endothelin-1 and 1-2-dioctanoylglycerol

The amphotericin B-perforated whole-cell patch clamp technique was used to determine the modulation of L-type Ca2+ channels by protein kinase C (PKC)-mediated pathways in adult rat ventricular myocytes. Application of 10 nM endothelin-1 (ET-1) increased peak Ca2+ current (ICa) by 28.2 +/- 2.5 % (n = 13) and slowed current decay. These effects were prevented by the endothelin receptor antagonist PD145065 (10 microM) and by the PKC inhibitor chelerythrine (8 microM). To establish if direct activation of PKC mimicked the ET-1 effect, the active and inactive phorbol esters (phorbol-12-myristate-13-acetate and 4alpha-phorbol-12, 13-didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (approximately 10%) increase in ICa, suggesting PKC-independent effects. Bath application of dioctanoylglycerol (diC8), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak ICa (50.4 +/- 6.2 %, n = 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 microM). Intracellular photorelease of caged diC8 with 3 or 10 s exposure to UV light produced a concentration-dependent increase in peak ICa (20. 7 +/- 8.5 % (n = 8) for 3 s UV and 60.8 +/- 11.4 % (n = 13) for 10 s UV), which could be inhibited by chelerythrine. Our results demonstrate that both ET-1 and intracellularly photoreleased diC8 increase ICa by a PKC-mediated pathway, which is in direct contrast to the PKC-independent inhibition of ICa produced by bath-applied diC8. We conclude that specific cellular pools of DAG are crucially important in the regulation of ICa by PKC.

Topics: Alkaloids; Animals; Benzophenanthridines; Cadmium; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Carcinogens; Diglycerides; Electrophysiology; Endothelin-1; Enzyme Inhibitors; Male; Membrane Potentials; Muscle Fibers, Skeletal; Myocardium; Nifedipine; Phenanthridines; Photochemistry; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Ultraviolet Rays

To evaluate the possible role of the antioxidant ebselen in the treatment of cerebral vasospasm, we examined the effects of ebselen on the vasoactive mechanisms induced by endothelin (ET)-1, oxyhemoglobin, and oxygen-derived radicals.. Isolated rabbit basilar arteries with intact endothelium were fixed in a perfusion system and perfused intraluminally. Contraction of the artery was detected as an increase in perfusion pressure.. Ebselen, in a certain concentration range (3 x 10(-6) and 10(-5) mol/L), significantly reduced the contractile response to ET-1 (10(-10) to 10(-8) mol/L) but not the contraction induced by 40 mmol/L potassium. It reduced the contraction induced by 10(-4) mol/L 1,2-dioctanoyl-sn-glycerol, a protein kinase C activator. Addition of 10(-5) mol/L dithiothreitol, a sulfhydryl-reducing agent, partially reversed the inhibitory effects of ebselen on ET-1- and 1,2-dioctanoyl-sn-glycerol-induced contractions. Ebselen (10(-5) mol/L) as well as a combination of catalase (1000 units/mL) and superoxide dismutase (150 units/mL) inhibited the potentiating effects of oxyhemoglobin (10(-5) mol/L) on ET-1-induced contraction. Both ebselen and catalase inhibited the contractile response to hydroxyl radical generated by ferrous ion (10(-3) mol/L) plus hydrogen peroxide (10(-2) mol/L). Ebselen reduced the response to potassium when a high dose (3 x 10(-5) mol/L) was applied and failed to preserve contractility of the preparation after exposure to hydroxyl radical.. Ebselen suppressed ET-1-induced contraction and synergetic interaction between oxyhemoglobin and ET-1, where free radical formation was involved. These effects may result from modification of the intracellular regulatory system including protein kinase C, as well as from protection against free radicals.

Topics: Animals; Antioxidants; Azoles; Basilar Artery; Diglycerides; Dithiothreitol; Drug Synergism; Endothelin-1; Hydroxyl Radical; In Vitro Techniques; Isoindoles; Male; Organoselenium Compounds; Osmolar Concentration; Oxidoreductases; Oxyhemoglobins; Perfusion; Potassium; Rabbits; Sulfhydryl Reagents; Vasoconstriction; Vasoconstrictor Agents