Ca2+ buffering action of sarcoplasmic reticulum on Bay k 8644-induced Ca2+ influx in rat femoral arterial smooth muscle

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Abstract

We examined the Ca2+ buffering action of sarcoplasmic reticulum during the stimulation of arterial smooth muscle with Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate]. The effects of Bay k 8644 on tension and cellular Ca2+ level were first determined in endothelium-denuded strips of rat femoral artery. The Ca2+ buffering action was examined by using cyclopiazonic acid and thapsigargin to inhibit Ca2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca2+ stored in sarcoplasmic reticulum. The addition of Bay k 8644 (0.3–300 nM) to the resting strips almost failed to cause a contraction. When the strips were preincubated with 10 μM cyclopiazonic acid, Bay k 8644 induced a concentration-dependent contraction that is antagonized by nifedipine. The maximum contraction induced by Bay k 8644 in the presence of cyclopiazonic acid was comparable to the maximum contraction induced by 65.9 mM K+-depolarization and the ED50 value for Bay k 8644 was around 5 nM. Similar results were obtained when the strips were preincubated with 30 nM thapsigargin or 10 μM ryanodine. Bay k 8644 also induced a strong contraction when the extracellular K+ concentration was elevated. During the stimulation with 100 nM Bay k 8644, the Ca2+ influx was increased. We conclude that in rat femoral arterial smooth muscle, (1) the Ca2+ influx induced by Bay k 8644 is completely buffered by Ca2+ uptake into the sarcoplasmic reticulum, and (2) this sarcoplasmic reticulum can buffer a large amount of Ca2+ that induces a maximum contraction.

Introduction

L-type Ca2+ channels are heteromultimetric proteins that play important roles in the cardiovascular system (Triggle, 1990; McDonald et al., 1994; Hockerman et al., 1997). These channels are activated at relatively high voltages, conduct large, relatively long-lasting currents and are sensitive to dihydropyridines. The dihydropyridines act on the specific receptors of L-type Ca2+ channels as either antagonists favoring the inactivated states or agonists favoring the open state. Nifedipine is a typical dihydropyridine receptor antagonist that inhibits Ca2+ influx via L-type Ca2+ channels and thus used as a valuable tool to investigate the function of these channels in a variety of tissues. Due to the potent vasodilator properties, nifedipine and related dihydropyridines have already gained wide use in the treatment of hypertension. Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate] is a typical dihydropyridine receptor agonist that increases the Ca2+ influx through the increase in the open probability of the channel. This agonist is therefore expected to possess potent vasoconstrictor properties that are antagonized by nifedipine. However, the first report by Schramm et al. (1983)showed that Bay k 8644 did not induce a contraction in rabbit aortic strips unless the K+ concentration in the bathing solution was increased to 15 mM. A need of such preactivation has been confirmed later in many studies, while some studies clearly showed the Bay k 8644-induced strong contraction without the preactivation (for details, see Asano et al., 1987; Wanstall and O'Donnell, 1989). Thus, the inability of Bay k 8644 to induce a contraction may indicate two opposite possibilities: (1) Ca2+ influx induced by Bay k 8644 is small, or (2) the influx is large but the entered Ca2+ is completely buffered by Ca2+ uptake into the sarcoplasmic reticulum.

By measuring the 45Ca2+ influx, Hwang and Van Breemen (1985)noted a marked disparity between Bay k 8644-induced tension and net Ca2+ content; under the resting condition (5 mM K+), Bay k 8644 did not induce tension but did induce net 45Ca2+ gain in the caffeine-releasable sarcoplasmic reticulum. These authors concluded that the superficial sarcoplasmic reticulum accumulates Ca2+ entering through the L-type Ca2+ channels before it reaches the myofilaments. To examine the Ca2+ buffering action of sarcoplasmic reticulum, several valuable tools are now available; cyclopiazonic acid and thapsigargin to inhibit Ca2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca2+ stored in sarcoplasmic reticulum. However, such examinations have never been evaluated. We therefore determined the Ca2+ buffering action of sarcoplasmic reticulum during the stimulation with Bay k 8644 in rat femoral artery. The prediction is that if sarcoplasmic reticulum of the artery is a buffer barrier to the Ca2+ influx induced by Bay k 8644, the functional elimination of sarcoplasmic reticulum Ca2+ buffering by each of the agents will augment the contractions to Bay k 8644. Here, we show that under the inhibition of sarcoplasmic reticulum Ca2+ buffering, Bay k 8644 induced a strong contraction that was comparable to the maximum contraction induced by K+-depolarization. A preliminary account of these findings was presented to the 71st Annual Meeting of the Japanese Pharmacological Society (Nomura and Asano, 1998).

Section snippets

Preparation of arterial smooth muscle strips and measurement of isometric tension

Male Wistar–Kyoto rats at 12 to 14 weeks of age (270–315 g) were used. They were inbred in our laboratory. Rats were decapitated, and femoral arteries (0.6–0.8 mm o.d.) were excised and placed in a Krebs solution of the following composition (in mM): NaCl 115.0, KCl 4.7, CaCl2 2.5, MgCl2 1.2, NaHCO3 25.0, KH2PO4 1.2 and dextrose 10.0. Arteries were cut into helical strips (0.7–0.8 mm in width) as described previously (Asano et al., 1988; Nomura et al., 1996). To avoid the possible influences of

Effects of cyclopiazonic acid and thapsigargin on the responses to Bay k 8644

The addition of Bay k 8644 (0.3–300 nM) to the resting state of the strips almost failed to cause a contraction (Fig. 1A). Even at a high concentration (300 nM), Bay k 8644 induced a contraction of 2.8±0.5% (n=11) of the maximum contraction induced by 65.9 mM K+ (Table 1A). To examine the Ca2+ buffering action of sarcoplasmic reticulum during the responses to Bay k 8644, effects of cyclopiazonic acid on these responses were first determined (Fig. 1B, Fig. 2A, Table 1B). The preincubation of the

Discussion

The present study clearly showed that in rat femoral artery, Bay k 8644 itself almost failed to induce a contraction, but the inhibition of sarcoplasmic reticulum Ca2+ buffering by cyclopiazonic acid, thapsigargin or ryanodine markedly augmented the contractions to Bay k 8644. We consider that these responses to Bay k 8644 can be explained by the `superficial buffer barrier' hypothesis proposed by Van Breemen and his colleagues (Van Breemen and Saida, 1989; Chen et al., 1992; Van Breemen et

Acknowledgements

We thank Drs. Yuji Imaizumi and Minoru Watanabe (Faculty of Pharmaceutical Sciences, Nagoya City University) and Dr. Katsuaki Ito (Faculty of Agriculture, Miyazaki University) for helpful comments and discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. Generous gifts of Bay k 8644 (Bayer) and nifedipine (Bayer Yakuhin) are gratefully acknowledged.

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