Pathways through which glucose induces a rise in [Ca2+]i of polymorphonuclear leukocytes of rats

Basal levels of [Ca2+]i are elevated in diabetes mellitus. Such an abnormality is most likely due to both increased calcium influx into cells and decreased efflux of this ion out of the cells. The present study examined the cellular pathways that are responsible for hyperglycemia-induced acute rise in polymorphonuclear leukocytes (PMNL), and explored whether such a rise is due to increased calcium entry into PMNL and/or to calcium release from their intracellular stores. There were dose dependent and time dependent rises in the [Ca2+]i of PMNL exposed to high concentrations of glucose. Similar effects were observed when the PMNL were exposed to high concentrations of choline chloride or mannitol. A substantial part of the rise in [Ca2+]i was inhibited when the media contained verapamil or nifedipine or when the PMNL were placed in calcium free media, and the rise in [Ca2+]i was completely abolished when the PMNL were placed in calcium free media containing ryanodine. GDP beta S or pertussis toxin almost completely prevented the glucose-induced rise in [Ca2+]i of PMNL. Rp-cAMP, H-89 or staurosporine produced significant inhibition of the rise in [Ca2+]i. High concentrations of glucose produced a dose dependent shrinkage of PMNL volume over a period of two hours. The volume of PMNL, however, was normal after 24 hours in vitro incubation studies as well as after 1, 2 and 12 days of streptozotocin-induced hyperglycemia in rats. The results are consistent with the formulation that the osmotic activity (cell shrinkage) of the high glucose concentrations activates G protein(s) which then stimulates the adenylate-cAMP-protein kinase A pathway, phospholipase C system and calcium channels. The stimulation of these cellular pathways permits both calcium influx into the PMNL as well as mobilization of calcium from their intracellular stores. Both of these events contribute to the acute rise in their [Ca2+]i. It is possible that the rise in [Ca2+]i is critical for the stimulation of the events that lead to the generation and accumulation of inorganic osmolytes to restore cell volume to normal.     

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+.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

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.     

A protein kinase C agonist, selective for the beta I isozyme, induces E-selectin and VCAM-1 expression on HUVEC but does not translocate PKC

A protein kinase C (PKC) agonist selective for the beta I isozyme, 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), induced NF-kappa B-like binding activity and surface expression of E-selectin and VCAM-1 in human umbilical vein endothelial cells (HUVEC), similar to the effects of tumor necrosis factor-alpha (TNF-alpha). Induction of E-selectin and VCAM-1 expression by dPPA was completely inhibited by the PKC inhibitors staurosporine and Ro31-7549. The PKC inhibitors also reduce TNF-alpha-induced VCAM-1 expression. However, neither dPPA nor TNF-alpha translocated PKC from the cytosolic to the plasma or nuclear membrane particulate fractions in HUVEC. These results indicate that activation of the beta I PKC isozyme is sufficient for expression of E-selectin and VCAM-1, and suggest that PKC may mediate the effects of TNF-alpha and dPPA without requiring the translocation normally associated with activation of PKC.     

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.     

Protective ability of acetylsalicylic acid (aspirin) to scavenge radiation induced free radicals in J774A.1 macrophage cells

Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular calcium ([Ca2+]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained concerning the effect of PKC inhibition on progesterone-stimulated [Ca2+]i increase. In the present study, we investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC inhibitors on progesterone-stimulated [Ca2+]i increase and tyrosine phosphorylation of proteins. We found that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors (sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca2+]i increase. Neither short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effect of progesterone on sperm [Ca2+]i. Nor was responsiveness to progesterone affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors, sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid.     

Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.     

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.     

Lysophosphatidic acid-induced proliferation-related signals in astrocytes

Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, G? 6976. The increase in [Ca2+]i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.     

Disrupted [Ca2+]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 microM) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.     

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.     

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.     

Differential modulation of protein kinase C isozymes in rat parotid acinar cells. Relation to amylase secretion

We investigated the expression, distribution, and activation parameters of protein kinase C (PKC) isozymes in isolated rat parotid acinar cells. By analyzing cellular extracts by western blot analysis and for isozyme-specific RNA, the Ca(2+)-independent PKC-delta, -epsilon, and -zeta were detected in the cytosolic, particulate (plasma membrane), and nuclear fractions of unstimulated cells, whereas the Ca(2+)-dependent PKC-alpha was confined to the cytosolic and particulate fractions. The expressed isozymes showed distinct responses to phorbol 12-myristate 13-acetate (PMA), thymeleatoxin, and cell surface receptor agonists with respect to translocation from cytosol to particulate fraction and nucleus, as well as sensitivity to down-regulation caused by prolonged exposure to PMA (3-20 hr). The marked susceptibility to down-regulation displayed by PKC-alpha and -delta was accompanied by an enhanced secretory response to norepinephrine as compared with control cells. Further, the selective PKC inhibitors Ro 31-8220 and CGP 41,251 also produced a concentration-dependent enhancement of norepinephrine-induced amylase secretion. Our findings suggest that PKC-alpha or -delta plays a negative modulatory role, rather than an obligatory role, in amylase secretion. Also, the localization and redistribution of PKC-epsilon and -delta to the nucleus by PKC activators imply that one or both of these isozymes may regulate such processes as cellular proliferation and/or differentiation.     

Nuclear morphometry of the myocardial cells as a diagnostic tool in cases of sudden death due to coronary thrombosis

One general signalling mechanism used to transfer the information delivered by agonists into appropriate intracellular compartments involves the rapid redistribution of ionised calcium throughout the cell, which results in transient elevations of the cytosolic free Ca2+ concentration. Various physiological stimuli increase [Ca2+]i transiently and, thereby, induce cellular responses. However, under pathological conditions, changes of [Ca2+]i are generally more pronounced and sustained. Marked elevations of [Ca2+]i activate hydrolytic enzymes, lead to exaggerated energy expenditure, impair energy production, initiate cytoskeletal degradation, and ultimately result in cell death. Such Ca(2+)-induced cytotoxicity may play a major role in several diseases, including neuropathological conditions such as chronic neurodegenerative diseases and acute neuronal losses (e.g. in stroke).     

Lysophosphatidylcholine potentiates vascular contractile responses by enhancing vasoconstrictor-induced increase in cytosolic free Ca2+ in rat aorta

We investigated the effects of palmitoyl-L-alpha-lysophosphatidylcholine on the contractile responses of the endothelium-denuded rat aorta to high K+, noradrenaline, UK14,304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (a selective alpha2 adrenoceptor agonist) and phorbol 12-myristate 13-acetate (PMA). Lysophosphatidylcholine at concentrations from 10(-6) M to 10(-4) M did not contract aortic strips. However, lysophosphatidylcholine strongly potentiated the UK14,304-induced contraction. High K+ - and PMA-induced contractions were also potentiated. In contrast, the noradrenaline-induced contraction was only slightly potentiated by 10(-5) M lysophosphatidylcholine. In fura PE-3-loaded aortic strips, lysophosphatidylcholine (10(-5) M) markedly augmented the increase in both cytosolic free Ca2+ ([Ca2+]i) and contractile tension induced by UK14,304, high K+ and PMA. Nicardipine (10(-7) M) and 10(-6) M Ro-31-8220 (?1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-++ +indoyl-3-yl)-maleimide-methane sulfate) strongly inhibited the increase in [Ca2+]i and contractile tension induced by UK14,304 and in the presence of these inhibitors, the enhancing effects of lysophosphatidylcholine were attenuated. However, the enhancing effect on high K+ -induced contraction was not affected by Ro-31-8220. These results suggest that lysophosphatidylcholine may cause an augmentation of the increase in [Ca2+]i induced by UK14,304 which response is depend on protein kinase C activation and in this way potentiate contractile responses in the rat aorta. Protein kinase C independent mechanisms may also be involved in the enhancing effect of lysophosphatidylcholine on smooth muscle contraction.     

Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells

Previous studies indicated that rat basophilic RBL-2H3 cells contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C (PKC); of these, PKC beta and delta were the most potent transducers of signals for exocytosis in antigen-stimulated permeabilized cells. Exocytosis, nevertheless, was still dependent on an elevated free Ca2+. (Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756). We now demonstrate that PKC alpha and epsilon, exclusively, inhibit antigen-induced hydrolysis of inositol phospholipids in the same permeabilized RBL-2H3 cells. Unlike secretion, the inhibitory actions occurred at a basal concentration (0.1 microM) of free Ca2+. The inhibitory actions of the two isozymes were potentiated by 20 nM phorbol 12-myristate 13-acetate. As indicated by the effects of the phorbol ester, the probable mechanism was reduced tyrosine phosphorylation of phospholipase C gamma 1. The negative regulation of phospholipase C was apparent in intact cells, because the PKC inhibitor Ro31-7549 or down-regulation of PKC with phorbol ester enhanced antigen-induced hydrolysis of inositol phospholipids. The concentrations of the various isozymes of PKC in RBL-2H3 cells, as estimated by immunoblotting studies, were sufficient for promotion of exocytosis (i.e. beta and delta) and inhibition of phospholipid hydrolysis (i.e. alpha and epsilon).     

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.     

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.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.