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Inhibition of phorbol ester-induced contraction by calmodulin antagonists in rat aorta
J K Chuprun1, E Bazan, K C Chang
1Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Abstract:
The purpose of the present study was to investigate the relative roles of protein kinase C (PKC) and myosin light chain kinase (MLCK) in phorbol ester-induced contraction of vascular smooth muscle through the use of PKC and calmodulin antagonists. Prior exposure to PKC antagonists staurosporine (0.03 microM) and H-7 (10 microM) had relatively little effect on contractions to phorbol 12-myristate 13-acetate (PMA), while contractions to norepinephrine and KCl were greatly inhibited. Prior exposure to the calmodulin antagonists calmidazolium (3 and 10 microM) and W-7 (10 microM) inhibited contractions to PMA in the presence and absence of extracellular Ca2+, while contractions to norepinephrine and KCl remained relatively unaffected. Calmidazolium and W-7 were relatively weak relaxants when applied during the PMA contraction, and the magnitudes of relaxation were similar to those observed in norepinephrine- and KCl-contracted tissues. Calmidazolium partially inhibited the PMA-induced translocation of PKC. These results suggest that 1) the calmodulin antagonists inhibit the development of PMA-induced contraction, at least in part, through inhibition of PKC translocation; 2) the mechanisms of phorbol ester- and agonist-induced translocation of PKC are distinct; 3) the potencies and inhibitory mechanisms of these agents depend on whether the agents are added before or during the contraction; and 4) the selectivity of these agents, as evaluated in enzyme preparations, may not be consistent with their cellular actions.
Insights
Calmodulin antagonists, not protein kinase C antagonists, inhibit phorbol ester-induced vascular smooth muscle contraction, suggesting distinct PKC translocation mechanisms. These findings impact understanding of cellular signaling pathways.
Area of Science:
- Vascular pharmacology
- Cell signaling
- Biochemistry
Background:
- Protein kinase C (PKC) and myosin light chain kinase (MLCK) are key regulators of vascular smooth muscle contraction.
- Phorbol esters, like phorbol 12-myristate 13-acetate (PMA), activate PKC and induce muscle contraction.
- The precise roles of PKC and calmodulin in PMA-induced contractions require further elucidation.
Purpose of the Study:
- To investigate the relative contributions of PKC and MLCK in PMA-induced vascular smooth muscle contraction.
- To differentiate the signaling pathways activated by phorbol esters versus traditional agonists (norepinephrine, KCl).
- To examine the effects of PKC and calmodulin antagonists on PMA-induced cellular responses.
Main Methods:
- Utilized PKC antagonists (staurosporine, H-7) and calmodulin antagonists (calmidazolium, W-7) in isolated vascular smooth muscle preparations.
- Administered antagonists prior to or during contraction induced by PMA, norepinephrine, or KCl.
- Assessed the impact of antagonists on muscle contraction magnitude and PKC translocation.
- Investigated the role of extracellular calcium (Ca2+) in PMA-induced contractions.
Main Results:
- PKC antagonists minimally affected PMA-induced contractions but significantly inhibited norepinephrine- and KCl-induced contractions.
- Calmodulin antagonists potently inhibited PMA-induced contractions, irrespective of extracellular Ca2+ presence.
- Calmodulin antagonists showed weak relaxation effects when applied during PMA contraction, similar to their effects on other agonists.
- Calmidazolium partially inhibited PMA-induced PKC translocation, suggesting a link between calmodulin antagonism and PKC signaling.
Conclusions:
- Calmodulin antagonists inhibit PMA-induced vascular smooth muscle contraction, partly by interfering with PKC translocation.
- Distinct mechanisms govern phorbol ester- and agonist-induced PKC translocation.
- The timing of antagonist administration (before vs. during contraction) influences their efficacy and inhibitory mechanisms.
- In vitro enzyme selectivity of antagonists may not accurately predict their cellular actions in complex systems like vascular smooth muscle.