Vasoreactions to acute hypoxia, whole lungs and isolated vessels compared: modulation by NO.

We aimed to explain diverse pulmonary vascular responses to hypoxia in different preparations and their modulation by NO. In rats we compared isolated perfused lungs (IPL), small vessels in vitro (PRVs) and in vivo preparations. In IPL and in vivo, acute and chronic nitric oxide synthase (NOS) blockade with L-NAME left normoxic pulmonary artery pressure unchanged but enhanced hypoxic vasoconstriction, hypoxia-induced pulmonary vasoconstriction (HPV). PRVs in vitro, precontracted with PGF(2alpha), showed four tension changes in acute hypoxia: dilatation, contraction, dilatation, contraction. Acute and chronic NOS blockade reduced the first two phases. In non-precontracted PRVs (from other laboratories), NOS inhibition enhanced HPV as in vivo and IPL; attenuation of HPV seems associated with precontraction. Thus reduced NOS activity does not cause pulmonary hypertension but exaggerates HPV. In IPL, prolonged severe hypoxia caused biphasic vasoconstriction separated by dilatation; the time course resembled that seen in PRVs. We suggest that the sequence of events during hypoxia in PRVs can be detected in whole lung preparations.

Novel gain-of-function mechanism in K(+) channel-related long-QT syndrome: altered gating and selectivity in the HERG1 N629D mutant.

The N629D mutation, adjacent to the GFG signature sequence of the HERG1 A K(+) channel, causes long-QT syndrome (LQTS). Expression of N629D in Xenopus oocytes produces a rapidly activating, noninactivating current. N629D is nonselective among monovalent cations; permeation of K(+) was similar to that of Na(+) or Cs(+). During repolarization to potentials between -30 and -70 mV, N629D manifested an inward tail current, which was abolished by replacement of extracellular Na(+) (Na(+)(e)) with extracellular N-methyl-D-glucamine (NMG(e)). Because LQTS occurs in heterozygous patients, we coexpressed N629D and wild type (WT) at equimolar concentrations. Heteromultimer formation was demonstrated by analyzing the response to 0 [K(+)](e). The outward time-dependent current was nearly eliminated for WT at 0 [K(+)](e), whereas no reduction was observed for homomultimeric N629D or for the equimolar coexpressed current. To assess physiological significance, dofetilide-sensitive currents were recorded during application of simulated action potential clamps. During phase 3 repolarization, WT manifested outward currents, whereas homomultimeric N629D manifested inward depolarizing currents. During coexpression studies, variable phenotypes were observed ranging from a reduction in outward repolarizing current to net inward depolarizing current during phase 3. In summary, N629D replaces the WT outward repolarizing tail current with an inward depolarizing sodium current, which is expected to delay later stages of repolarization and contribute to arrhythmogenesis. Thus, the consequences of N629D resemble the pathophysiology seen in LQT3 Na(+) channel mutations and may be considered the first LQTS K(+) channel mutation that exhibits gain of function.

Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: involvement of S6 sites.

This study reports that the affinity of HERG1 A for dofetilide is decreased from 0.125 +/- 0.003 microM for wild-type (WT) channels to 15 +/- 3 microM for F656V, a mutation in the COOH-terminal half of the S6. Similarly, the IC(50) for quinidine was increased from 8 +/- 4 microM for WT to 219 +/- 65 microM for the F656V mutation, whereas affinity for external tetraethylammonium was similar for WT (51 +/- 10 mM) and F656V (36 +/- 10 mM, NS). Kinetics of onset of inactivation of F656V was similar to WT but kinetics of deactivation, activation, and recovery from inactivation differed from WT. However, mutations in nearby amino acids in the S6 more strikingly altered deactivation, activation, and recovery from inactivation but had little effect on affinity for dofetilide. To assess the effects of disruption of inactivation, the S631A mutation was made. The S631A mutation altered the IC(50) for dofetilide to 20 +/- 3 microM, but the IC(50) for quinidine was unchanged at 8 +/- 4 microM for WT and 10 +/- 1 microM for S631A. To address whether the F656V mutation alters the IC(50) for dofetilide in a channel that does not inactivate, the double mutation S631A/F656V was made. The IC(50) for dofetilide of the double mutation was 32 +/- 3 microM, which is not substantially different than that of S631A. These data support the notion that allosteric changes occurring during the process of inactivation are necessary for high-affinity dofetilide binding. In conclusion, the Phe-656 residue of HERG is a molecular determinant of high-affinity dofetilide binding.

Effects of maturation, artery size, and chronic hypoxia on 5-HT receptor type in ovine cranial arteries.

To test the hypothesis that variations in cerebrovascular reactivity to 5-HT among arteries of different size or type, during maturation, or during acclimatization to high altitude involve differences in serotonergic receptor subtype, we determined relative agonist potency orders and antagonist affinities in common carotid (Com), main branch middle cerebral (Main), and second branch middle cerebral (2BR) arteries from term fetal lambs and nonpregnant adult sheep acclimatized at sea level or at an altitude of 3,820 m for approximately 110 days. In normoxic adult Com segments, agonist potency order was 5-hydroxytryptamine (5-HT) > 5-carboxamidotryptamine (5-CT) >/= 8-hydroxy-2(di-n-propylamino)tetraline (8-OH-DPAT); sumatriptan (Suma) produced no contractile response; and antagonist dissociation constant (pKb) values were 9.4 and 9.5 for ketanserin against 5-HT and 5-CT, 7.5 for GR-127935 against 5-HT, and 7.2 for SB-206553 against 5-HT. In normoxic adult Main segments, agonist potency order was 5-HT > 5-CT >/= Suma >/= DPAT, and pKb values were 9.1 and 9.2 for ketanserin against 5-HT and 5-CT and 7.4 and 8.5 for GR-127935 against 5-HT and Suma, respectively. In the 2BR segments from normoxic adults, agonist potency order was 5-CT > 5-HT > Suma > DPAT and pKb values were 7.4 and 7.2 for ketanserin against 5-HT and 5-CT and 10.0 and 8.7 for GR-127935 against 5-HT and Suma, respectively. Compared with normoxic adults, none of these values were significantly different in hypoxic adults and in fetuses only the pKb values for ketanserin against 5-HT in the 2BR segments (8.8) were greater. From these results we propose that the ratio of 5-HT2 to 5-HT1 receptors is greatest in the Com and decreases progressively to its smallest values in 2BR or smaller segments. Because this gradient appears stable and relatively resistant to the effects of maturation and chronic hypoxia, changes in reactivity associated with these perturbations may involve alterations in receptor density and/or coupling efficiency for 5-HT in ovine cranial arteries.

In vitro responses of lung arteries to acute hypoxia after NO synthase blockade or chronic hypoxia.

Responses to hypoxia of lung arteries (200-350 microns) from control (C) and chronically hypoxic (CH) rats were compared in a myograph before and after blockade of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME). After precontraction with prostaglandin F2 alpha (PGF2 alpha), hypoxia caused a four-phase tension change: brief dilation, transient contraction, prolonged dilation, and slow contraction (we studied the first three phases). In CH rats, the first dilation and first contraction were significantly reduced. After L-NAME, the first dilation was reduced in C rats and abolished in CH rats; thus the first phase is attributable to NO release and is affected by chronic hypoxia. The first contractile phase was significantly reduced by L-NAME in C but not in CH rats, where it was small. Thus NO synthase inhibition inhibits hypoxic constriction in isolated vessels, whereas it enhances hypoxic constriction in perfused lungs. The third dilator phase was unaffected by chronic hypoxia; it was increased after L-NAME in CH rats. Thus, in vitro, responses to hypoxia are complex; there is a balance between two dilator and two constrictor processes.