Chronic Hypoxia Rats Research Articles

Age-dependent vulnerability to ischemia-reperfusion injury of cyanotic myocardium in a chronic hypoxic rat model.

This study evaluated the effects of chronic hypoxia from birth on the resistance of rat hearts to global ischemia, with special emphasis on the duration of hypoxia. Male Wistar rats were housed from birth for 4 weeks or 8 weeks either in a hypoxic environment (FiO2 = 0.12) or in ambient air (8 animals for each group). Isolated rat hearts were perfused for 40 min with oxygenated Krebs-Henseleit buffer, subjected to 20 min global no-flow ischemia at 37, and then underwent 40 min of reperfusion. A non-elastic balloon was inserted into the left ventricle and inflated until the pre-ischemic LVEDP rose to 8 mmHg. Cardiac function was measured before and after ischemia. The post-ischemic percent recovery of LVDP in hypoxic hearts was worse than in normoxic hearts (4 weeks:55+/-7 vs. 96+/-3%, p0.01;8 weeks:40+/-5 vs. 92+/-4%, p0.01), and was worst in the 8-week-hypoxic hearts. Similarly, the percent recovery of dP/dt in the hypoxic hearts was lower than in the normoxic hearts (4 weeks:51+/-5 vs. 96+/-7%, p0.01;8 weeks:31+/-6 vs. 92+/-7%, p0.01), and was lowest in the 8-week-hypoxic hearts. In conclusion, cyanotic myocardium revealed an age-dependent vulnerability to ischemia-reperfusion injury in a chronic hypoxic rat model.

Changes in pulmonary blood flow distribution in monocrotaline compared with hypoxia-induced models of pulmonary hypertension: assessed using synchrotron radiation

We have previously described anatomical changes in pulmonary blood flow distribution in chronic hypoxic rats, associated with pulmonary arterial hypertension (PAH). In this study, we utilized synchrotron radiation microangiography to compare these changes in pulmonary blood flow with a PAH-model induced with monocrotaline (MCT), as the etiology for these two models of PAH is different. Three weeks after a subcutaneous injection of MCT (60 mg/kg) or vehicle (control), Sprague-Dawley rats were anesthetized, and microangiography was performed on the left lung to assess branching distribution of pulmonary blood flow and changes in vessel diameter during acute (8% O2 for 4 min) hypoxic pulmonary vasoconstriction--before and after sympathetic beta-adrenoceptor blockade (propranolol, 2 mg/kg, intravenous). Comparisons were made with chronic hypoxic rats using data previously published. We observed that adverse changes in pulmonary blood flow were comparable for both chronic hypoxia and MCT models of PAH. Specifically, the number of opaque third and fourth generation vessels was significantly and equally fewer than that of control rats. The acute hypoxic pulmonary vasoconstriction was not altered in the hypertensive lung, though sympathetic modulation of pulmonary vasoreactivity was enhanced by chronic hypoxia, but not MCT. In summary, we have demonstrated comparable adverse changes in pulmonary blood flow for chronic hypoxia and MCT models of PAH. In contrast, modulation of the hypoxic pulmonary vasoconstriction differs between the two PAH models, likely due to the impact that different pathological pathways have on the physiology of the whole organism. Such differences between models of PAH should be considered in future studies.

Pulmonary apelin levels and effects in rats with hypoxic pulmonary hypertension

The peptide apelin is localised in the vascular endothelium and highly expressed in pulmonary tissue. The aim of this study was to investigate whether apelin could be a potential lung-derived plasma marker for pulmonary hypertension, and study the effect of apelin in pulmonary arteries. Apelin protein levels were measured in the lung, right ventricle, and plasma from normoxic and chronic hypoxic rats with pulmonary hypertension. Isolated intrapulmonary arteries were mounted in microvascular myographs and the effect of apelin investigated. Finally, the distribution of apelin receptors in pulmonary tissue was visualised by immunohistochemistry. Total pulmonary apelin content was not changed by hypoxia. Right ventricular apelin concentrations and content were lower than in the lung, but increased substantially in hypoxia in correlation with right ventricular pressure. Plasma apelin did not reflect pulmonary or right ventricular apelin levels. In pulmonary arteries from normoxic rats, apelin inhibited vasoconstriction to endothelin-1 and angiotensin-II. However, in arteries from hypoxic rats, apelin failed to inhibit contraction to angiotensin-II and endothelin-1. No difference in immunoreaction for apelin receptors was found in lung sections and arteries from normoxic versus chronic hypoxic rats. Apelin changes in the right ventricle seem more specific for pulmonary hypertension than do changes in pulmonary tissue, which does not speak in favour of apelin as a lung-derived marker for this disease. During normoxic conditions, apelin has a modulating effect on vasoconstriction which is lost in chronic hypoxia. This may reflect alterations in the signal transduction downstream of the apelin receptor.

Chronic hypoxia decreases response to central chemoreceptor stimulation in the nucleus tractus solitarius (NTS)

One hallmark of acclimatization to chronic hypoxia (CH) is hyperventilation and a lower arterial carbon dioxide (CO2) level. These effects persist upon return to normoxia, suggesting a change in the regulation of CO2 during CH. We tested the hypothesis that plasticity in one central chemoreceptor site, the NTS, contributes to altered CO2 regulation in CH. In urethane‐anesthetized rats, we stimulated chemoreceptors in the NTS by a unilateral microinjection of acetazolamide (ACZ, 1 nl of 0.3 μM), to produce focal acidosis. The product of rectified, integrated phrenic nerve amplitude and frequency (Phr) was measured 30 min after ACZ in normoxic control (N; n=5) and CH rats (n=5). ACZ increased Phr significantly more in N rats than CH rats (N=263±14%; CH=83±18%; p<0.001). ACZ in adjacent brainstem regions had no effect on Phr. All rats showed the capacity to increase Phr to a 10% inspired CO2 challenge following ACZ, suggesting that the dose of ACZ did not produce a maximal phrenic response. These results indicate plasticity in chemosensitivity in the NTS but they cannot easily explain the lower CO2 set point with CH.Supported by NIH HL81823 (FLP) and AHA 0615069Y (KAW).

Heme Oxygenase Induced Endothelial BKCa Channel Activity Following Chronic Hypoxia

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist‐induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reversed by either removal of the endothelium or by blockade of the CO producing enzyme heme oxygenase (HO). Interestingly, endothelial cell (EC) specific blockade of large conductance Ca2+‐activated K+ (BKCa) channels also restores vasoreactivity in arteries from CH rats. Patch clamp studies demonstrate EC BKCa activity following CH, but not in ECs from control rats, although channels are observed by immunofluorescence (IF) in cells from both groups. Since HO‐derived CO is an endogenous activator of BKCa channels, we hypothesized that HO and BKCa form a functional unit that results in enhanced channel activity following CH. In support of the hypothesis, the HO inhibitor zinc protoporphyrin IX (ZnPPIX) significantly decreased K+ currents in ECs from CH rats, but was without effect in EC from control rats. In addition, the HO substrate hemin increased K+ currents only in the CH group. HO‐2, but not HO‐1, was found to co‐localize with BKCa in cells from each group. These results suggest that although HO and BKCa channels are expressed in EC from each group, HO‐derived CO only stimulates channel activity following CH.

Caveolin Dysfunction Impairs Pulmonary Endothelial Calcium Entry Following Chronic Hypoxia

Agonist‐induced Ca2+ entry into the pulmonary endothelium is dependent upon activation of both store‐operated Ca2+ entry (SOCE) and receptor operated Ca2+ entry (ROCE) pathways. In addition, there is recent evidence that trafficking of the relevant ion channels to the cell membrane by caveolin‐1 (cav‐1) is necessary for both forms of Ca2+ entry. We have previously reported that pulmonary endothelial cell (EC) SOCE and ROCE are reduced in the chronic hypoxia (CH) model of pulmonary hypertension. Therefore, we hypothesized that diminished endothelial Ca2+ entry following CH is due to impaired cav‐1 mediated trafficking of agonist‐induced Ca2+ entry pathways. To test this hypothesis we measured Ca2+ influx by fura‐2 fluorescence following the application of ATP (20 μM) in ECs pretreated with the caveolar disrupting agent methyl‐beta cyclodextrin (mβcd; 10 mM). We found that cholesterol depletion with mβcd attenuated agonist‐induced Ca2+ entry in control ECs to the level of ECs from CH rats. Furthermore, cholesterol repletion with a mβcd‐cholesterol (1:5) mixture or the introduction of the cav‐1 scaffolding peptide (AP‐CAV, 10 μM) rescued ATP‐induced Ca2+ entry in ECs from CH hypertensive arteries. These data suggest that CH‐induced pulmonary hypertension leads to impaired cav‐1 trafficking of ion channels necessary for receptor‐activated Ca2+ entry.

Reduced basal Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats is restored by administration of the scaffolding domain of caveolin‐1

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with impaired endothelial Ca2+ influx in response to store depletion. Molecular candidates for store‐operated and receptor operated Ca2+ entry (SOCE/ROCE) such as transient receptor potential (TRP) channels may also be involved in basal influx and appear to be trafficked to the plasma membrane by caveolin‐1 (cav‐1). We hypothesized that impaired basal Ca2+ entry occurs following CH and is due to deficient cav‐1 mediated trafficking of relevant ion channels. Ca2+ influx was assessed in isolated pressurized intrapulmonary arteries from control and CH rats (4 weeks at 0.5 atm) by Mn2+ quenching of fura‐2 fluorescence in the presence of a cav‐1 scaffolding domain peptide (AP‐CAV) or scrambled control. AP‐CAV increased basal Ca2+ influx in arteries from CH animals, and had no effect in the controls. In additional studies, we observed reduced immunofluorescent membrane localization of TRPC4 and cav‐1 in cultured bovine pulmonary artery endothelial cells (BPAECS) exposed to hypoxia (2% O2, 4 days) compared to controls. These studies suggest that hypoxia per se impairs cav‐1 mediated trafficking of ion channels that may be responsible for basal endothelial Ca2+ influx.

TRPC1 contribute to hypoxic pulmonary hypertension and right heart hypertrophy: evidence from TRPC1 knockout mice

Prolonged exposure to aveolar hypoxia causes pulmonary hypertension (PAH) and right heart hypertrophy. Hypoxic PAH is associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that the store‐operated TRPC1 and receptor‐operated TRPC6 channels are upregulated in PASMCs of chronic hypoxic rats; and the enhanced store‐operated Ca2+ entry is responsible for the elevated [Ca2+]i and vascular tone in pulmonary arteries (PAs). In this study, we used trpc1−/− and trpc6−/− mice to examine whether these channels are required for the development of hypoxic PAH. Real‐time RT‐PCR showed that TRPC1, TRPC2, TRPC3, TRPC4 and TRPC6 channels were expressed in wild‐type mouse PASM. There was no significant shift in the expression of other TRPC subtypes in PAs, except the absence of TRPC1 and TRPC6 transcripts in the trpc1−/− and trpc6−/− mice, respectively. In PA and aorta of trpc1−/− mice, store‐operated Ca2+ entry induced by cyclopiazonic acid was slightly reduced, suggesting that TRPC1 and other channels were involved. Phenylephrine and serotonin induced vasoconstriction were unaltered in trpc1−/− PAs and aorta. However, when trpc1−/− mice were exposed to 4 weeks of hypoxia (10% or 8.5% O2), right ventricular systolic pressure (RVSP) and right heart mass ratio (RV/(LV+S)) was significantly attenuated. There was no significant difference in RVSP and RV/(LV+S) between chronic hypoxic trpc6−/− and wild‐type mice. These results, for the first time, suggest that TRPC1 might play a significant role in the development of hypoxic PAH and right heart hypertrophy.

Intermittent hypoxia: keeping it real

in this issue of the Journal of Applied Physiology , Tamisier and colleagues ([20][1]) describe a novel method for studying the effects of chronic intermittent hypoxia (CIH) in healthy human subjects. To investigate the effects of CIH on sleep, ventilatory control, and blood pressure, the authors

Doppler echocardiographic assessment of left ventricular diastolic function in chronic hypoxic rats.

Little information is available on the mechanism of diastolic left ventricular (LV) dysfunction in patients with chronic respiratory disease complicated by hypoxia. The purpose of this study was to investigate how chronic hypoxia impairs LV diastolic function in an hypoxic animal model. Thirty-six male Wistar rats 8 weeks old were assigned to normoxia (N), chronic hypoxia (CH), and re-normoxia (RN) groups, 12 rats per group. The N group rats were kept in ambient air for 8 weeks, while the CH group was kept hypoxic for 8 weeks. After 8 weeks of hypoxia the RN group rats were kept for a further 8 weeks in ambient air. LV systolic and diastolic functions, as well as right ventricular (RV) function, were analyzed using Doppler echocardiography;we also measured the hematocrit, and weighed the LV and RV. Hematocrit, RV weight/body weight, and RV weight/LV weight were higher in the CH group than in the other 2 groups. However, most of these parameters returned to normoxia levels after re-normoxia. In the CH group, LV dimension and area were smaller than in the other 2 groups. LV systolic function was preserved in all groups;however, in the CH group, mitral flow showed a restrictive pattern, while pulmonary flow demonstrated a pulmonary hypertensive pattern with prolonged RV ejection time. In conclusion, chronic hypoxia induced pulmonary hypertension and RV hypertrophy. Although LV systolic function was preserved, diastolic function was impaired in hypoxia. Ventricular interaction may impair LV diastolic function.

Antioxidant and Vasodilatory Effects of Heme Oxygenase on Mesenteric Vasoreactivity Following Chronic Hypoxia

Chronic hypoxia (CH) results in impaired vasoconstriction associated with increased expression of heme oxygenase (HO). We hypothesized that enhanced HO activity minimizes reactive oxygen species (ROS) in arteries from CH rats, thereby normalizing endothelium-dependent vasodilation and concurrently produces carbon monoxide (CO), resulting in tonic vasodilation. ROS were quantified in mesenteric arteries from control and CH Sprague-Dawley rats. Reactivity to the endothelium-dependent vasodilator, acetylcholine (ACh), and the vasoconstrictor, phenylephrine (PE), were also assessed. Basal ROS levels did not differ between groups and were similarly increased by HO inhibition. In contrast, catalase inhibition increased ROS in CH rats only. Vasodilatory responses to ACh were not different between groups. Combined inhibition of catalase and HO impaired PE-induced vasoconstriction in both groups. CH-induced impairment of vasoconstriction was reversed by either catalase or HO inhibition supporting the protective roles of the HO and catalase pathways following CH. Increased vascular smooth muscle calcium was observed with inhibition in the CH group, suggesting that catalase and HO-derived CO elicit reduced calcium influx, leading to the impaired vasoconstriction. Our data suggest that although the HO pathway is an important antioxidant influence, impaired vasoconstriction following CH appears to be due to effects of ROS and HO-derived CO.

Adaptative modifications of right coronary myocytes voltage-gated K+ currents in rat with hypoxic pulmonary hypertension

Chronic hypoxia (CH)-induced pulmonary hypertension (PHT) is well known to alter K+ channels in pulmonary myocytes. PHT induces right ventricle hypertrophy that increases oxygen demand; however, coronary blood flow and K+ channel adaptations of coronary myocytes during PHT remain unknown. We determined whether CH and PHT altered K+ currents and coronary reactivity and what impact they might have on right myocardial perfusion. Right ventricle perfusion, as attested by microspheres, was redistributed toward hypertrophied right ventricle [RV/LV (%)=0.59+/-0.07% in CH rats vs. 0.29+/-0.03 in control rats, P<0.05]. Whole-cell patch clamping showed a reduction of global outward current in hypoxic right coronary artery myocytes (H-RCA), whereas hypoxic left coronary artery myocytes exhibited an increase. K+ channel blockers revealed that a 4-aminopyridine (4AP)-sensitive current (Kv current) was decreased in H-RCA (14.3+/-1.1 vs. 23.4+/-2.5 pA/pF at 60 mV in control RCA, P<0.05) and increased in hypoxic left coronary artery myocytes (H-LCA; 26.4+/-3.8 vs. 11.8+/-1.6 pA/pF at 60 mV in control LCA, P<0.05). Constriction to 4AP was decreased in H-RCA when compared to normoxic control and increased in H-LCA when compared to LCA. Finally, we observed that the expression of Kv1.2 and Kv1.5 were lower in H-RCA than that in H-LCA. This study reveals that CH differentially regulates Kv channels in coronary myocytes. Hypoxia decreases Kv currents and therefore reduces vasoreactivity that contributes to an adaptative response leading to right hypertrophied ventricle perfusion enhancement at rest.

Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia

Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca(2+)](i)). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca(2+) sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endothelium-denuded, Ca(2+)-permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidium-loaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca(2+) sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca(2+) sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1(Thr696) phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca(2+) sensitization following CH. We conclude that CH augments ET-1-induced Ca(2+) sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.

Atrial natriuretic peptide gene transfection with a novel envelope vector system ameliorates pulmonary hypertension in rats

A novel hemagglutinating virus of Japan (HVJ, a murine parainfluenza virus) envelope vector system, in which DNA is incorporated into an inactivated viral particle deprived of its genome, was recently developed as a ready-to-use vector for gene therapy. We therefore investigated whether intratracheal gene transfer using this vector can induce transgene expression in the lung and whether atrial natriuretic peptide gene transfer ameliorates pulmonary hypertension in rats. Rats transfected intratracheally with beta-galactosidase vector, atrial natriuretic peptide vector, or mock vector were investigated for the evaluation of beta-galactosidase expression, atrial natriuretic peptide mRNA expression, and inflammatory cell infiltration. Rats were divided into 5 treatment groups (n = 73): normoxic rats treated intratracheally with mock vector or atrial natriuretic peptide gene and chronic hypoxic rats treated similarly with mock vector, atrial natriuretic peptide, or a reporter gene, beta-galactosidase. Pulmonary hypertension and transfected gene expression were evaluated. Beta-galactosidase gene transfer induced its intense enzymatic activity in bronchial and alveolar epithelial cells but not in other organs in normoxic rats. Transfected lungs were not associated with inflammatory cell infiltration. Atrial natriuretic peptide gene transfection inhibited pulmonary hypertension, which is associated with its mRNA expression in the lungs. Indices of right ventricular hypertrophy and pulmonary vascular diseases induced by chronic hypoxia were significantly but incompletely ameliorated. HVJ-envelope vector is an efficient, relatively safe, and ready-to-use gene delivery system for pulmonary vascular diseases. Atrial natriuretic peptide gene transfer to lungs by using this vector could be a promising therapeutic approach against pulmonary hypertension.

Hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy is unaltered by long-term oral L-arginine administration

Nitric oxide has been shown to reduce the development of chronic hypoxic pulmonary hypertension. L-arginine is the substrate for endogenous nitric oxide synthesis. The aim of this study was to investigate whether oral L-arginine prevents the development of pulmonary vascular and right ventricular hypertrophy in adult chronic hypoxic rats. Male rats were maintained in either normoxic or hypobaric hypoxic (10% O 2) chambers for two weeks as controls or treated with L-arginine (2 g kg − 1 day − 1 in the drinking water). Both in vehicle and L-arginine-treated rats, chronic hypoxia caused right ventricular hypertrophy, increased media to lumen ratio and increased lung weight. Contraction to the thromboxane analogue, U46619, was increased in intrapulmonary arteries, while systemic blood pressure was unaltered. Relaxations induced by the nitric oxide donor, S-nitroso- N-acetylpenicillamine (SNAP), were increased in arteries from L-arginine-treated normoxic and hypoxic animals. In conclusion, long-term oral L-arginine administration fails to prevent development of right ventricular hypertrophy and vascular media hypertrophy in adult chronic hypoxic rats.

Attenuated PLC‐Dependent Activation of Voltage‐Gated Ca2+ Entry in Pulmonary Endothelium Following Chronic Hypoxia

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with reduced endothelial cell (EC) receptor‐operated Ca2+ (ROC) entry. Voltage‐gated Ca2+ channels (VGCC) are present in pulmonary ECs and are activated by protein kinase C (PKC). Although VGCCs may participate in ROC entry, neither the role of phospolipase C (PLC) nor the effect of CH on this entry pathway are known. We hypothesized that CH reduces phospholipase C (PLC)‐induced Ca2+ influx through VGCCs in pulmonary ECs. Thus, we assessed both KCl and ATP‐induced Ca2+ entry in freshly isolated, fura‐2 loaded ECs obtained from control and CH rats (4 wk at 0.5 atm). Consistent with earlier findings, KCl‐induced Ca2+ entry was diminished following CH. Furthermore, KCl‐mediated Ca2+ influx in control ECs was attenuated by the T‐type VGCC inhibitor, mibrefradil (MBF; 10 μM), but not by the L‐type inhibitor diltiazem (50 μM). Similarly, ATP‐induced Ca2+ entry in control ECs was reduced to the level of CH ECs by MBF and was partially inhibited by the ROC blocker SKF96365 (20 μM). Neither of these antagonists affected Ca2+ entry in CH ECs. Finally, the PLC inhibitor U73122 (1 μM) abolished ATP‐induced Ca2+ influx in ECs from both control and CH rats, but the inactive analog U73343 was without effect. These data suggest that purinergic receptor stimulation in pulmonary ECs activates T‐type VGCCs through a PLC‐dependent mechanism that is diminished following CH.

Contribution of ASIC to Increased Pulmonary Vascular Smooth Muscle Resting [Ca 2+]i and Store‐Operated Ca2+ Entry Following Chronic Hypoxia

Increases in basal pulmonary vascular smooth muscle (VSM) intracellular Ca2+([Ca2+]i) and store‐operated Ca2+ entry (SOCE) have been implicated in mediating the vasoconstrictor and arterial remodeling components of chronic hypoxia (CH)‐induced pulmonary hypertension. The goal of the present study was to characterize a role for the unique class of ion channels, acid sensing ion channels (ASICs), in regulating basal VSM [Ca2+]i and SOCE in the hypertensive pulmonary circulation. We hypothesized that enhanced Ca2+ entry through ASIC contributes to elevated basal VSM [Ca2+]i and increased SOCE following CH. Inhibition of ASIC with amiloride or benzamil attenuated SOCE and associated vasoconstriction in fura‐2‐loaded, endothelium‐disrupted, pressurized small pulmonary arteries from control rats. Furthermore, siRNA knockdown of ASIC1, but not ASIC2 or ‐3, diminished SOCE in primary cultures of rat pulmonary VSM cells. Resting [Ca2+]i and SOCE were enhanced in isolated small pulmonary arteries from CH rats (4 wk at 0.5 atm) vs. controls. Benzamil reduced basal [Ca2+]i in arteries from both groups, but caused a greater reduction in resting [Ca2+]i in arteries from CH rats compared to controls. In addition, benzamil attenuated the increase in SOCE in CH arteries and normalized responses between groups. Consistent with these findings, CH upregulated pulmonary vascular ASIC1, but not ASIC2 or ‐3 expression as determined by Western blotting. These studies support a novel role of ASIC to mediate elevated basal VSM [Ca2+]i and increased SOCE following CH.

Interaction of nitric oxide and reactive oxygen species in the development of hypoxic pulmonary hypertension

Chronic hypoxia damages peripheral pulmonary vessels and causes hypoxic pulmonary hypertension (HPH). According to our hypothesis, the release of reactive oxygen species (ROS), nitric oxide (NO) and products of their interaction contribute to the pathogenesis of HPH. Experimenting with chronic hypoxic adult male rats, we tested the effect of NO and ROS interaction. Hypoxic rats were treated solely with SOD mimetic Tempol (group T, n=6) and NO donor Molsidomine (group M, n=9) or in combination (group MT, n=6) during first 7 days of exposure to 3 weeks hypoxia (FiO2=0.1). Seven untreated hypoxic rats (group H) and seven normoxic controls (group C) were used.Pulmonary artery blood pressure (PAP), and the weight of RV, LV+S were measured. PAP was significantly decreased in group M (P&lt;0.05) and group T (P&lt;0.05) but not in group MT compared with group H. In all hypoxic groups the right‐to‐left heart weight ratio was significantly increased compared to group C, and groups receiving Tempol (T, MT) had significantly (P&lt;0.01 and P&lt;0.01 respectively) less increase in weight of RV or whole heart compared with group H.Molsidomine or Tempol added alone (but not in combination) at the beginning of exposure to chronic hypoxia reduces HPH. Tempol alone or in a combination reduces of hypoxia‐induced right heart hypertrophy.Supported by Cardiovascular Research Center MSMT 1M 0510, Grants: GACR 305/05/0672 and GAUK 7780/2007

Chronic hypoxia (CH)‐induced inflammation down‐regulates soluble guanylate cyclase (sGC) in rat carotid body

sGC in oxygen‐sensitive carotid body type I cells is activated by the diffusible gas, nitric oxide (NO). In rats exposed to CH, sGC is down‐regulated, with a corresponding drop in NO‐stimulated production in cGMP. Moreover, CH induces an invasion of activated macrophages and the production of inflammatory cytokines in chemosensory tissue. The present study investigates the relationship between inflammation and the down‐regulation of sGC in type I cells. Quantitative PCR (qPCR) assays of carotid bodies from 7‐day CH (380 Torr) rats demonstrated a 50–500% increase in expression of cytokines interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNFα). CH (7 days) elicited a &gt;90% reduction in the expression of sGC. In contrast, animals concurrently treated with anti‐inflammatory drugs, ibuprofen (4mg/kg/day) or bosentan (200mg/kg/day), expressed normal levels of the sGC gene. sGC expression was reduced by 50% in normal type I cells cultured for 48 hrs in the presence of TNFα (50 ng/ml). However, exposure to TNFα did not alter the expression of the gene for tyrosine hydroxylase (TH). Our data indicate that a CHinduced increase in cytokine production directly influences the expression of sGC, a signaling molecule which mediates type I cell inhibition. Inflammation appears to be an important factor in the readjustment of chemoreceptor sensitivity following CH. USPHS Grants NS 12636 and NS 07938.

Characterization of receptor‐ and store‐operated cation currents in rat pulmonary arterial smooth muscle cells (PASMCs)

Store‐ and receptor‐operated cation channels (SOC and ROC) in PASMCs have been implicated to play important roles in many physiological functions, including hypoxia‐ and agonist‐induced vasoconstriction, and pulmonary hypertension. Here we characterized the SOC and ROC currents in rat intralobar PASMCs. PASMCs were whole‐cell voltage‐clamped. K+ and Ca2+ currents were eliminated by substituting K+ with Cs+, and by nifedipine. Depletion of SR Ca2+ stores with cyclopiazonic acid activated a small inward current at −60 mV. The current was elicited with a time‐delay, exhibited typical bimodal dependence to extracellular Ca2+ due to the effect of anomalous mole fraction, and was blocked by La3+ or substitution of Na+ with N‐methyl‐D‐glucamine. I‐V relationship of La3+‐sensitive SOC current was quasi‐linear with a reverse potential close to 0 mM, indicative of its non‐selective nature. ROC agonist 1‐oleoyl‐2‐acetyl‐sn‐glycerol also activated a La3+ (1 mM) sensitive non‐selective cation current with slight outward rectification. Moreover, ROC currents recorded in PASMCs of chronic hypoxic rats (10% O2, 4 weeks) were significantly larger than those of normoxic rats. These results are consistent with the notion that functional SOC and ROC are present in PASMCs and their activities are up‐regulated in hypoxic pulmonary hypertension. (Supported by NIH R01HL074134, NSFC 30670772 and NSF(Fujian) C0620002)