscarinic-stimulated⁸⁶Rb influx, it seems that a portion of the carbachol-stimulated⁸⁶Rb flux (i.e. that portion not inhibited by PMA) might occur independently of the Ca²⁺ signal. PMA fails to inhibit the A23187-stimulated⁸⁶Rb fluxes, however, suggesting that PKC regulates Ca²⁺-sensitive K⁺ channel activity by regulating the Ca²⁺ signal, and not steps distal to this event. 4-α-Phorbol-12,13-didecanoate, a phorbol ester which fails to activate PKC, fails to inhibit either the carbachol-stimulated increase in intracellular free Ca²⁺, or carbachol-stimulated⁸⁶Rb fluxes.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.     

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.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Shigella dysenteriae type 1 toxin induced lipid peroxidation in enterocytes isolated from rabbit ileum

To evaluate the role of reactive oxygen species (ROS) in Shigella dysenteriae 1 toxin (STx) mediated intestinal infection, the ligated rabbit small intestinal loops were injected with STx. The enterocytes isolated from STx treated rabbit ileal loops had a significantly higher level of lipid peroxidation as compared to enterocytes isolated from control rabbit ileum. To study the role of second messengers in STx mediated intestinal damage, the in vivo and in vitro effects of modulators of lipid peroxidation of enterocytes were used. The presence of Ca²⁺-ionophore A23187 enhanced the extent of lipid peroxidation in enterocytes isolated from the control and STx treated rabbit ileum. However, l-verapamil only marginally decreased the lipid peroxidation level of enterocytes isolated from STx treated rabbit ileum. The in vitro effect of modulators was in agreement with in vivo studies. Dantrolene significantly decreased the extent of lipid peroxidation of enterocytes isolated from STx treated rabbit ileum. PMA significantly increased the lipid peroxidation level of enterocytes isolated from control ileum. However, PMA could not further enhance the lipid peroxidation level of enterocytes isolated from STx treated rabbit ileum. The presence of H-7 significantly decreased the extent of lipid peroxidation of enterocytes isolated from STx treated rabbit ileum. In vitro effect of PMA and H-7 was in agreement with that of in vivo findings. The role of arachidonic acid metabolites, prostaglandins (PGs), in mediating STx induced lipid peroxidation was also studied. The presence of indomethacin (a PG synthesis inhibitor) significantly decreased the lipid peroxidation induced by STx. These findings suggest that lipid peroxidation induced by STx is mediated through cytosolic calcium. The increase in (Ca²⁺)_i leads to activation of PKC.A significant decrease in the enterocyte levels of antioxidant enzymes superoxide dismutase, catalase and reduced glutathione in STx treated rabbit ileum as compared to control was seen. A significant decrease in vitamin E levels was also observed. This suggests that there is decreased endogenous intestinal protection against ROS in STx mediated intestinal infection which could contribute to enterocyte membrane damage that ultimately leads to changes in membrane permeability and thus to fluid secretion.     

Protein Kinase C Modulates Calcium Channels in Isolated Presynaptic Nerve Terminals of Rat Hippocampus

Abstract: Nerve terminals (“synaptosomes”) isolated from rat brain hippocampus were loaded with the fluorescent Ca²⁺ indicator fura-2 and were subjected to depolarization with an elevated K⁺ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca²⁺ channels in the presynaptic membrane. Three components of Ca²⁺ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca²⁺ channels with different inactivation kinetics (τ of ～60 ms and 1 s, respectively) and Na⁺/Ca²⁺ exchange working in the “reverse” mode. The activity of both classes of voltage-dependent Ca²⁺ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca²⁺ channel activity, but the effect of DHS could be showed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca²⁺ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

The dynamics of PKC‐induced phosphorylation triggered by Ca2+ oscillations in mouse eggs

Fertilization of mammalian eggs is characterized by a series of Ca²⁺ oscillations triggered by a phospholipase C activity. These Ca²⁺ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC‐induced phosphorylation in mouse eggs, alongside Ca²⁺ oscillations, using fluorescent C‐kinase activity reporter (CKAR) probes. Ca²⁺ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr²⁺ all caused repetitive increases in PKC‐induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca²⁺ transients. High frequency oscillations caused by PLCζ led to an integration of PKC‐induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca²⁺ oscillations using an isoform‐specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ‐specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC‐induced phosphorylation outlasts each Ca²⁺ increase in mouse eggs but that signal integration only occurs at a non‐physiological, high Ca²⁺ oscillation frequency. The results also suggest that Ca²⁺‐induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.     

The exploration of effect of terfenadine on Ca2+ signaling in renal tubular cells

Terfenadine, an antihistamine used for the treatment of allergic conditions, affected Ca²⁺-related physiological responses in various models. However, the effect of terfenadine on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) and its related physiology in renal tubular cells is unknown. This study examined whether terfenadine altered Ca²⁺ signaling and caused cytotoxicity in Madin–Darby canine kidney (MDCK) renal tubular cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Terfenadine at concentrations of 100–1000?μM induced [Ca²⁺]_i rises concentration dependently. The response was reduced by approximately 35% by removing extracellular Ca²⁺. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited terfenadine-evoked [Ca²⁺]_i rises. Conversely, treatment with terfenadine abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 95% of terfenadine-induced Ca²⁺ release. Terfenadine-induced Ca²⁺ entry was supported by Mn²⁺-caused quenching of fura-2 fluorescence. Terfenadine-induced Ca²⁺ entry was partly inhibited by an activator of protein kinase C (PKC), phorbol 12-myristate 13 acetate (PMA) and by three modulators of store-operated Ca²⁺ channels (nifedipine, econazole, and SKF96365). Terfenadine at 200–300?μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in MDCK cells, terfenadine induced [Ca²⁺]_i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Furthermore, terfenadine caused cell death that was not triggered by preceding [Ca²⁺]_i rises.