This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

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A relaxation effect of sodium nitroprusside on smooth muscle cells of rat aorta due to intracellular Ca2+-store refilling is demonstrated using the double sucrose gap technique. It is suggested that sodium nitroprusside-induced repolarization of the smooth muscle cell membrane is associated with inhibition of Ca permeability and/or Ca-dependent K § permeability of the plasma membrane. Key words: sodium nitroprusside; Ca ions; smooth muscle cellsNitrate derivatives, in particular sodium nitroprusside (SN), activate cytosolic guanylate cyclase, which leads to a rise of intracellular cyclic GMP and triggers a cascade of cGMP-dependent events [4,9,10]. Activation of single potassium channels in smooth muscle cells (SMC) in the presence of cGMP and cGMPdependent protein kinases is extensively discussed in the literature [9,15]. Some authors suggest that this mechanism underlies the nitrate-induced relaxation of vascular SMC [3,7,15], although this is in controversy [8-121. It has been shown that cGMP inhibits both voltageoperated [8,14] and receptor-operated Ca 2 § channels [7]. The possible effect of cGMP on Ca 2 § utilization from the cytosol [2,6,13] and on the activity of contractile proteins in SMC [10,11] is intensively discussed. At the same time, simultaneous effects of nitrates on Ca 2 § influx through membrane ion channels and CaZ+release from SMC stores are disputable.The aim of the present work was to study membrane and intracellular mechanisms by which cyclic MATERIALS AND METHODSThe membrane potential and muscle tension in SMC were simultaneously measured using the double sucrose gap method [1]. Electrical potentials were recorded via nonpolarizable electrodes using a C1-83 oscilloscope coupled to a KSP-4 recorder. Contractile activity was recorded on a 6MKhlB mechanotron in a nearly isometric regimen.Male random-bred albino rats (200-250 g) were sacrificed and endothelium-denuded smooth muscle strips (0.6-0.7 mm wide, 10-12 mm long) were isolated from the middle portion of the thoracic aorta.Isolated smooth muscle strips were kept in Krebs solution (36.8~ pH 7.35) containing (in mM): 120.4 NaC1, 5.6 KCI, 15.5 NaHCO3. 1.2 MgCI 2, 1.2 NaH2PO 4, 2.5 CaC12, and 11.5 glucose for 40-45 min, and then treated with nigh-potassium (40 mM KC1) Krebs solution (Fig. 1). Test solutions were prepared on the basis of Krebs saline containing verapamil, SN (Serva), and caffeine; in the Ca-free solutions CaCI: was replaced with EGTA.

The involvement of cAMP- and cGMP-dependent signal systems into the regulation of the contractile reaction of smooth muscles of the rabbit pulmonary arteries was studied using a mechanographic method. For modulation of intracellular level of cyclic nucleotides we used adenylate cyclase activators beta-adrenoceptor agonist isadrin, guanylate cyclase inhibitor methylene blue, penetrating analog dibutyryl-cAMP, and phosphodiesterase inhibitors. The mechanisms of cAMP- and cGMP-dependent regulation of smooth muscle contractile activity were realized in close interrelationship, and the key component of this was cyclic nucleotide phosphodiesterases. The ratio of activities of phosphodiesterase subtypes in smooth-muscle cells can essentially modulate the adrenergic effects in the pulmonary artery wall and even invert them.

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