Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids

1998 ◽  
Vol 76 (12) ◽  
pp. 1110-1119 ◽  
Author(s):  
Celia A Kanashiro ◽  
Raouf A Khalil
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Coronary Artery ◽  
Protein Kinase ◽  
Calcium Influx ◽  
Surface Membrane ◽  
Smooth Muscle Contraction ◽  
Specific Protein ◽  
Porcine Coronary Artery ◽  
Kinase C

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2α (PGF2α) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 µM [Ca2+]e, both PGF2α and U46619 (10-5 M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 µM [Ca2+]e, PGF2α and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx - PKC activity - contraction relationship showed a 45Ca2+-influx threshold of ~7 µmol·kg-1·min-1 for maximal PKC activation by PGF2α and U46619. 45Ca2+ influx > 10 µmol·kg-1·min-1 was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed α-, δ-, ε-, and ζ-PKC in porcine coronary artery. In unstimulated tissues, α- and ε-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of ε-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of α-PKC was observed at 600 µM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent ε-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent α-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.Key words: protein kinase C, isoform, calcium influx, eicosanoids, vascular smooth muscle, contraction.

Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle

1992 ◽  
Vol 263 (3) ◽  
pp. C714-C719 ◽  
Author(s):  
R. A. Khalil ◽  
C. Lajoie ◽  
M. S. Resnick ◽  
K. G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Surface Membrane ◽  
Smooth Muscle Contraction ◽  
Nuclear Space ◽  
Control Of Gene Expression ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Independent Protein

It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.

Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKCζ

1996 ◽  
Vol 74 (1) ◽  
pp. 51-65 ◽  
Author(s):  
Odile Clément-Chomienne ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Specific Activity ◽  
Regulation Of Gene Expression ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Partial Purification ◽  
Chicken Gizzard ◽  
Kinase C

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1 and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙min−1∙mg−1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.

2-Chloroadenosine prevents phorbol ester-induced depletion of protein kinase C in porcine coronary artery

1993 ◽  
Vol 264 (5) ◽  
pp. H1465-H1471 ◽  
Author(s):  
R. B. Marala ◽  
K. Ways ◽  
S. J. Mustafa
Keyword(s):  
Protein Kinase C ◽  
Coronary Artery ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Chronic Exposure ◽  
Porcine Coronary Artery ◽  
Kinase C ◽  
Adenosine Analogue ◽  
Presence And Absence

In this study we investigated the role of the adenosine analogue 2-chloroadenosine (CAD) in the regulation of protein kinase C (PKC) in porcine coronary artery. Arterial rings were contracted with endothelin-1 (ET-1; 10(-10) to 10(-7) M) and phorbol 12,13-dibutyrate (PDBu; 10(-7) M) after incubating them for 1 and 2 days with PDBu (200 nM) in the presence and absence of CAD (10(-4) M). Chronic exposure to PDBu alone attenuated ET-1-induced contractions, while inclusion of CAD during incubation protected against the PDBu-induced blunting of ET-1-induced contraction. Similarly, PDBu (10(-7) M)-induced contraction of the arterial rings was attenuated upon chronic incubation with PDBu, and once again, inclusion of CAD showed an improved response to PDBu-induced contraction when compared with PDBu alone. Incubation with PDBu (200 nM) for 20 min caused the PKC translocation from cytosol to membrane, whereas CAD totally blocked this translocation. Chronic (1 and 2 days) incubation with PDBu caused a substantial depletion of PKC activities in cytosol and membrane. The presence of CAD protected the PDBu-induced depletion of PKC in both cytosol and membrane. To replete PKC, after incubation with the drugs, the arteries were incubated in the absence of drugs for another 2 days. Arteries incubated with PDBu in the presence and absence of CAD recovered significantly in their response to ET-1 as well as PDBu. These results indicate that CAD protects against the PDBu-induced activation and depletion of PKC in porcine coronary artery.

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