Deoxyglucose and reduced glutathione mimic effects of hypoxia on K+ and Ca2+ conductances in pulmonary artery cells

Abstract

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.

Relationship between membrane potential, delayed rectifier K+ currents and hypoxia in rat pulmonary arterial myocytes

“…However, no previous isolated cell studies have shown that a 'priming' depolarization is a prerequisite for subsequent depolarization in response to hypoxia. In some studies (for example, Yuan et al 1993Yuan et al , 1994) the membrane potential in normoxic conditions was more depolarized than that observed in our study, thereby (perhaps) rendering cells sensitive to hypoxia. This difference, and the possibility that cells from different species may respond to hypoxia in a subtly different manner, could explain the discrepancy between our results and those of others.…”

“…In our studies hypoxia was defined as an oxygen tension of between 20 and 30 mmHg. Others have on occasion used a greater degree of hypoxia (8 mmHg; Yuan et al 1994) or added dithionite (e.g. Salvaterra & Goldman, 1993;Yuan et al 1993) to scavenge and reduce PO2 levels in solution in order to obtain depolarization (note that dithionite causes a reduction of 02 with a concomitant production of superoxide anion, which itself has been reported to inhibit vascular reactivity in response to hypoxia (Archer, Hampl, Nelson, Sidney, Peterson & Weir, 1995).…”

“…However, the actual mechanism underlying hypoxia-induced inhibition of Kv channels is not fully understood and several mechanisms could be responsible for this effect. These include changes in the metabolic status of cells (Yuan et al 1994), decreases in intracellular pH (Krampetz & Rhoades, 1991) and changes in the redox status of cells …”

Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms

“…The concept of K v channels being directly regulated by redox-changes has been proposed by POST et al [39]. The K v channels may be regulated by: 1) reducing agents [45][46][47][48], such as glutathione (GSH) [49]; 2) an associated haemoprotein; 3) membrane depolarisation; or 4) a calcium increase triggered by intracellular calcium release from intracellular stores or via NSCCs. Thus NSCCs may be key channels for hypoxic signalling, for example, in the regulation of potassium channels, of L-type calcium channels or the calcium level.…”

Patho-, physiological roles of voltage-dependent K+ channels in pulmonary arterial smooth muscle cells

“…Others have alternatively shown that ROS may increase during hypoxia (Waypa et al, 2006). Inhibition of Kv currents by mETC inhibitors, particularly those acting at complexes I and III, supports this concept; at the same time, mitochondrial uncouplers, which decrease mitochondrial membrane potential (ΔΨm) (Yuan et al, 1996), and metabolic inhibition of PASMCs with 2-deoxyglucose (Yuan et al, 1994) also inhibit Kv currents, suggesting the presence of multiple, presently undetermined, mitochondria-dependent mechanisms that affect Kv currents in PASMCs. Data supporting the mitochondria as an O2 sensor in PASMCs were recently published and demonstrated a mitochondrial-dependent regulation of Kv channels in PASMC, a mechanism that was absent in MASMCs and involves increased cellular Mg 2+ (Firth et al, 2008;Firth et al, 2009).…”