Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells.
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Short-term regulation of sodium metabolism is dependent on the modulation of the activity of sodium transporters by first and second messengers. In understanding diseases associated with sodium retention, it is necessary to identify the coupling between these messengers. We have examined whether dopamine, an important first messenger in tubular cells, activates and translocates various protein kinase C (PKC) isoforms. We used a proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was found to inhibit Na(+)-K(+)-ATPase in a PKC-dependent manner. Translocation of PKC isoforms was studied with both subcellular fractionation and confocal microscopy. Both techniques revealed a dopamine-induced translocation from cytosol to plasma membrane of PKC-alpha and -epsilon, but not of PKC-delta, -gamma, and -zeta. The process of subcellular fractionation resulted in partial translocation of PKC-epsilon. This artifact was eliminated in confocal studies. Confocal imaging permitted detection of translocation within 20 s. Translocation was abolished by a phospholipase C inhibitor and by an antagonist against the dopamine 1 subtype (D(1)) but not the 2 subtype of receptor (D(2)). In conclusion, this study visualizes in renal epithelial cells a very rapid activation of the PKC-alpha and -epsilon isoforms by the D(1) receptor subtype. (+info)
Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Ca(2+) channels.
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Limited information is available regarding the effects of protein kinase C (PKC) isozyme(s) in the regulation of L-type Ca(2+) channels due to lack of isozyme-selective modulators. To dissect the role of individual PKC isozymes in the regulation of cardiac Ca(2+) channels, we used the recently developed novel peptide activator of the epsilonPKC, epsilonV1-7, to assess the role of epsilonPKC in the modulation of L-type Ca(2+) current (I(Ca,L)). Whole cell I(Ca,L) was recorded using patch-clamp technique from rat ventricular myocytes. Intracellular application of epsilonV1-7 (0.1 microM) resulted in a significant inhibition of I(Ca,L) by 27.9 +/- 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The inhibitory effect of epsilonV1-7 on I(Ca,L) was completely prevented by the peptide inhibitor of epsilonPKC, epsilonV1-2 [5.2 +/- 1.7%, not significant (NS), n = 5] but not by the peptide inhibitors of cPKC, alphaC2-4 (31.3 +/- 2.9%, P < 0.01, n = 6) or betaC2-2 plus betaC2-4 (26.1 +/- 2.9%, P < 0.01, n = 5). In addition, the use of a general inhibitor (GF-109203X, 10 microM) of the catalytic activity of PKC also prevented the inhibitory effect of epsilonV1-7 on I(Ca,L) (7.5 +/- 2.1%, NS, n = 6). In conclusion, we show that selective activation of epsilonPKC inhibits the L-type Ca channel in the heart. (+info)
Role for PKC in the adenosine-induced decrease in shortening velocity of rat ventricular myocytes.
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We previously demonstrated that both adenosine receptor activation and direct activation of protein kinase C (PKC) decrease unloaded shortening velocity (V(max)) of rat ventricular myocytes. The goal of this study was to further investigate a possible link among adenosine receptors, phosphoinositide-PKC signaling, and V(max) in rat ventricular myocytes. We determined that the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 100 microM) and the alpha-adrenergic receptor agonist phenylephrine (Phe, 10 microM) increased turnover of inositol phosphates. PKC translocation from the cytosol to the sarcolemma was used as an indicator of PKC activation. Western blot analysis demonstrated an increased PKC-epsilon translocation after exposure to R-PIA, Phe, and the PKC activators dioctanoylglycerol (50 microM) and phorbol myristate acetate (1 microM). PKC-alpha, PKC-delta, and PKC-zeta did not translocate to the membrane after R-PIA exposure. Finally, PKC inhibitors blocked R-PIA-induced decreases in V(max) as well as Ca(2+)-dependent actomyosin ATPase in rat ventricular myocytes. These results support the conclusions that adenosine receptors activate phosphoinositide-PKC signaling and that adenosine receptor-induced PKC activation mediates a decrease in V(max) in ventricular myocytes. (+info)
Hypoxia increases the sensitivity of the L-type Ca(2+) current to beta-adrenergic receptor stimulation via a C2 region-containing protein kinase C isoform.
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The effects of hypoxia on the L-type Ca(2+) current (I:(Ca-L)) in the absence and presence of the ss-adrenergic receptor agonist isoproterenol (Iso) were examined. Exposing guinea pig ventricular myocytes to hypoxia alone resulted in a reversible inhibition of basal I:(Ca-L). When cells were exposed to Iso in the presence of hypoxia, the K:(0.5) for activation of I:(Ca-L) by Iso was significantly decreased from 5.3+/-0.7 to 1.6+/-0.1 nmol/L. The membrane-impermeant thiol-specific oxidizing compound 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) attenuated the inhibition of basal I:(Ca-L) by hypoxia 81.3+/-9.4% but had no effect on the increase in sensitivity of I:(Ca-L) to Iso. In addition, DTT mimicked the effects of hypoxia on basal I:(Ca-L) and the increase in sensitivity to Iso. Neither the inhibitors of guanylate cyclase LY-83583 or methylene blue nor the NO synthase inhibitor N:(G)-monomethyl-L-arginine monoacetate had any effect on the basal inhibition of I:(Ca-L) or the decrease in K:(0.5) for activation of I:(Ca-L) by Iso during hypoxia. However, the protein kinase C (PKC) inhibitors bisindolylmaleimide I and Go 7874 significantly attenuated the increase in sensitivity of I:(Ca-L) to Iso. More specifically, the response was attenuated when cells were dialyzed with a peptide inhibitor of the C2 region-containing classical PKC isoforms. The same effect was not observed with the PKCepsilon peptide inhibitor. These results suggest that hypoxia regulates I:(Ca-L) through the following 2 distinct mechanisms: direct inhibition of basal I:(Ca-L) and an indirect effect on the sensitivity of the channel to ss-adrenergic receptor stimulation that is mediated through a classical PKC isoform. (+info)
Signalling pathways regulating the dephosphorylation of Ser729 in the hydrophobic domain of protein kinase Cepsilon upon cell passage.
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We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729). (+info)
Ethanol acts synergistically with a D2 dopamine agonist to cause translocation of protein kinase C.
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Ethanol and other drugs of abuse increase synaptic dopamine levels; however, little is known about how ethanol alters dopaminergic signaling. We have reported that ethanol induces translocation of delta and epsilon protein kinase C (PKC) in neural cells in culture. Using NG108-15 and Chinese hamster ovary cell lines that express the dopamine D2 receptor (D2R), we show here that the D2R agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (NPA) also causes translocation of delta and epsilon PKC to the same sites as ethanol-induced translocation. D2R agonist and ethanol-induced translocation of delta and epsilon PKC share a common pathway that is blocked by pertussis toxin and requires phospholipase C (PLC) activity. These data suggest that both D2R agonists and ethanol activate PLC via a trimeric G protein leading to production of diacylglycerol with subsequent activation and translocation of delta and epsilon PKC. Moreover, ethanol and NPA, when present together at low concentrations that alone are ineffective, act synergistically to cause translocation of delta and epsilon PKC. Our data suggest that ethanol causes translocation of delta and epsilon PKC but cells expressing the D2R, such as neurons in the nucleus accumbens, may be particularly sensitive to low concentrations of ethanol. (+info)
Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.
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Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading. (+info)
A Ca(2+)-dependent transgenic model of cardiac hypertrophy: A role for protein kinase Calpha.
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BACKGROUND: Calcium imbalances have been implicated as an underlying mechanism of human cardiac dysfunction. The voltage-dependent calcium channel plays a critical role in calcium regulation in the heart. Thus, aberrant calcium signaling arising from this channel could initiate the calcium imbalances observed in heart failure. In the present study, we used a transgenic mouse with an increased number of L-type calcium channels to identify the role of an increased, sustained ingress of calcium as an initiator of hypertrophy. METHODS AND RESULTS: Whole-heart histology and electrophysiology in isolated cardiomyocytes identified calcium-channel overexpression in the hearts of transgenic mice. Calcium-channel density was increased in 2-, 4-, and 8-month-old transgenic cardiomyocytes. Ventricular fibrosis, damage, and remodeling became more pronounced as the transgenic mice aged. Apoptosis was also present in transgenic hearts at 8 months of age. Increased protein kinase Calpha activation was elevated before the development of hypertrophy and failure. CONCLUSIONS: Transgenic mice developed hypertrophy and severe cardiomyopathy as a function of age, thus confirming that changes in channel density are sufficient to induce disease. The small, sustained increase in the ingress of Ca(2+) through the calcium channel elevated protein kinase Calpha before the development of hypertrophy, suggesting that protein kinase Calpha plays an important role in triggering hypertrophy. (+info)