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.
High-ceiling diuretics (HCD), known potent inhibitors of housekeeping Na(+),K(+),2Cl cotransporter (NKCC1) and renal-specific NKCC2, decrease [Cl(-)](i), hyperpolarize vascular smooth muscle cells (VSMC), and suppress contractions evoked by modest depolarization, phenylephrine, angiotensin II, and UTP. These actions are absent in nkcc1 (/) knock-out mice, indicating that HCD interact with NKCC1 rather than with other potential targets. These findings also suggest that VSMC-specific inhibitors of NKCC1 may be considered potential pharmacological therapeutic tools in treatment of hypertension. It should be underlined that side by side with attenuation of peripheral resistance and systemic blood pressure, HCD blocked myogenic tone (MT) in renal afferent arterioles. Keeping this in mind, attenuation of MT might be a mechanism underlying the prevalence of end-stage renal disease documented in hypertensive African-Americans with decreased NKCC1 activity and in hypertensive patients subjected to chronic HCD treatment. The role of NKCC1-mediated MT in protection of the brain, heart, and other encapsulated organs deserves further investigation.
The article contains the current understanding of gas communication in smooth muscle cells, the basic mechanisms of action of gaseous transmitters, analyzes the different views on the nature of the endothelial relaxing and endothelial hyperpolarizing factors. We discuss the controversial issues of the mechanisms of endothelium-dependent relaxation of vascular smooth muscle.
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.
Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca2+ channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca2+-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca2+ channel-mediated contraction of swollen vascular SMC and Ca2+o-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.
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