Translocation of protein kinase C isoenzymes by elevated extracellular Ca2+ concentration in cells from a human giant cell tumor of bone

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -alpha, -delta, and -epsilon, PKC isoforms, but not the -beta isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-alpha, -delta, and -epsilon are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca2+ concentration ([Ca2+]o) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). An increase of [Ca2+]o stimulated the translocation of PKC-alpha from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+]o sensing. By contrast, PKC-delta was not altered by exposure to elevated [Ca2+]o, whereas PKC-epsilon underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca2+]o-induced [Ca2+]i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.     

The role of immune complex in otitis media with effusion

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.     

Applying task analysis in design specification: current problems and a solution

The immune and endocrine mediators that are released during sepsis (e.g., tumor necrosis factor [TNF] alpha, interleukin [IL]-1, IL-6, transforming growth factor [TGF] beta, prostaglandin [PG] E2, catecholamines, vasopressin, glucagon, insulin, and glucocorticoids) can produce inappropriate detrimental cellular responses contributing to exacerbation of septic injury. Examples of such sepsis-related inappropriate responses are: exaggerated hepatic acute-phase protein (APP) expression and release skeletal muscle insulin resistance, and suppressed T-lymphocyte proliferation. The studies discussed in this article present evidence that the generation of the sepsis-related hepatic, skeletal muscle, and T-lymphocyte responses emanate from alterations in intracellular Ca2+ (Ca2+i) homeostasis. In hepatocytes, there is indication of a sepsis-mediated increase in Ca2+ influx from the extracellular milieu leading to a sustained increase in the apparent resting cell Ca2+i concentration ([Ca2+]i) and its depressed elevation on stimulation with Ca2+-mobilizing hormones such as catecholamines and vasopressin. These Ca(2+)- related changes can affect not only the signaling pathways in which Ca2+i itself serves as a signaling component, but also the signaling systems turned on by other sepsis-induced agonists which may not be dependent on Ca2+ signaling. TGF-beta, IL-1, TNF alpha, and IL-6 activate a primarily protein kinase C (PKC)-dependent intracellular signal system for the elicitation of a normal hepatic APP response (APPR). The increased apparent basal [Ca2+]i in sepsis can hypersensitize PKC activation and thus lead to an exaggerated APPR. In the skeletal muscle, an evident increase in Ca2+ membrane flux during sepsis pointed to an increase in the basal [Ca2+]i resulting from a plausible cytokine-mediated overactivation of the voltage-sensitive Ca2+ channels. The increased basal [Ca2+]i can negatively modulate the insulin-mediated stimulation of GLUT4-dependent glucose transport despite the possibility that Ca2+i might not participate as a component in the insulin-receptor-regulated signaling pathway. Increased [Ca2+]i in skeletal myocytes can either directly promote the phosphorylation of GLUT4 or prevent its dephosphorylation, both of which effectively block insulin stimulation of glucose uptake, thereby contributing to insulin resistance. In T lymphocytes, septic injury appears to induce an attenuation in the mitogen and, thus, presumably a T-cell antigen receptor (TCR)-mediated elevation in [Ca2+]i without affecting the basal [Ca2+]i. This decrease in TCR-related Ca2+i mobilization evidently contributes to the suppression of T lymphocyte proliferation during sepsis, probably via an in vivo action of prostaglandin (PG) E2 on the T cells during sepsis. The blockade of PGE2 production after indomethacin administration to septic animals prevents alterations in both T-cell Ca2+i mobilization and proliferation. PGE2 probably acts through its second messenger, cyclic adenosine 3'5'-monophosphate, which can antagonize Ca2+i signaling in T cells.     

Modulation of calcium responses by altered peptide ligands in a human T cell clone

To determine whether altered peptide ligands (APL) affect calcium signaling events, we investigated changes in intracellular calcium concentration ([Ca2+]i) in human T cell clone stimulated with either the fully agonistic peptide M12p54-68, the partially agonistic analogue E63V or the simple antagonistic analogue E58M. Both E63V and E58M stimulated a Ca2+ response in approximately 40% of T cells, whereas M12p54-68 did so in approximately 70% of T cells. The most predominant pattern of a Ca2+ increase induced by M12p54-68 was a small sinusoidal peak followed by a sustained high response. The most frequent pattern of calcium response induced by E63V was a continuous high response without a preceding sinusoidal peak, whereas that induced by E58M was large with frequent oscillations. Genistein, an inhibitor of the protein tyrosine kinases (PTK), markedly inhibited the wild-type peptide-induced increase in [Ca2+]i, whereas it marginally inhibited the response induced by E63V or E58M. In contrast, GF109203X, a protein kinase C (PKC)-specific inhibitor, markedly inhibited the E63V- or E58M-induced Ca2+ response, whereas it marginally affected the wild peptide-induced Ca2+ response. Furthermore, in nominal Ca2+-free medium, the E58M-induced Ca2+ response was almost completely blocked, while the M12p54-68- or E63V-induced responses were only partially inhibited. Our results suggest that the Ca2+ response induced by the fully agonistic peptide depends on activation of the genistein-sensitive signaling pathway, including PTK, whereas the Ca2+ response to a simple antagonistic APL completely depends on extracellular Ca2+ and activation of the GF109203X-sensitive signaling pathway, including PKC. These differences in the CA2+i response in recognition of different APL may parallel the unique T cell activation patterns induced by APL in human T cells.     

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.     

Adenosine A1 receptor stimulation antagonizes the negative inotropic effects of the PKC activator dioctanoylglycerol

It has been suggested that adenosine cardioprotection occurs via adenosine A1 receptor-mediated activation of protein kinase C (PKC). However, adenosine has well-known vasodilatory effects in the myocardium, whereas PKC is a vasoconstrictor. This study examined whether adenosine A1 receptor activation alters the effects of the PKC activator. 1,2-dioctanoyl-s,n-glycerol (DOG) in isolated perfused rat hearts (left-ventricular developed pressure) and rat ventricular myocytes ([Ca2+]i and cell shortening). Exposure to DOG decreased left-ventricular developed pressure by 30%, an effect that was completely reversible. Pretreatment of isolated hearts with either the PKC inhibitor chelerythrine or the adenosine A1 agonist 2-chloro-N6-cyclo-cyclo-isolated pentlyadenosine (CCPA) attenuated the negative inotropic effects of DOG. In the isolated myocytes, DOG decreased [Ca2+]i and cell shortening by 25 and 28%, respectively, effects that were attenuated by both chelerythrine and CCPA. The CCPA attenuation of the DOG-induced decrease in [Ca2+]i and cell shortening was blocked by pretreating the myocytes with the adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results indicate that in rat ventricular myocardium, adenosine A1 receptor activation attenuates the apparent PKC-dependent negative inotropic effects of DOG via preservation of [Ca2+]i levels.     

Effects of a protein kinase C inhibitor, Ro 31-7549, on the activation of human leukocytes by particulate stimuli

1. A new specific protein kinase C (PKC) inhibitor, Ro 31-7549, was used to explore the mechanisms by which particulate stimuli, quartz and chrysotile, stimulate human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM). Also soluble stimuli, formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and phorbol myristate acetate (PMA) were used. 2. Ro 31-7549 inhibited chrysotile-induced free intracellular calcium ([Ca2+]i) elevations but did not have an effect on quartz-induced elevations of [Ca2+]i. Both quartz and chrysotile induced production of ROM were partially inhibited by Ro 31-7549. fMLP-induced elevation of [Ca2+]i was inhibited by Ro 31-7549 whereas PMA did not affect [Ca2+]i. Ro 31-7549 strongly inhibited fMLP-induced ROM production, and completely abolished that induced by PMA. 3. These result suggest that PKC may have an important role in the activation of PMNL to produce ROM by particulate and soluble stimuli. However, the inhibition of chrysotile-, but not of quartz-induced [Ca2+]i elevations by Ro 31-7549 provides evidence that both PKC-dependent and -independent mechanisms may play a role in the activation of human leukocytes to produce ROM.     

Genetic relationship between measures of HDL phenotypes and insulin concentrations

Stimulation of T cells via the T cell receptor (TCR) leads to an increase intracellular in free Ca2+ levels ([Ca2+]i) and the activation of the MAP kinase signaling pathway. This study analyzes for the first time Ca2+ fluxes in naive cytotoxic T cells stimulated with full agonists, partial agonists, or antagonists. Four different types of Ca2+ responses could be observed. Full agonists triggered a strong and sustained increase in [Ca2+]i. In contrast, partial T cell agonists induced either a strong but transient Ca2+ flux or very low to no increases in [Ca2+]i, while T cell antagonists failed to induce any measurable Ca2+ flux. The ability of peptides to induce elevated [Ca2+]i perfectly paralleled their ability to trigger TCR internalization and T cell activation. Thus, stimulation of naive cytotoxic T cells with a panel of defined altered peptide ligands reveals a consistent picture, where Ca2+ fluxes predict agonist, partial agonist and antagonist properties of peptides.     

Evidence for the involvement of protein kinase C isoforms in alpha-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells

BACKGROUND: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of alpha-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2 (PLA2). METHODS: Norepinephrine (NE, 10 mumol/L) was used as an alpha-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo-1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. RESULTS: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-alpha, -beta I, -beta II, -gamma, delta, -epsilon, -zeta, and -eta isoforms were identified. Norepinephrine induced translocation of PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon isoforms but not -zeta and -eta from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-alpha. CONCLUSIONS: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in alpha-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-alpha translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-alpha, to regulate AA generation is an intriguing question that remains to be clarified.     

The rapid inhibitory effect of glucocorticoid on cytosolic free Ca2+ increment induced by high extracellular K+ and its underlying mechanism in PC12 cells

The effect of glucocorticoid(GC) on peak cytosolic free calcium net increment (delta[Ca2+]i) induced by high-K+ was detected with MiraCal Image System. The main results were as follows: (1) Corticosterone(B) could inhibit delta[Ca2+]i in a time-dependent and concentration-dependent manner. (2) The inhibitory effect of B could be mimicked by bovine-serum albumin conjugated corticosterone (B-BSA) also in a dose-dependent manner. (3) G-protein inhibitor, either PTX or GDP beta S significantly reduced the inhibitory effect of B and B-BSA on delta[Ca2+]i (4) PMA, a stimulator for protein kinase C(PKC), could inhibit delta[Ca2+]i. (5) Although the inhibitors of PKC, chelerythrine chloride and bisindolylamide I per se had no influence on delta[Ca2+]i, but they significantly antagonized the inhibitory effect of B and B-BSA on delta[Ca2+]i. It is postulated that GC inhibit delta[Ca2+]i induced by high-K+ through a membrane mechanism and by a pathway involving G-protein and PKC.     

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.     

Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genus Helenium, caused a marked potentiation of the increases in intracellular free Ca2+ concentration ([Ca2+]i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca2+ partly attenuated the increased [Ca2+]i responses caused by helenalin. The increased [Ca2+]i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca2+]i responses. No change in resting [Ca2+]i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca2+]i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca2+]i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca2+ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca2+]i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca2+ from these stores.     

Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.     

The effect of brief illumination on intracellular free calcium concentration in cells with 5-aminolevulinic acid-induced protoporphyrin IX synthesis

The effect of illumination on intracellular free calcium concentration, [Ca2+]i, was studied in a cell line (WiDr cells) derived from a primary adenocarcinoma of the rectosigmoid colon. In these cells the biosynthesis of protoporphyrin IX was stimulated by 5-aminolevulinic acid to reach levels of 600-700 pmol of protoporphyrin IX per mg cell protein. A brief (1-min) exposure of the cells to light (70% of light energy at 340-380 nm) resulted in an increase in [Ca2+]i. This increase was not reversible over a period of at least 20 min following illumination. Elevation of [Ca2+]i most probably represented an influx of calcium ions from the medium to the cell, since it was completely abolished in the presence of extracellular EGTA. The increased [Ca2+]i did not reflect general membrane damage, as determined by trypan blue staining as well as measurement of the intercalation of ethidium bromide into cellular DNA, and neither did the sustained elevation of [Ca2+]i lead to any substantial loss of clonogenicity following illumination of protoporphyrin-containing cells. Together these results indicate that an increased [Ca2+]i level is not per se a cause of cell death during photodynamic therapy.     

Modulations of early and late secretory processes by activation of protein kinases in the rat adrenal medulla

Modulatory effects of the activation of either protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) or protein kinase A (PKA) by forskolin on stimulant-evoked secretory processes in the perfused rat adrenal medulla were studied. PDBu or forskolin was applied during repetitive stimulation (30 s each at 10-min intervals) with nicotine, bradykinin, muscarine or histamine, and changes in [Ca2+]i (fura-2 microfluorometry) and catecholamine secretions (electrochemical detection) were simultaneously measured. PDBu markedly potentiated the nicotine-evoked secretion without altering the [Ca2+]i response. PDBu partially inhibited the muscarine-evoked secretion and almost completely blocked the histamine-evoked secretion, concomitantly with extensive suppressions of the [Ca2+]i responses to these stimulants. The bradykinin-evoked secretion was enhanced by PDBu despite a slight attenuation of the [Ca2+]i response. PDBu reduced bradykinin-induced intracellular Ca2+ release in a Ca2+-free medium but enhanced the secretion associated with the released Ca2+. These results suggest that PDBu-activated PKC modulates secretory processes at, at least, two different stages. An early-stage modulation may downregulate receptor/G protein systems, which accounts for the inhibitory effect of PDBu on the muscarine- and histamine-evoked responses. A late-stage modulation may generally promote Ca2+-triggered exocytosis after elevation of [Ca2+]i, which explains the potentiation of the nicotine-evoked secretion by PDBu. The late-stage modulation may counteract the early-stage modulation in bradykinin-stimulated cells. Forskolin potentiated the secretory responses to the four secretagogues without increasing the [Ca2+]i responses. PKA may modulate secretory process at a step(s) distal to the rise in [Ca2+]i as is the case with the late-stage modulation by PKC.     

Fluoride activates G protein-dependent and -independent pathways in dispersed intestinal smooth muscle cells

The existence of G protein-dependent and -independent mechanisms activated by sodium fluoride was examined in muscle cells isolated separately from the circular and longitudinal layers of guinea pig intestine. The cells were transiently permeabilized by incubation with Trans. Port Reagent in the presence or absence of GDP beta S (100 microM) and then re-sealed. In the absence of GDP beta S, NaF (1 mM) induced contraction and caused an increase in [Ca2+]i, IP3 and diacylglycerol levels and in protein kinase C (PKC) activity in both cell types. In the presence of GDP beta S, the increases in IP3, DAG and PKC were abolished whereas contraction and the increase in [Ca2+]i were partly inhibited. Residual contraction and [Ca2+]i were abolished by the Ca2+ channel blocker, methoxyverapamil. We conclude that contraction and Ca2+ mobilization induced by NaF is mediated by G protein activation as well as by a G protein-independent mechanism involving activation of plasmalemmal Ca2+ channels.