K(V)2.1 channels mediate hypoxic inhibition of I(KV) in native pulmonary arterial smooth muscle cells of the rat.

OBJECTIVE: To determine whether, in native pulmonary arterial smooth muscle cells (PASMC), K(V)2.1 delayed-rectifying K(+) channels are central to the process of hypoxic pulmonary vasoconstriction. METHODS: In this study, we tested for the presence of K(V)2.1 channel transcripts in rat small pulmonary arteries using RT-PCR, and for the protein itself using immunolocalisation. The contribution of K(V)2.1 channels to whole-cell K(V) currents (I(KV)) and their role in hypoxic inhibition of I(KV) in native PASMC was investigated utilising patch-clamp recordings. RESULTS: K(V)2.1 mRNA expression and AbK(V)2.1 (anti-K(V)2.1 antibody) protein immunoreactivity were both present in small pulmonary arteries. Dialysis of PASMC with AbK(V)2.1 significantly attenuated I(KV) by 67% at +50 mV. Hypoxia ( approximately 20-30 mmHg) inhibited I(KV) by approximately 70% at +50 mV. Ablation of currents associated with K(V)2.1 using AbK(V)2.1 caused a marked reduction in the amplitude of I(KV). Hypoxia in the presence of the antibody did not affect the magnitude of I(KV). CONCLUSIONS: These results indicate that K(V)2.1 channel subunits exist within small pulmonary arteries and conduct a significant part of I(KV) within native PASMC. Furthermore, application of AbK(V)2.1 abolishes hypoxic inhibition of I(KV) in native PASMC suggesting that K(V)2.1 channels play a pivotal role in mediating hypoxic pulmonary vasoconstriction.

Hypoxia activates a background conductance in freshly isolated pulmonary arterial endothelial cells of the rat.

Utilising the patch-clamp recording technique we have demonstrated for the first time the effects of hypoxia on the background current in pulmonary arterial endothelial cells. Electrophysiological studies revealed the presence of a novel oxygen-sensitive, non-selective cation conductance (I(NSC)) in these cells. The inward component of I(NSC) was significantly potentiated by hypoxia. Both the inward and outward components of I(NSC) were inhibited by both La(3+) and Gd(3+). Hypoxic activation of I(NSC) may provide an important Ca(2+) influx pathway essential for the release of a pulmonary-selective vasoconstrictor pivotal to the sustained phase of hypoxic pulmonary vasoconstriction.

Actions of the anti-oestrogen agent clomiphene on outward K+ currents in rat ventricular myocytes.

1. The effects of clomiphene (CLM) on cardiac outward K+ current components from rat isolated ventricular myocytes were investigated using the whole-cell patch-clamp technique. Clomiphene (10 micromol/L) significantly inhibited both peak (Ipeak) and end-pulse (Ilate) outward currents (elicited by a 500 msec voltage step from -40 to +50 mV in the presence of K+-containing intracellular and extracellular solutions) by approximately 37% (n = 6; P < 0.01) and 49% (n = 6; P < 0.01), respectively. In contrast, CLM had no effect on outward currents when K+-free solutions were used. 2. A double-pulse protocol and Boltzmann fitting were used to separate individual K+ current components on the basis of their voltage-dependent inactivation properties. At potentials positive to -80 mV, two inactivating transient outward components (Ito) and (IKx) and a non-inactivating steady state component (Iss) could be distinguished. 3. Clomiphene inhibited both Ito and Iss. The maximal block of Ito and Iss induced by CLM (100 micromol/L) was approximately 61% (n = 5) and 43% (n = 5) with IC50 values of 1.54 +/- 0.39 and 2.2 +/- 0.4 micromol/L, respectively. In contrast, the peak magnitude of IKx was unaltered by CLM, although its time-course of inactivation was accelerated. 4. Further experiments whereby myocytes were superfused with the vasoactive peptide endothelin (ET)-1 (20 nmol/L) revealed that CLM (10 micro mol/L) completely abolished the ET-1-sensitive component of Iss. 5. Our findings demonstrate, for the first time, the effects of CLM on distinct cardiac K+ current components and show that CLM modulates the voltage-gated K+ current components Ito and IKx and inhibits the steady state outward current Iss in rat ventricular myocytes.