Subcellular distribution and immunocytochemical localization of protein kinase C in myocardium, and phosphorylation of troponin in isolated myocytes stimulated by isoproterenol or phorbol ester

doi:10.1016/0006-291X(89)90787-0

Biochemical and Biophysical Research Communications

Volume 162, Issue 3, 15 August 1989, Pages 1105-1110

https://doi.org/10.1016/0006-291X(89)90787-0 Get rights and content

Abstract

Protein kinase C (PKC) catalytic activity was found in the cytosol, sarcolemma and sarcoplasmic reticulum, and PKC immunoreactivity was found in the striated regions and sarcolemma of rat hearts. Enhanced phosphorylation of troponin T and, to a lesser extent, troponin I was noted in isolated rat cardiac myocytes incubated with PKC activator phorbol ester, but only the phosphorylation of troponin I was stimulated by isoproterenol. It is suggested that PKC-mediated phosphorylation of troponin might be involved in regulation of myocardial function or in pathophysiology of the heart.

References (17)

J.T. Whitmer et al.
J. Biol Chem
(1978)
N. Katoh et al.
Biochem. Biophys. Res. Commun
(1982)
B.C. Wise et al.
J. Biol. Chem
(1982)
C. Frelin
J. Mol. Cell. Cardiol
(1980)
Y. Nishizuka
Science
(1986)
N. Katoh et al.
Biochem. J
(1981)
N. Katoh et al.
R.A. Minow et al.
Cancer Chemother. Rep
(1975)

There are more references available in the full text version of this article.

Cited by (39)

State-specific Monoclonal Antibodies Identify an Intermediate State in Epsilon Protein Kinase C Activation
2004, Journal of Biological Chemistry
Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of ϵPKC, was raised using the first variable (V1) domain of ϵPKC as the immunogen. 14E6 binding is specific for ϵPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated ϵPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, ϵRACK, the receptor for activated ϵPKC, inhibits 14E6 binding to ϵPKC, suggesting that the 14E6 epitope is lost or hidden when active ϵPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored ϵPKC. Moreover, binding of 14E6 to ϵPKC only after activation suggests that lipid-dependent conformational changes associated with ϵPKC activation precede binding of the activated isozyme to its specific RACK, ϵRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of ϵPKC from cytosolic to membrane localization on activation.
An εPKC-selective inhibitor attenuates back phosphorylation of a low molecular weight protein in cardiac myocytes
2003, Cellular Signalling
We have studied εPKC-mediated phosphorylation events in neonatal cardiac myocytes using back phosphorylation. 3 nM 4-β 12-myristate-13-acetate (PMA)-intact cell treatment preferentially activates εPKC in these cells (Circ. Res. 76 (1995) 654) and caused decreased ³²P incorporation (back phosphorylation) into an ∼18-kDa protein. This response required physiological levels of free Mg²⁺ and short (3–5 min) incubation periods in back phosphorylation assays. Introduction of a selective εPKC translocation inhibitor (εV1) into these cells attenuated the 3 nM PMA-induced back phosphorylation response while translocation inhibitors to the classical PKC or δPKC isozymes were without effect. Pretreatment of our cells with endothelin-1 (ET1) had similar effects to 3 nM PMA albeit the magnitude of the ET1 back phosphorylation response was about one-half that of 3 nM PMA. Our results suggest that εPKC phosphorylates an ∼18-kDa protein found in the particulate cell fraction of neonatal cardiac myocytes. εPKC modulates diverse cardiac responses including contraction, ion channel functions, hypertrophy, and ischemic preconditioning. Characterization of εPKC-selective phosphotransferase events may reveal novel regulatory mechanisms for this enzyme in neonatal cardiac myocytes.
Distribution of active protein kinase C in smooth muscle
1999, Biophysical Journal
To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 μM carbachol for 1 min, 1 μM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 μm 4-α phorbol, 12,13-didecanoate (4-α PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-α PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.
Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes
1998, Journal of Molecular and Cellular Cardiology
A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca²⁺]_i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca²⁺]_i transient was reduced following treatment with 100 nm phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5μm) and calphostin C (10μm) increased [Ca²⁺]_i transient amplitude, elevated basal [Ca²⁺]_i and slowed the decay of the [Ca²⁺]_i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca²⁺ channels was monitored indirectly as the rate of Mn²⁺ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn²⁺ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca²⁺ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca²⁺ content may be secondary to alterations of sarcolemmal Ca²⁺ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca²⁺ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca²⁺]_i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca²⁺ influx, which may play a key role in setting the basal tone of cardiac muscle.
Positive inotropic effect of angiotensin II. Increases in intracellular Ca2<sup>+</sup> or changes in myofilament Ca<sup>2</sup>+ responsiveness?
1997, Journal of Pharmacological and Toxicological Methods
Although it is well known that Angiotensin II (Ang II) has a direct positive inotropic effect in several species, the mechanisms of this action are still poorly understood. The aim of this review is to analyze the possible subcellular mechanisms underlying Ang II-induced positive inotropic action. The binding of Ang II to its receptor triggers a complex signal transduction cascade that stimulates the intracellular formation of two second messengers, inositol 1,4,5-triphosphate (IP₃), and 1,2, diacylglycerol (DAG). IP₃ triggers the release of Ca²⁺ from intracellular stores in several cell types and has been shown to increase myofilament Ca²⁺ sensitivity. DAG activates protein kinase C (PKC), an enzyme that catalyzes the phosphorylation of different cellular proteins, including several proteins of the myofibrils. Distinct ionic transporters, like the $Na^{+} H^{+}$ antiporter and the Na⁺-independent $Cl^{−} HCO_{3}^{−}$ exchanger, implicated in the regulation of intracellular pH, and the $Na^{+} Ca^{2+}$ exchanger which contribute to the intracellular Ca²⁺ homeostasis, have been shown to be activated by a PKC-dependent mechanism. Thus, either one of the Ang II-induced second messengers, that is, IP₃ and DAG, has the potential to affect myocardial contractility by modifying either intracellular Ca²⁺, myofilament Ca²⁺ responsiveness, or both. As described herein, the available data do not allow a definitive single model to explain the mechanism of the Ang II-induced positive inotropic effect. Moreover, it is possible that the final action of Ang II on myocardial inotropism is the end product of a complex interaction of several of the mechanisms triggered by the hormone.
A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function
1996, Journal of Biological Chemistry
Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of ϵPKC (ϵV1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of ϵPKC. Inhibition of ϵPKC translocation but not inhibition of δ or βPKC translocation specifically blocked phorbol ester- or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells.

View all citing articles on Scopus

View full text

Subcellular distribution and immunocytochemical localization of protein kinase C in myocardium, and phosphorylation of troponin in isolated myocytes stimulated by isoproterenol or phorbol ester

Abstract

J. Biol Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Mol. Cell. Cardiol

Science

Biochem. J

Cancer Chemother. Rep