To explore possible mechanisms underlying hypoxia-induced pulmonary vasoconstriction, the effect of hypoxia on outward K+ current (Iout) was evaluated in primary cultured rat pulmonary (PA) and mesenteric (MA) arterial smooth muscle cells using the whole cell patch-clamp technique. When the cells were bathed in standard physiological salt solution and the patch pipettes contained Ca(2+)-free media with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), virtually all of the Iout, including both the rapidly inactivating component (Irt) and the steady-state (noninactivating) component (Iss), was mediated by voltage-gated K+ channels. Reduction of O2 tension in the bath solution from 155 Torr to < 74 Torr with sodium dithionite reversibly inhibited both Irt and Iss in PA myocytes, but not in MA myocytes. The hypoxia-sensitive Iss was activated at about -50 mV; thus, some of the channels responsible for this current may be open at the resting membrane potential (-40 +/- 1 mV) of PA cells used in this study. Hypoxia also significantly depolarized PA cells bathed in PSS (1.8 mM Ca2+) from -40.7 +/- 1.3 to -24.0 +/- 2.4 mV, and PA cells bathed in Ca(2+)-free PSS (0.1 mM EGTA) from -38.4 +/- 1.3 to -26.1 +/- 3.9 mV. The hypoxia-induced inhibition of Iout in PA cells was accompanied by an apparent increase in inward Ca2+ current.(ABSTRACT TRUNCATED AT 250 WORDS)
The membrane potential (Em) of pulmonary arterial smooth muscle cells (PASMCs) regulates pulmonary arterial tone by controlling voltage-gated Ca2+ channel activity, which is a major contributor to [Ca2+]i. The resting membrane is mainly permeable to K+; thus, the resting Em is controlled by K+ permeability through sarcolemmal K+ channels. At least three K+ currents, voltage-gated K+ (KV) currents, Ca(2+)-activated K+ (KCa) currents, and ATP-sensitive (KATP) currents, have been identified in PASMCs. In this study, both patch-clamp and quantitative fluorescent microscopy techniques were used to determine which kind(s) of K+ channels (KV, KCa, and/or KATP) is responsible for controlling Em and [Ca2+]i under resting conditions in rat PASMCs. When the bath solution contained 1.8 mmol/L Ca2+ and the pipette solution included 0.1 mmol/L EGTA, depolarizations (-40 to +80 mV) elicited both KCa and KV currents. Removal of extracellular Ca2+ and increase of intracellular EGTA concentration (to 10 mmol/L) eliminated the Ca2+ influx-dependent KCa current. 4-Aminopyridine (4-AP, 5 to 10 mmol/L) but not charybdotoxin (ChTX, 10 to 20 nmol/L) significantly reduced KV current under these conditions. In current-clamp experiments, 4-AP decreased Em (depolarization) and induced Ca(2+)-dependent action potentials; this depolarization increased [Ca2+]i in intact PASMCs. Neither ChTX nor the specific blocker of KATP channels, glibenclamide (2 to 10 mumol/L), caused membrane depolarization and the increase in [Ca2+]i. However, pretreatment of PASMCs with ChTX enhanced the 4-AP-induced increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of hypoxia on resting and K-stimulated tension were tested on small rings of rat pulmonary and mesenteric resistance arteries (SPA and SMA, respectively) and on the large branches of the main pulmonary artery (LPA). Reduction of PO2 from approximately 135 Torr to less than 40 Torr slowly increased SPA and LPA resting tension but did not affect SMA tension. The increases in SPA and LPA tension during hypoxia were reversible and were dependent on external Ca2+. Verapamil, 10(-6) M, inhibited the hypoxic pulmonary vasoconstriction by 53-78%. The hypoxia-contracted SPA and LPA were relaxed by 2-4 microM cromakalim; these relaxations were reversed by 2 microM glibenclamide. Hypoxia attenuated the K-stimulated tension (delta TK) in both SPA and SMA at all external K+ concentrations ([K+]o = 10-100 mM) without affecting the shapes of the respective [K+]o-tension curves. However, the SPA curve was located much farther to the left on the [K+]o axis than the SMA curve. [K+]o congruent to 13 mM evoked a half-maximal increase in SPA tension; maximal delta TK was observed at [K+]o greater than or equal to 30 mM. In contrast, [K+]o less than 20 mM induced a negligible increase in SMA tension, whereas 35-40 mM K+ activated about one-half of the increase in tension elicited by 100 mM K+. The LPA [K+]o-tension curve in normoxia was intermediate between the SMA and SPA curves, but hypoxia shifted the LPA curve to the left: delta TK was augmented at [K+]o less than 20 mM and attenuated at high [K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypoxia-induced pulmonary vasoconstriction (HPV) is triggered by a rise in cytosolic Ca2+ concentration ([Ca2+]i) that is partially controlled by voltage-gated Ca2+ channels. Hypoxia inhibits voltage-gated K+ (KV) channels in pulmonary artery (PA) myocytes. This depolarizes the cells, opens voltage-gated Ca2+ channels, thereby increases [Ca2+]i, and initiates HPV. In intact animals and isolated perfused lungs, metabolic inhibitors and reducing agents augment HPV. We compared the effects of hypoxia with the glycolysis inhibitor, 2-deoxy-D-glucose (2-DOG), and the reducing agent, reduced glutathione (GSH), on voltage-gated steady-state K+ currents (IK,ss) and membrane potential (Em) in cultured rat pulmonary and mesenteric arterial (MA) smooth muscle cells. Bath application of 10 mM 2-DOG (glucose-free) or 5-10 mM GSH reversibly reduced IK,ss by 25-35% in PA myocytes, with 5 mM ATP present in the pipette solution. Neither hypoxia nor 2-DOG significantly affected IK,ss in MA myocytes, but GSH did reduce IK,ss in these cells. Furthermore, hypoxia, 2-DOG, and GSH depolarized PA cells in the absence as well as in the presence of external Ca2+. Hypoxia, 2-DOG, and GSH also evoked action potentials on the top of the steady depolarization in 36-50% of PA myocytes but not in any MA myocytes; removal of external Ca2+ abolished the action potentials without affecting the steady depolarization. These effects were comparable to those produced by 4-aminopyridine (5-10 mM), a blocker of KV channels. This implies that the action potentials are attributable to Ca2+ influx through voltage-gated Ca2+ channels opened by the steady depolarization due to KV channel inhibition. In the presence of 2-DOG or GSH, hypoxia had no further effect on IK,ss or Em in PA cells; this implies that hypoxia, 2-DOG, and GSH all block the same K+ channels. The data suggest that 1) the hypoxia-induced decrease of IK,ss and the resultant depolarization in PA myocytes may be related to a local decrease of intracellular ATP level and/or a change in redox status of the membrane or cytosol and 2) extracellular Ca(2+)-dependent action potentials may be responsible for at least part of the increase in [Ca2+]i during HPV. Similarities between the effects of hypoxia, 2-DOG, and GSH on IK,ss and Em in PA myocytes, along with the dissimilar responses of PA and MA myocytes, suggest that a common mechanism may underlie the responses of PA cells to these treatments.
The electrophysiological properties of cultured single vascular smooth muscle (VSM) cells from rat pulmonary (PA) and mesenteric (MA) arteries were studied using the whole cell patch-clamp technique. Cells were studied at 3-7 days as primary cultures, or were replated after 10-20 days and subcultured for 2-5 days. In the standard physiological bath solution (containing 1.8 mM Ca2+), and with 125 mM K+ + 10 mM ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA)-filled pipettes, both PA and MA primary cultured cells had high input resistances (mean = 2-3 G omega) and resting membrane potentials of about -40 mV. The cells were clamped at a holding potential of -70 mV. Depolarization to -20 mV or more evoked a transient inward current (Iin) that was eliminated in Ca(2+)-free bath solution; this indicates that Iin was carried by Ca2+. Iin was substantially smaller in subcultured cells from both PA and MA. Depolarization also activated three components of outward current (Iout) in primary cultured PA and MA cells: a rapidly inactivating transient component (Irt), a slowly inactivating transient component (Ist), and a steady-state (noninactivating) component (Iss). All three components of Iout were inhibited to varying degrees by 5 mM 4-aminopyridine and were eliminated by replacing intracellular K+ with Cs+, but were only minimally affected by removal of extracellular Ca2+. These results suggest that this Iout was carried by K+ and was voltage gated. Little external Ca(2+)-dependent Iout was observed under these conditions, but a substantial Ca(2+)-dependent component was seen when the EGTA concentration in the pipettes was reduced to 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
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