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

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

Activation of protein kinase C is not required for glyceraldehyde-stimulated insulin secretion from rat islets

Glyceraldehyde-induced insulin release from rat islets of Langerhans was not affected following down-regulation of protein kinase C (PKC) by prolonged exposure to the tumour-promoting phorbol ester, 4 beta-phorbol myristate acetate (PMA). Glyceraldehyde did not cause translocation of islet PKC under conditions in which PMA stimulated redistribution of enzyme activity. These results indicate that activation of PKC is not required for glyceraldehyde stimulation of insulin secretion from normal rat islets.     

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.     

Protein kinase CbetaI interacts with the beta1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates beta1-adrenergic receptor (beta1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms alpha and betaI, but not betaII, delta, epsilon, or zeta, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform alpha increased, while expression of PKCdelta decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCbetaI/betaII inhibitor LY 333531, but not by the PKCbetaII inhibitor CGP 53353 or the PKCdelta inhibitor rottlerin. These data suggest that PKCbetaI interacts with beta1-AR signaling and attenuates lipolysis in rat adipocytes.     

Translocation of protein kinase C in rat islets of Langerhans. Effects of a phorbol ester, carbachol and glucose

In unstimulated rat islets (2 mM glucose), most of the ion-exchange purified protein kinase C (PKC) activity was associated with the cytosolic fraction. Both carbachol and phorbol myristate acetate caused a significant translocation of PKC activity from cytosolic to membrane fractions, but under the same conditions, glucose (20 mM) did not cause such a redistribution of PKC activity. PMA-induced translocation of PKC to the membrane fraction was also observed in electrically permeabilised islets, in which recovery of the enzyme activity was enhanced by buffering the intracellular Ca2+ concentration to 50 nM and supplying the permeabilised islets with protease inhibitors.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca²⁺ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca²⁺ were monitored by using single channel recordings of the high conductance Ca²⁺-activated K⁺ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na⁺-saline, UTP (>30 nm) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 m. Cells bathed in K⁺ saline (150 m) were less sensitive to UTP (5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K⁺ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca²⁺ entry blockers or by removal of extracellular Ca²⁺. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca²⁺ ionophores (<5 m) to that of UTP indicate that both agents elevate cytosolic Ca²⁺ by mobilization of this ion from intracellular pools. However, the Ca²⁺ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca²⁺ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues. PKC does not appear to be directly involved, even though the UTP response is blocked by low nm levels of PMA. While the latter data implicate PKC in diminishing the UTP response, agents that inhibit either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.     

The mTORC2/PKC pathway sustains compensatory insulin secretion of pancreatic β cells in response to metabolic stress

BackgroundCompensation of the pancreatic β cell functional mass in response to metabolic stress is key to the pathogenesis of Type 2 Diabetes. The mTORC2 pathway governs fuel metabolism and β cell functional mass. It is unknown whether mTORC2 is required for regulating metabolic stress-induced β cell compensation.MethodsWe challenged four-week-old β-cell-specific Rictor (a key component of mTORC2)-knockout mice with a high fat diet (HFD) for 4 weeks and measured metabolic and pancreatic morphological parameters. We performed ex vivo experiments to analyse β cell insulin secretion and electrophysiology characteristics. Adenoviral-mediated overexpression and lentiviral-ShRNA-mediated knocking down proteins were applied in Min6 cells and cultured primary mouse islets.ResultsβRicKO mice showed a significant glucose intolerance and a reduced plasma insulin level and an unchanged level β cell mass versus the control mice under HFD. A HFD or palmitate treatment enhanced both glucose-induced insulin secretion (GIIS) and the PMA (phorbol 12-myristate 13-acetate)-induced insulin secretion in the control islets but not in the βRicKO islets. The KO β cells showed similar glucose-induced Ca^2 + influx but lower membrane capacitance increments versus the control cells. The enhanced mTORC2/PKC proteins levels in the control HFD group were ablated by Rictor deletion. Replenishing PKCα by overexpression of PKCα-T638D restored the defective GIIS in βRicKO islets.ConclusionsThe mTORC2/Rictor pathway modulates β cell compensatory GIIS under nutrient overload mediated by its phosphorylation of PKCα.General significanceThis study suggests that the mTORC2/PKC pathway in β cells is involved in the pathogenesis of T2D.     

Characterization of protein kinase C in rat and human prostates

The properties of protein kinase C (PKC) activity have been studied in cytosolic and membrane fractions from rat and human prostate. Ion exchange chromatography indicated the existence of different PKC isoforms, PKC from rat ventral prostate behaved as a classical Ca²⁺- and phospholipid-dependent enzyme and was activated by 1,2-diacylglycerol as well as by high concentrations of arachidonic acid. PKC activity in the cytosolic fraction was higher and presented different cofactor requirements than that in the membrane fraction. PKC from human benign hyperplastic prostate was also phospholipid dependent, activated by tumor-promotong phorbol esters, and appeared to belong to the group of PKC isozymes which lack Ca²⁺ sensitivity. Human prostatic PKC activity appeared to be of similar nature in both membrane and cytosolic fractions but the specific activity was higher in the particulate preparation which could be related to the stage of endogenous activation of the enzyme. These results extend previous observations in rat ventral prostate and present evidences on the human counterpart. Forthcoming experiments are needed to establish the exact nature of PKC isozymes and their physiological and pathophysiological role in this gland.     

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.     

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca(2+)-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in l-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 micro M). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 micro M), which activates novel PKC isoforms, did not affect l-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 micro M; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.     

Changes in islet plasma membrane calcium-ATPase activity and isoform expression induced by insulin resistance

We studied the effect of insulin resistance (IR) induced by administration of a fructose-rich diet (FRD) to normal Wistar rats for 21 days, upon islet plasma membrane calcium ATPases (PMCAs) and insulin secretion. FRD rats showed significantly higher triglyceride and insulin levels, insulin:glucose ratio and HOMA-IR index than controls. FRD islets released significantly more insulin in response to glucose and showed (a) marked changes in PMCA isoform protein content (decreased PMCA 2 and increased PMCA 3), (b) a decrease in total PMCAs activity, and (c) higher levels of cytosolic calcium [Ca²⁺]_i. The lower PMCAs activity with the resultant increase in [Ca²⁺]_i would favor the compensatory greater release of insulin necessary to cope with the IR state present in FRD rats and to maintain normal glucose homeostasis. Thus, changes in PMCAs activity and isoform expression play a modulatory role upon insulin secretion during long-term adaptation to an increased hormone demand.     

Differential Regulation of Histamine- and Bradykinin-Stimulated Phospholipase C in Adrenal Chromaffin Cells: Evidence for Involvement of Different Protein Kinase C Isoforms

Abstract: In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-α, ε, and ζ were identified. To characterize down-regulation of these enzymes, cells were incubated for 6–48 h with 1 µM phorbol myristate acetate (PMA). PKC-ε down-regulated more rapidly than PKC-α. At 12 h, PMA pretreatment, for example, PKC-ε was maximally down-regulated (23 ± 4% of controls), whereas PKC-α was unchanged. PKC-α showed partial down-regulation by 24 h of PMA pretreatment. PKC-ζ did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-ε than for PKC-α. The accumulation of total ³H-inositol (poly)phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 µM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-ε, whereas the restoration of the histamine response corresponds to the loss of PKC-α. This picture was confirmed with further studies on cytosolic Ca²⁺. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.     

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.     

PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

Protein kinase C (PKC) and the actin cytoskeleton are criticaleffectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either theapical or basolateral membrane is poorly defined. In the present study,phorbol 12-myristate 13-acetate (PMA) and other activators of PKCselectively enhanced basolateral but not apical fluid-phase endocytosisin human T84 intestinal epithelia. Stimulation of basolateralendocytosis was blocked by the conventional and novel PKC inhibitorGö-6850, but not the conventional PKC inhibitor Gö-6976,and correlated with translocation of the novel PKC isoform PKC-. PMAtreatment induced remodeling of basolateral F-actin. The actindisassembler cytochalasin D stimulated basolateral endocytosis andenhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin andjasplakinolide. PMA induced membrane-to-cytosol redistribution of theF-actin cross-linking protein myristoylated alanine-rich C kinasesubstrate (MARCKS). Cytochalasin D also induced MARCKS translocationand enhanced PMA-stimulated translocation of MARCKS. A myristoylatedpeptide corresponding to the phosphorylation site domain of MARCKSinhibited both MARCKS translocation and PMA stimulation of endocytosis.MARCKS translocation was inhibited by Gö-6850 but notGö-6976. The results suggest that a novel PKC isoform, likelyPKC-, stimulates basolateral endocytosis in model epithelia by amechanism that involves F-actin and MARCKS.

     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Stimulus-induced selective association of actin-associated proteins (alpha-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils.

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein alpha-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and actin. Increased association of PKCbetaI and betaII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, alpha-actinin, and PKCbetaII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal alpha-actinin and PKCbetaII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 microM) completely blocked PMA-induced increases in cytoskeletal alpha-actinin but reduced cytoskeletal recruitment of PKCbetaII only by 16%. Higher concentrations of latrunculin A (4 microM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCbetaII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

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.