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Title: The effect of the PKC inhibitor calphostin C and the PKC agonist phorbol 12-myristate 13-acetate on regulation of cytosolic Ca{sup 2+} in mammalian skeletal muscle cells

Abstract

Protein kinase C (PKC) has been shown to exert broad actions in modulating Ca{sup 2+} in cardiac myocytes, however, the effect of PKC in skeletal muscle cells is largely unknown. In this study, we examined the effect of the PKC inhibitor calphostin C (CC) and the PKC agonist phorbol 12-myristate 13-acetate (PMA) on intracellular Ca{sup 2+} handling in C2C12 skeletal myotubes and skinned skeletal muscle fibers of the rat. CC (250 nM) significantly prolonged (P = 0.01, n = 6), and the PKC agonist PMA (500 nM; P = 0.03, n 6) significantly shortened the decay phase of electrically induced Ca{sup 2+} transients in C2C12 myotubes without affecting the amplitude or the time to peak of the transients. Skinned fiber studies showed that CC significantly inhibits SR Ca{sup 2+} uptake in skeletal muscle cells. PMA had no effect. CC also increased the peak of ATP-induced Ca{sup 2+} transients release by 94.2% (P < 0.0001) in the presence of extracellular Ca{sup 2+} and 54.5% (P = 0.04) without external Ca{sup 2+} via IP{sub 3}-Ca{sup 2+} release pathway in C2C12 myotubes, while PMA had no effect, suggesting that CC may modulate IP{sub 3}-induced Ca{sup 2+} release via a PKC-independent mechanism. CC atmore » a concentration of 1 {mu}M was able to induce a large sustained elevation in basal [Ca{sup 2+}]{sub i} that was blocked by Ca{sup 2+} store depletion and the IP{sub 3} receptor blocker 2-APB. These results indicate that PKC plays a role in modulation of SR function in skeletal muscle cells, and the PKC inhibitor CC may alter Ca{sup 2+} handling via both PKC-dependent and PKC-independent pathways.« less

Authors:
 [1];  [2]
  1. Physiology, School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009 (Australia). E-mail: renzhi-han@uiowa.edu
  2. Physiology, School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009 (Australia)
Publication Date:
OSTI Identifier:
20783471
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 212; Journal Issue: 3; Other Information: DOI: 10.1016/j.taap.2005.07.023; PII: S0041-008X(05)00440-0; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACETATES; ATP; CALCIUM IONS; MUSCLES; RATS; RECEPTORS; SARCOPLASMIC RETICULUM; UPTAKE

Citation Formats

Han Renzhi, and Bakker, Anthony J. The effect of the PKC inhibitor calphostin C and the PKC agonist phorbol 12-myristate 13-acetate on regulation of cytosolic Ca{sup 2+} in mammalian skeletal muscle cells. United States: N. p., 2006. Web.
Han Renzhi, & Bakker, Anthony J. The effect of the PKC inhibitor calphostin C and the PKC agonist phorbol 12-myristate 13-acetate on regulation of cytosolic Ca{sup 2+} in mammalian skeletal muscle cells. United States.
Han Renzhi, and Bakker, Anthony J. Mon . "The effect of the PKC inhibitor calphostin C and the PKC agonist phorbol 12-myristate 13-acetate on regulation of cytosolic Ca{sup 2+} in mammalian skeletal muscle cells". United States. doi:.
@article{osti_20783471,
title = {The effect of the PKC inhibitor calphostin C and the PKC agonist phorbol 12-myristate 13-acetate on regulation of cytosolic Ca{sup 2+} in mammalian skeletal muscle cells},
author = {Han Renzhi and Bakker, Anthony J.},
abstractNote = {Protein kinase C (PKC) has been shown to exert broad actions in modulating Ca{sup 2+} in cardiac myocytes, however, the effect of PKC in skeletal muscle cells is largely unknown. In this study, we examined the effect of the PKC inhibitor calphostin C (CC) and the PKC agonist phorbol 12-myristate 13-acetate (PMA) on intracellular Ca{sup 2+} handling in C2C12 skeletal myotubes and skinned skeletal muscle fibers of the rat. CC (250 nM) significantly prolonged (P = 0.01, n = 6), and the PKC agonist PMA (500 nM; P = 0.03, n 6) significantly shortened the decay phase of electrically induced Ca{sup 2+} transients in C2C12 myotubes without affecting the amplitude or the time to peak of the transients. Skinned fiber studies showed that CC significantly inhibits SR Ca{sup 2+} uptake in skeletal muscle cells. PMA had no effect. CC also increased the peak of ATP-induced Ca{sup 2+} transients release by 94.2% (P < 0.0001) in the presence of extracellular Ca{sup 2+} and 54.5% (P = 0.04) without external Ca{sup 2+} via IP{sub 3}-Ca{sup 2+} release pathway in C2C12 myotubes, while PMA had no effect, suggesting that CC may modulate IP{sub 3}-induced Ca{sup 2+} release via a PKC-independent mechanism. CC at a concentration of 1 {mu}M was able to induce a large sustained elevation in basal [Ca{sup 2+}]{sub i} that was blocked by Ca{sup 2+} store depletion and the IP{sub 3} receptor blocker 2-APB. These results indicate that PKC plays a role in modulation of SR function in skeletal muscle cells, and the PKC inhibitor CC may alter Ca{sup 2+} handling via both PKC-dependent and PKC-independent pathways.},
doi = {},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 212,
place = {United States},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
}
  • The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 {mu}M of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. These results indicated that the inhibition of arborization by PMA wasmore » mediated through the activation of protein kinase C. Colchicine at 5.0 {mu}M also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. The observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.« less
  • The whole-cell patch-clamp technique has been used to analyze the properties of the dihydropyridine-sensitive Ca/sup 2 +/ channel in rat skeletal muscle cells (myoballs) in culture. The potential dependence of Ca/sup 2 +/ -channel activation is similar to that observed in cardiac cells. However, the skeletal muscle Ca/sup 2 +/ channel is activated more slowly. The voltage dependence of Ca/sup 2 +/-channel inactivation indicates a half-maximal inactivation (V/sub h0.5/) at -72 mV as compared to V/sub h0.5/ = -35 mV for cardiac cells. Blockade of the skeletal muscle Ca/sup 2 +/ channel by the dihydrophyridine (+)-PN 200-110 is voltage dependent,more » with a half-maximal effect of 13 nM for an application of the drug to the myoball membrane held at -90 mV and of 0.15 nM for an application at a potential of -65 mV. The 100-fold difference in apparent affinity is interpreted as a preferential association of PN 200-110 with the inactivated form of the Ca/sup 2 +/ channel. The K/sub 0.5/ value found from electrophysiological experiments for the binding to the inactivated state is nearly identical to the equilibrium dissociation constant found from binding experiments with (+)-(/sup 3/H)PN 200-110 using transverse-tubular membranes. The dihydropyridine activator Bay K8644 acts by increasing Ca/sup 2 +/ current amplitude and by slowing down deactivation.« less
  • Sphingosine and other long-chain (sphingoid) bases inhibit protein kinase C, the putative cellular receptor for the tumor promoter phorbol 12-myristate 13-acetate (PMA) and exert potent effects on diverse cell functions. The authors tested the ability of long-chain bases to modulate multistage carcinogenesis in mouse C3H/10T 1/2 cells exposed to {gamma}-rays and PMA. Sphingosine and sphinganine completely blocked the enhancement of radiation-induced transformation by PMA (promotion) and partially suppressed transformation by radiation alone. N-Acetylspingosine, a ceramide analog, did not inhibit transformation. Sphingosine was rapidly taken up by the cells and metabolized; hence, the long-chain bases were added daily to achieve prolongedmore » inhibition. Long-chain bases inhibited protein kinase C activity in C3H/10T 1/2 cells and suppressed the down-regulation of this enzyme by PMA. The results establish that long-chain bases are highly effective inhibitors of carcinogenesis in this model. The results also indicate that the suppressive effects may be mediated, in part, by inhibition of protein kinase C. The data suggest that sphingosine and other long-chain bases derived from complex sphingolipids may act as cancer-preventative agents.« less
  • The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotidesmore » against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.« less
  • An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was conducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes (9p- and t(10;13)), which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13-acetate-induced cell differentiation contained double minute chromatine bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of doublemore » minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cell differed also in a number of other chromosomal alteration, including gains or losses involving chromosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes.« less