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Stimulation of canine cardiac sarcoplasmic reticulum Ca2+ uptake by dihydropyridine Ca2+ antagonists
Abstract:
We examined the effects of four Ca2+ antagonists that possess the ability to bind to calmodulin-felodipine, nitrendipine, prenylamine, and verapamil--as well as the effect of the calmodulin antagonist trifluoperazine on Ca2+ uptake and Ca2+ + Mg2+/ATPase activity in canine cardiac sarcoplasmic reticulum. In the presence of 20-30 microM felodipine and 100-200 microM nitrendipine, Ca2+ uptake increased from 69 nmoles X mg-1 X min-1 to 107 and 108 nmoles X mg-1 X min-1, respectively, with half-maximal stimulation occurring at 7.5 and 28 microM respectively. Ca2+ + Mg2+/ATPase activity was unchanged over the same concentration ranges. In contrast, both Ca2+ uptake and Ca2+ + Mg2+/ATPase activities were inhibited in the presence of 10-100 microM trifluoperazine (IC50 = 25 microM), 10-100 microM prenylamine (IC50 = 35 microM) and 100-200 microM verapamil (inhibition insufficient for IC50 determination). None of the drugs affected membrane permeability to Ca2+ as determined by passive 45Ca2+ efflux in the presence of ethyleneglycol bis(beta-amenoethyl ether)N,N,N1-tetraacetic acid (EGTA). Drug inhibition of calmodulin-dependent turkey gizzard myosin light chain kinase activation in a purified protein system was used as a direct measure of calmodulin antagonism, and felodipine, nitrendipine, trifluoperazine, prenylamine, and verapamil blocked this activation at IC50 values of 9.8, 55, 6.4, 31, and 93 microM respectively. None of the drugs studied, however, had any effect upon endogenous phospholamban phosphorylation in our cardiac sarcoplasmic reticulum preparations. These observations indicate that dihydropyridine Ca2+ antagonists stimulate cardiac sarcoplasmic reticulum Ca2+ uptake in vitro either by increasing the efficiency of the transport process or by inhibiting Ca2+-dependent Ca2+ release, and suggest that these effects do not result from interference with calmodulin-mediated processes.
Insights
Calcium (Ca2+) antagonists like felodipine and nitrendipine enhance cardiac sarcoplasmic reticulum Ca2+ uptake. These effects on Ca2+ transport are not mediated by calmodulin, suggesting alternative mechanisms in cardiac function.
Area of Science:
- Biochemistry
- Cardiovascular Physiology
Background:
- Cardiac sarcoplasmic reticulum (SR) is crucial for regulating intracellular calcium (Ca2+) levels during muscle contraction and relaxation.
- Calcium (Ca2+) uptake and Ca2+ + Mg2+/ATPase activity in the SR are key determinants of cardiac contractility.
- Calmodulin is a calcium-binding protein that modulates various cellular processes, including ion transport.
Purpose of the Study:
- To investigate the effects of specific Ca2+ antagonists (felodipine, nitrendipine, prenylamine, verapamil) and a calmodulin antagonist (trifluoperazine) on canine cardiac SR Ca2+ uptake and Ca2+ + Mg2+/ATPase activity.
- To determine if the observed effects of these drugs on SR function are mediated through calmodulin antagonism.
- To assess the impact of these drugs on membrane permeability and phospholamban phosphorylation.
Main Methods:
- Assessed Ca2+ uptake and Ca2+ + Mg2+/ATPase activity in canine cardiac sarcoplasmic reticulum (SR) preparations.
- Utilized purified turkey gizzard myosin light chain kinase (MLCK) to directly measure calmodulin antagonism.
- Measured passive 45Ca2+ efflux to evaluate drug effects on membrane permeability.
- Examined endogenous phospholamban phosphorylation in SR preparations.
Main Results:
- Felodipine and nitrendipine significantly increased Ca2+ uptake in cardiac SR, with half-maximal stimulation at 7.5 and 28 microM, respectively. Ca2+ + Mg2+/ATPase activity remained unchanged.
- Trifluoperazine, prenylamine, and verapamil inhibited both Ca2+ uptake and Ca2+ + Mg2+/ATPase activities in a dose-dependent manner.
- Direct calmodulin antagonism was confirmed for all tested drugs, with varying IC50 values.
- No significant effects were observed on membrane permeability or phospholamban phosphorylation.
Conclusions:
- Dihydropyridine Ca2+ antagonists (felodipine, nitrendipine) stimulate cardiac SR Ca2+ uptake in vitro.
- The stimulation of Ca2+ uptake by these antagonists may occur via increased transport efficiency or inhibition of Ca2+-dependent Ca2+ release.
- The observed effects on cardiac SR Ca2+ handling are likely independent of calmodulin antagonism.