Cystamine Treatment Fails to Prevent the Development of Pulmonary Hypertension in Chronic Hypoxic Rats.

INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.

Effect of sodium nitrite on renal function and sodium and water excretion and brachial and central blood pressure in healthy subjects: a dose-response study.

Sodium nitrite (NaNO2) is converted to nitric oxide (NO) in vivo and has vasodilatory and natriuretic effects. Our aim was to examine the effects of NaNO2 on hemodynamics, sodium excretion, and glomerular filtration rate (GFR). In a single-blinded, placebo-controlled, crossover study, we infused placebo (0.9% NaCl) or 0.58, 1.74, or 3.48 µmol NaNO2·kg-1·h-1 for 2 h in 12 healthy subjects, after 4 days of a standard diet. Subjects were supine and water loaded. We measured brachial and central blood pressure (BP), plasma concentrations of renin, angiotensin II, aldosterone, arginine vasopressin (P-AVP), and plasma nitrite (P-[Formula: see text]), GFR by Cr-EDTA clearance, fractional excretion of sodium (FENa) free water clearance (CH2O), and urinary excretion rate of guanosine 3',5'-cyclic monophosphate (U-cGMP). The highest dose reduced brachial systolic BP (5.6 mmHg, P = 0.003), central systolic BP (5.6 mmHg, P = 0.035), and CH2O (maximum change from 3.79 to 1.27 ml/min, P = 0.031) and increased P-[Formula: see text] (from 0.065 to 0.766 µmol/l, P < 0.001), while reducing U-cGMP (from 444 to 247 pmol/min, P = 0.004). GFR, FENa, P-AVP, and the components in the renin-angiotensin-aldosterone system did not change significantly. In conclusion, intravenous NaNO2 induced a dose-dependent reduction of brachial and central BP. The hemodynamic effect was not mediated by the renin-angiotensin-aldosterone system. NaNO2 infusion resulted in a vasopressin-independent decrease in CH2O and urine output but no change in urinary sodium excretion or GFR. The lack of increase in cGMP accompanying the increase in [Formula: see text] suggests a direct effect of nitrite or nitrate on the renal tubules and vascular bed with little or no systemic conversion to NO.

K(Ca)3.1 channel downregulation and impaired endothelium-derived hyperpolarization-type relaxation in pulmonary arteries from chronically hypoxic rats.

Calcium-activated potassium channels of small (K(Ca)2, SK) and intermediate (K(Ca)3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of K(Ca)2 and K(Ca)3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The K(Ca) channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na(+),K(+)-ATPase with ouabain or blocking K(Ca)2 and K(Ca)3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel K(Ca)2 and K(Ca)3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of K(Ca)2.3 and K(Ca)3.1 were unaltered, whereas K(Ca)2.3 protein expression was upregulated and K(Ca)3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of K(Ca)2.3 channels. Since impaired EDH-type relaxation was accompanied by K(Ca)3.1 channel protein downregulation, these findings suggest that K(Ca)3.1 channels are important for the maintenance of EDH-type relaxation.

Genetic deficit of KCa 3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation.

BACKGROUND AND PURPOSE: The intermediate conductance calcium/calmodulin-regulated K+ channel KCa 3.1 produces hyperpolarizing K+ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl- and fluid movements. We investigated whether a deficiency in KCa 3.1 (KCa 3.1-/- ) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels. EXPERIMENTAL APPROACH: An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and KCa 3.1-/- mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC). KEY RESULTS: In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, KCa 3.1-/- mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of KCa 3.1-/- mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and KCa 3.1-/- mice, which co-activated KCa 3.1 and disrupted membrane resistance in wt PAEC, but not in KCa 3.1-/- PAEC. CONCLUSIONS AND IMPLICATIONS: Our findings show that a genetic deficiency of KCa 3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of KCa 3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.

Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels.

OBJECTIVE: In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. APPROACH AND RESULT: Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. CONCLUSION: Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.

Alterations of N-3 polyunsaturated fatty acid-activated K2P channels in hypoxia-induced pulmonary hypertension.

Polyunsaturated fatty acid (PUFA)-activated two-pore domain potassium channels (K2P ) have been proposed to be expressed in the pulmonary vasculature. However, their physiological or pathophysiological roles are poorly defined. Here, we tested the hypothesis that PUFA-activated K2P are involved in pulmonary vasorelaxation and that alterations of channel expression are pathophysiologically linked to pulmonary hypertension. Expression of PUFA-activated K2P in the murine lung was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), by patch clamp (PC) and myography. K2P -gene expression was examined in chronic hypoxic mice. qRT-PCR showed that the K2P 2.1 and K2P 6.1 were the predominantly expressed K2P in the murine lung. IHC revealed protein expression of K2P 2.1 and K2P 6.1 in the endothelium of pulmonary arteries and of K2P 6.1 in bronchial epithelium. PC showed pimozide-sensitive K2P -like K(+) -current activated by docosahexaenoic acid (DHA) in freshly isolated endothelial cells as well as DHA-induced membrane hyperpolarization. Myography on pulmonary arteries showed that DHA induced concentration-dependent instantaneous relaxations that were resistant to endothelial removal and inhibition of NO and prostacyclin synthesis and to a cocktail of blockers of calcium-activated K(+) channels but were abolished by high extracellular (30 mM) K(+) -concentration. Gene expression and protein of K2P 2.1 were not altered in chronic hypoxic mice, while K2P 6.1 was up-regulated by fourfold. In conclusion, the PUFA-activated K2P 2.1 and K2P 6.1 are expressed in murine lung and functional K2P -like channels contribute to endothelium hyperpolarization and pulmonary artery relaxation. The increased K2P 6.1-gene expression may represent a novel counter-regulatory mechanism in pulmonary hypertension and suggest that arterial K2P 2.1 and K2P 6.1 could be novel therapeutic targets.