Václav Hampl

Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes.

Hypoxia initiates pulmonary vasoconstriction (HPV) by inhibiting one or more voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells (PASMCs) of resistance arteries. The resulting membrane depolarization increases opening of voltage-gated calcium channels, raising cytosolic Ca2+ and initiating HPV. There are presently nine families of Kv channels known and pharmacological inhibitors lack the specificity to distinguish those involved in control of resting membrane potential (Em) or HPV. However, the Kv channels involved in Em and HPV have characteristic electrophysiological and pharmacological properties which suggest their molecular identity. They are slowly inactivating, delayed rectifier currents, inhibited by 4-aminopyridine (4-AP) but insensitive to charybdotoxin. Candidate Kv channels with these traits (Kv1.5 and Kv2.1) were studied. Antibodies were used to immunolocalize and functionally characterize the contribution of Kv1. 5 and Kv2.1 to PASMC electrophysiology and vascular tone. Immunoblotting confirmed the presence of Kv1.1, 1.2, 1.3, 1.5, 1.6, and 2.1, but not Kv1.4, in PASMCs. Intracellular administration of anti-Kv2.1 inhibited whole cell K+ current (IK) and depolarized Em. Anti-Kv2.1 also elevated resting tension and diminished 4-AP-induced vasoconstriction in membrane-permeabilized pulmonary artery rings. Anti-Kv1.5 inhibited IK and selectively reduced the rise in [Ca2+]i and constriction caused by hypoxia and 4-AP. However, anti-Kv1.5 neither caused depolarization nor elevated basal pulmonary artery tone. This study demonstrates that antibodies can be used to dissect the whole cell K+ currents in mammalian cells. We conclude that Kv2. 1 is an important determinant of resting Em in PASMCs from resistance arteries. Both Kv2.1 and Kv1.5 contribute to the initiation of HPV.

Diversity in Mitochondrial Function Explains Differences in Vascular Oxygen Sensing

Renal arteries (RAs) dilate in response to hypoxia, whereas the pulmonary arteries (PAs) constrict. In the PA, O2 tension is detected by an unidentified redox sensor, which controls K+ channel function and thus smooth muscle cell (SMC) membrane potential and cytosolic calcium. Mitochondria are important regulators of cellular redox status and are candidate vascular O2 sensors. Mitochondria-derived activated oxygen species (AOS), like H2O2, can diffuse to the cytoplasm and cause vasodilatation by activating sarcolemmal K+ channels. We hypothesize that mitochondrial diversity between vascular beds explains the opposing responses to hypoxia in PAs versus RAs. The effects of hypoxia and proximal electron transport chain (pETC) inhibitors (rotenone and antimycin A) were compared in rat isolated arteries, vascular SMCs, and perfused organs. Hypoxia and pETC inhibitors decrease production of AOS and outward K+ current and constrict PAs while increasing AOS production and outward K+ current and dilating RAs. At baseline, lung mitochondria have lower respiratory rates and higher rates of AOS and H2O2 production. Similarly, production of AOS and H2O2 is greater in PA versus RA rings. SMC mitochondrial membrane potential is more depolarized in PAs versus RAs. These differences relate in part to the lower expression of proximal ETC components and greater expression of mitochondrial manganese superoxide dismutase in PAs versus RAs. Differential regulation of a tonically produced, mitochondria-derived, vasodilating factor, possibly H2O2, can explain the opposing effects of hypoxia on the PAs versus RAs. We conclude that the PA and RA have different mitochondria.

Anorexic Agents Aminorex, Fenfluramine, and Dexfenfluramine Inhibit Potassium Current in Rat Pulmonary Vascular Smooth Muscle and Cause Pulmonary Vasoconstriction

BACKGROUND The appetite suppressant aminorex fumarate is thought to have caused an epidemic of pulmonary hypertension in Europe in the 1960s. More recently, pulmonary hypertension has been described in some patients taking other amphetamine-like, anorexic agents: fenfluramine and its d-isomer, dexfenfluramine. No mechanism has been demonstrated that might account for the association between anorexic drugs and pulmonary hypertension. METHODS AND RESULTS Using the whole-cell, patch-clamp technique, we found that aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in smooth muscle cells taken from the small resistance pulmonary arteries of the rat lung. Dexfenfluramine causes reversible membrane depolarization in these cells. These actions are similar to those of hypoxia, which initiates pulmonary vasoconstriction by inhibiting a potassium current in pulmonary vascular smooth muscle. In the isolated, perfused rat lung, aminorex, fenfluramine, and dexfenfluramine induce a dose-related increase in perfusion pressure. When the production of endogenous NO is inhibited by N-nitro-L-arginine methyl ester, the pressor response to dexfenfluramine is greatly enhanced. CONCLUSIONS These observations indicate that anorexic agents, like hypoxia, can inhibit potassium current, cause membrane depolarization, and stimulate pulmonary vasoconstriction. They suggest one mechanism that could be responsible for initiating pulmonary hypertension in susceptible individuals. It is possible that susceptibility is the result of the reduced production of an endogenous vasodilator, such as NO, but this remains speculative.

Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5

Hypoxic pulmonary vasoconstriction (HPV) is initiated by the inhibition of several 4‐aminopyridine (4‐AP)‐sensitive, voltage‐gated, K+ channels (Kv). Several O2‐sensitive candidate channels (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) have been proposed, based on similarities between their characteristics in expression systems and the properties of the O2‐sensitive K+ current (IK) in pulmonary artery smooth muscle cells (PASMCs). We used gene targeting to delete Kv1.5 in mice by creating a SWAP mouse that is functionally a Kv1.5 knockout. We hypothesized that SWAP mice would display impaired HPV. The Kv1.5 α‐subunits present in the endothelium and PASMCs of wild‐type mice were absent in the lungs of SWAP mice, whereas expression of other channels Kv (1.1, 1.2, 2.1, 3.1, 4.3), Kir 3.1, Kir 6.1, and BKCa was unaltered. In isolated lungs and resistance PA rings, HPV was reduced significantly in SWAP versus wild‐type mice. Consistent with this finding, PASMCs from SWAP PAs were slightly depolarized and lacked IKv1.5, a 4‐AP and hypoxia‐sensitive component of IK that activated between ‐50 mV and ‐30 mV. We conclude that a K+ channel containing Kv1.5 α‐subunits is an important effector of HPV in mice.

NG-monomethyl-l-arginine causes nitric oxide synthesis in isolated arterial rings: Trouble in paradise☆

Arginine analogs are commonly used as inhibitors of the synthesis of endothelium-derived relaxing factor, nitric oxide. However, their effect on nitric oxide levels is rarely measured. Using a chemiluminescence assay for nitric oxide, we found that NG-monomethyl-L-arginine enhanced, rather than reduced, nitric oxide synthesis in pulmonary arterial and aortic rings. NG-monomethyl-L-arginine inhibited relaxation to the endothelium-dependent vasodilator A23187 in aortic but not pulmonary arterial rings. In contrast, N omega-nitro-L-arginine did not stimulate nitric oxide synthesis and it inhibited relaxation to A23187 in all rings. We conclude that NG-monomethyl-L-arginine is a partial agonist for nitric oxide synthesis.

Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension

OBJECTIVE Inhaled nitric oxide (NO) is a selective pulmonary vasodilator, but its use has been restricted almost exclusively to the intensive care setting due to the complexity of its delivery. NO/nucleophile adducts, such as diethylenetriamine/NO (DETA/NO), spontaneously release NO in aqueous solutions. We hypothesized that a nebulized DETA/NO (half-time of NO release > 20 h) would stay in the lower airways and continuously supply sufficient NO to achieve sustained vasodilation in chronic pulmonary hypertension. METHODS Chronic pulmonary hypertension was induced in rats by a monocrotaline injection. Nineteen days later, nebulizations of DETA/NO were given on 4 consecutive days (5 and 50 mu mol; 10 min/day). One day after the last nebulization, pulmonary and systemic arterial pressure and cardiac output were measured after thoracotomy. The lungs were isolated and perfused to study the pressure-flow relationship. The effect of DETA/NO nebulization on acute vasoconstrictor reactivity was studied in additional isolated lungs. RESULTS Total pulmonary, but not systemic, vascular resistance was significantly reduced by both DETA/NO doses, suggesting that DETA/NO, like NO, causes preferential dilation of the pulmonary circulation. The pulmonary perfusion pressure-flow curves were shifted downwards by DETA/NO treatment, indicating improved resistive properties of the pulmonary vasculature. DETA/NO nebulization into isolated lungs increased exhaled NO levels and progressively reduced vasoconstrictor responses to angiotensin II and acute hypoxia. These effects were not reversed by perfusate exchange. In intact rats, carotid artery pressure and plasma NO2- + NO3- levels did not change during and after DETA/NO nebulization. CONCLUSION DETA/NO nebulization offers a possibility of once a day, ambulatory delivery of NO and is a potential treatment for chronic pulmonary hypertension, although further studies are needed to establish safety and selectivity.

Dehydroepiandrosterone sulphate reduces chronic hypoxic pulmonary hypertension in rats

Pathogenesis of pulmonary hypertension includes vascular smooth muscle cell membrane depolarisation and consequent calcium influx. Usually, calcium-gated potassium channels are activated under such conditions and repolarise the membrane. However, in pulmonary hypertension they are downregulated. The authors hypothesised that pharmacological augmentation of these channels would reduce pulmonary hypertension. Dehydroepiandrosterone sulphate (DHEA‐S, 0.1 mg·mL−1), a recently characterised activator of calcium-gated potassium channels, was given to rats in drinking water. Pulmonary arterial blood pressure, increased by 4 weeks of hypoxia (from 15±0.2 to 29.4±2.5 mmHg), was selectively attenuated in rats treated with DHEA‐S for the whole duration of the hypoxic exposure (23.9±0.9 mmHg) and in rats given DHEA‐S only after pulmonary hypertension had fully developed (last 2 weeks of hypoxia; 24.4±1.4 mmHg). Pulmonary vascular remodelling and right ventricular hypertrophy associated with pulmonary hypertension were also reduced by DHEA‐S. Cardiac index and systemic arterial blood pressure did not differ among the groups. The authors conclude that treatment with an activator of calcium-gated potassium channels, dehydroepiandrosterone sulphate, known to be well tolerated by humans, reduces hypoxic pulmonary hypertension in rats.

Acute and chronic hypoxia as well as 7-day recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP-13 expression in rats

Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodelling of peripheral lung blood vessels. We hypothesize that vascular remodelling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP‐13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in rats exposed for 4 and 20 days to hypoxia as well as after 7‐day recovery from 20‐day hypoxia (10% O2). Mast cells were identified using Toluidine Blue staining, and MMP‐13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant increase in the number of mast cells and their MMP‐13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP‐13 positive mast cells accumulated within the walls of conduit arteries and subpleurally. In recovered rats, MMP‐13 positive mast cells gathered at the prealveolar arterial level as well as in the walls of small muscular arteries; these mast cells stayed also in the conduit part of the pulmonary vasculature. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodelling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase.

Prevention of Mast Cell Degranulation by Disodium Cromoglycate Attenuates the Development of Hypoxic Pulmonary Hypertension in Rats Exposed to Chronic Hypoxia

Background: Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. Vascular remodeling is associated with collagenolysis and activation of matrix metalloproteinases (MMPs). One of the possible sources of MMPs in hypoxic lung are mast cells. Objective: The role of lung mast cell collagenolytic activity in hypoxic pulmonary hypertension was tested by the inhibitor of mast cell degranulation disodium cromoglycate (DSCG). Methods: Rats were treated with DSCG in an early or later phase of isobaric hypoxia. Control groups were exposed to hypoxia only or to normoxia. Lung hemodynamics, muscularization and collagen metabolism in the walls of peripheral pulmonary vessels in the lungs were measured. Results: DSCG applied at an early phase of exposure to hypoxia reduced the development of pulmonary hypertension, inhibited muscularization in peripheral pulmonary arteries and decreased the amount of collagen cleavage fragments in prealveolar vessels. Conclusions: Mast cell degranulation plays a role in the initiation of hypoxic pulmonary vascular remodeling.

Chronic hypoxia increases fetoplacental vascular resistance and vasoconstrictor reactivity in the rat

An increase in fetoplacental vascular resistance caused by hypoxia is considered one of the key factors of placental hypoperfusion and fetal undernutrition leading to intrauterine growth restriction (IUGR), one of the serious problems in current neonatology. However, although acute hypoxia has been shown to cause fetoplacental vasoconstriction, the effects of more sustained hypoxic exposure are unknown. This study was designed to test the hypothesis that chronic hypoxia elicits elevations in fetoplacental resistance, that this effect is not completely reversible by acute reoxygenation, and that it is accompanied by increased acute vasoconstrictor reactivity of the fetoplacental vasculature. We measured fetoplacental vascular resistance as well as acute vasoconstrictor reactivity in isolated perfused placentae from rats exposed to hypoxia (10% O(2)) during the last week of a 3-wk pregnancy. We found that chronic hypoxia shifted the relationship between perfusion pressure and flow rate toward higher pressure values (by approximately 20%). This increased vascular resistance was refractory to a high dose of sodium nitroprusside, implying the involvement of other factors than increased vascular tone. Chronic hypoxia also increased vasoconstrictor responses to angiotensin II (by approximately 75%) and to acute hypoxic challenges (by >150%). We conclude that chronic prenatal hypoxia causes a sustained elevation of fetoplacental vascular resistance and vasoconstrictor reactivity that are likely to produce placental hypoperfusion and fetal undernutrition in vivo.