Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.

The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action

The effects of thiamylal on contractions induced by various mechanisms were investigated in mesenteric arteries isolated from dogs. Thiamylal (10−4 to 10−3 M) significantly inhibited contractions induced by KCl (20 mM) in normal media, and those induced by norepinephrine (10−5 M) in normal and Ca2+-free media. Caffeine-induced contraction was significantly inhibited by thiamylal in the concentrations greater than 3 × 10−5 M in intact fibers and 10−5 M in chemically skinned fibers. Chemically skinned fibers that were precontracted with Ca2+ were relaxed by thiamylal in concentrations lower than those required to relax intact fibers that were precontracted with KCl (20 mM); the ED50 was 1.52 × 10−5 M in skinned fibers and 5.50 × 10−4 M in intact fibers. These results suggest that intracellular mechanisms are involved in thiamylal-induced vasodilatation of dog mesenteric artery.Key words: barbiturates, thiamylal, mesenteric artery, vascular smooth muscle.

Na+-Ca2+ exchange in sarcolemmal membrane vesicles of dog mesenteric artery

The kinetic characteristics of a Na+-Ca2+ exchange system in the cell membrane of vascular smooth muscle were explored in vitro in isolated sarcolemmal membrane vesicles of dog mesenteric artery. Na+-loaded vesicles rapidly accumulated Ca2+ when an outwardly directed Na+ concentration gradient was created by suspension of the vesicles in a Na+-free medium. This Ca2+ uptake process was reversible depending on the direction and the magnitude of the Na+ concentration gradient across the membrane of the vesicles. Low temperature, monensin, and external Na+ drastically decreased Ca2+ uptake in Na+-loaded vesicles. Monovalent cations K+, Rb+, Li+, and Cs+ did not substitute for Na+ in the exchange process. Divalent cations Ba2+, Cd2+, Mg2+, Mn2+, and Sr2+ inhibited Ca2+ uptake in Na+-loaded vesicles. The order of potency of these divalent cations and concentration which produced 50% inhibition (IC50, microM) were Cd2+(38) greater than Sr2+(110) greater than Ba2+(405) greater than Mn2+(500) greater than Mg2+(greater than 2,500). The trivalent cation La3+ also inhibited Ca2+ uptake (IC50 = 0.175 microM). The apparent Km for free Ca2+ in vesicles loaded with 150 mM NaCl was 2.64 +/- 0.5 microM, and the apparent maximum velocity was 14.8 +/- 1.9 nmol.min-1.mg protein-1. The half of the apparent maximum rate (K0.5) of Ca2+ uptake was observed at 45.5 mM Na+ when loaded internally in the vesicles. Valinomycin in the presence of K+ increased the magnitude of Ca2+ uptake by 16% in Na+-loaded vesicles, indicating that the process may be electrogenic. These data indicate the existence and operation of a specific carrier-mediated Na+-Ca2+ exchange system in sarcolemmal membrane vesicles isolated from a small blood vessel.