cium homeostasis in rat cardiomyocytes during chronic hypoxia: a time course study. Am J Physiol Cell Physiol 285: C1420-C1428, 2003; 10.1152/ajpcell.00534.2002.-The present study determined Ca 2ϩ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14,21,28 Recently, we showed (27) that the electrically induced [Ca 2ϩ ] i transient of cardiomyocytes from rats subjected to 28 days of CH was reduced. Furthermore, the elevation in electrically induced [Ca 2ϩ ] i transient to ␤-adrenoceptor (␤-AR) stimulation in the same cardiomyocytes was also attenuated (26). The reduction in the electrically induced [Ca 2ϩ ] i transient in CH may result from impaired Ca 2ϩ handling by both the sarcolemma and the SR. To test this hypothesis we determined the activity and expression of the SR proteins SERCA and RyR, which are involved in handling of Ca 2ϩ , and the activity of NCX in the sarcolemma of ventricular myocytes from the right heart of rats subjected to CH for from 1 day to 2 mo. The changes were correlated with changes in [Ca 2ϩ ] i transients induced by electrical stimulation and caffeine were suppressed after CH. The altered handling of Ca 2ϩ by SR and sarcolemmal membrane correlated well with the attenuated Ca 2ϩ responses to ␤-AR stimulation. Four weeks is required for full adaptation to hypoxia. MATERIALS AND METHODSAll procedures in this study were approved by the Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong.Chronic hypoxia. Male Sprague-Dawley rats that weighed 100-150 g at the start of the experiment were randomly divided into two groups. One group was exposed to CH, and the control group was maintained in room air. All rats were kept in the same room with the same light-dark cycle. For CH, rats were given inspired oxygen (10% O 2) in a 300-liter

1. We have identified a neuronal nitric oxide synthase (NOS)-like constitutive form of NOS in vascular smooth muscle (VSM) using a functional contractility approach as well as immunohistochemical methods. 2. N(G)-Nitro-L-arginine methyl ester, N(G)-monomethyl-L- arginine and N(G)-nitro-L-arginine (L-NOARG), the competitive inhibitors of NOS, inhibited Mg(2+)-induced relaxation of de-endothelialized rat aorta precontracted with phenylephrine (PE). This Mg(2+) relaxation of VSM was not affected by inhibitors of inducible NOS. 3. Electrical field stimulation (EFS; 30-70 Hz) caused relaxation of rat aorta in the presence of tetrodotoxin (therefore not a neurogenic effect) and this EFS relaxation was effectively inhibited by L-NOARG, oxyhemoglobin and methylene blue. 4. Immunohistochemical studies of dog saphenous vein using antibodies raised against neuronal NOS indicated prominent staining along the plasmalemma in a punctate pattern similar to the distribution of antibodies against caveolin-1, a major constituent of the plasmalemmal caveolae. 5. We propose that a constitutive NOS of non-endothelial, non-neuronal origin is present in a special caveolae domain of VSM cell membranes and could be activated by an ionic mechanism yet to be characterized.

1 L-N G -nitro-arginine methyl ester (L-NAME; 100 mM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 mM acetylcholine (ACh) and 2 ± 10 mM Mg 2+ in endotheliumintact (+E) rat aortic rings precontracted with 1 mM phenylephrine (PE). In PE-precontracted endothelium-denuded (7E) rat aorta, 3 mM ACh did not, but Mg 2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response pro®les of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg 2+ and 0.2 mM ACh were not signi®cantly di erent. 3 L-NAME (100 mM) also reversed Mg 2+ -relaxation of 7E aorta pre-contracted with 20 mM KCl or 10 mM prostaglandin F 2a (PGF 2a ). L-N G -monomethyl-arginine (L-NMMA; 100 mM) was also e ective in reversing the Mg 2+ -relaxation. 4 Addition of 0.2 mM Ni 2+ , like Mg 2+ , caused relaxation of PE-pre-contracted 7E aorta, which was subsequently reversed by 100 mM L-NAME. 5 Reversal of the Mg 2+ -relaxation by 100 mM L-NAME in PE-precontracted 7E aorta persisted following pre-incubation with 1 mM dexamethasone or 300 mM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or 7E aortic rings with 100 mM L-NAME caused elevation of contractile responses to Ca 2+ in the presence of 1 mM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidenti®ed vascular smooth muscle plasma membrane protein(s), thus a ecting its reactivity to divalent cations leading to the reversal of relaxation. Such an e ect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

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