correolide and iberiotoxin

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E(M)), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (I(K)) and a more hyperpolarized E(M) and were uniquely O2- and correolide-sensitive. The O2-sensitive current (active at -65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I(K) 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I(K) (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2- and correolide-sensitive I(K) like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

Topics: 4-Aminopyridine; Acetylcholine; Animals; Cell Hypoxia; Cells, Cultured; Gene Expression Regulation; Humans; Hypoxia; Ion Channel Gating; Ion Transport; Kv1.5 Potassium Channel; Male; Membrane Potentials; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oxygen; Patch-Clamp Techniques; Peptides; Potassium; Potassium Channels, Voltage-Gated; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Scorpion Venoms; Shab Potassium Channels; Transduction, Genetic; Triterpenes; Vascular Resistance; Vasoconstriction

The role of voltage-dependent (I(K(v))) and large conductance Ca(2+)-activated (BK(Ca)) K(+) currents in the function of the rat aorta was investigated using specific BK(Ca) and K(V) channel inhibitors in single rat aortic myocytes (RAMs) with patch-clamp technique and in endothelium-denuded aortic rings with isometric tension measurements. The whole-cell K(+) currents were recorded in RAMs dialysed with 200 and 444 nm Ca(2+) and in perforated-patch configuration. Electrophysiological analysis demonstrated that I(K(v)) appeared at >/=-40 mV, while BK(Ca) (isolated using 1 microm paxilline) were seen positive to -20 mV in all conditions. Voltage-dependent characteristics, but not maximal conductance, of I(K(v)) was significantly altered in increased [Ca(2+)](i). Correolide (1 microm) (a K(V)1 channel blocker) did not inhibit the I(K(v)), whereas millimolar concentration of TEA (IC(50)=3.1+/-0.6 mm, n=5) and 4-aminopyridine (4-AP, IC(50)=5.9+/-1.9 mm, n=7) suppressed I(K(v)). These results and immunocytochemical analysis suggest the K(V)2.1 channel to be a molecular correlate for I(K(v)). In nonstimulated aortic rings 1-5 mm TEA and 4-AP (inhibitors of I(K(v))), but not paxilline (1 microm), caused contraction. The frequency of contractile responses to TEA and 4-AP was increased in the presence of 10 mm KCl, which itself did not significantly affect the aortic basal tone. Phenylephrine (15-40 nm) induced sustained tension with superimposed slow oscillatory contractions (termed OWs). OWs were blocked by diltiazem, ryanodine and cyclopiazonic acid, suggesting the involvement of L-type Ca(2+) channels and ryanodine-sensitive Ca(2+) stores in this process. TEA and 4-AP, but not IbTX, paxilline or correolide, increased the duration and amplitude of OWs, indicating that I(K(v)) is involved in the control of oscillatory activity. In conclusion, our findings suggest that the K(V)2.1-mediated I(K(v)), and not BK(Ca), plays an important role in the regulation of the excitability and contractility of rat aorta.

Topics: Animals; Aorta, Thoracic; Calcium Signaling; Electrophysiology; Immunochemistry; Indoles; Male; Membrane Potentials; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Peptides; Potassium Channels, Calcium-Activated; Potassium Channels, Voltage-Gated; Protein Isoforms; Rats; Rats, Wistar; Tetraethylammonium; Triterpenes; United Kingdom; Vasoconstriction