Forskolin fails to activate L-type calcium current in hypertrophied cardiomyocytes of chronically hypoxic rats

We have shown that the contractile, cytosolic calcium ([Ca2+]i) and cyclic AMP (cAMP) responses to beta-adrenoceptor stimulation are attenuated in ventricular myocytes of chronically hypoxic (CH) rats. The aim of this study was to examine the effect of forskolin on the L-type Ca2+ current in CH hypertrophied ventricular myocytes. Patch-clamp recording of the L-type Ca2+ current was measured in right ventricular myocytes of normoxic control and CH rats exposed to 10% inspired oxygen for 4 weeks. The breadth, but not the length, of CH myocytes was significantly greater than that of the control group. Activation of beta-adrenoceptor with isoproterenol (0.1 microM) increased the peak Ca2+ current by 83% in the normoxic control but the increase of peak Ca2+ current was not significant in the CH myocytes. Forskolin (0.1 - 1 microM), an activator of adenylyl cyclase, increased the peak Ca2+ current by 49% - 102% in the normoxic controls but it did not cause significant change of the peak Ca2+ current in CH myocytes. These results suggest an absence of forskolin-induced activation of Ca2+ current in hypertrophied ventricular myocytes during chronic hypoxia. The failure of activation of the Ca2+ current is consistent with the idea that adenylyl cyclase function is down-regulated in CH hypertrophied myocytes.     

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca(2+) concentration ([Ca(2+)](i)). We hypothesized that CH decreases EC [Ca(2+)](i) associated with membrane depolarization and reduced Ca(2+) entry. To test this hypothesis, we assessed 1) basal endothelial Ca(2+) in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (E(m)), 3) store-operated Ca(2+) current (I(SOC)), and 4) store-operated Ca(2+) (SOC) entry in arteries from control and CH rats. We found that basal EC Ca(2+) was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. I(SOC) activation by 1 muM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn(2+) quenching revealed a parallel reduction in Ca(2+) entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca(2+)](i). In contrast, basal EC Ca(2+) influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that E(m) may not play a major role in determining basal EC [Ca(2+)](i) following CH.     

Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats

Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10-100 nM) decreased the resting [Ca2+]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]i response induced by caffeine. Sildenafil (0.1 nM-1 microM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+ signaling pathway involved in this model of pulmonary hypertension.     

Mitochondrial potassium channels: from pharmacology to function

Mitochondrial potassium channels, such as ATP-regulated or large conductance Ca2+ -activated and voltage gated channels were implicated in cytoprotective phenomenon in different tissues. Basic effects of these channels activity include changes in mitochondrial matrix volume, mitochondrial respiration and membrane potential, and generation of reactive oxygen species. In this paper, we describe the pharmacological properties of mitochondrial potassium channels and their modulation by channel inhibitors and potassium channel openers. We also discuss potential side effects of these substances.     

Renal vasodilatory influence of endogenous carbon monoxide in chronically hypoxic rats

Chronic hypoxia (CH) attenuates systemic vasoconstriction to a variety of agonists in conscious rats. Recent evidence suggests that similarly diminished responses to vasoconstrictors in aortic rings from CH rats may be due to increased endothelial heme oxygenase (HO) activity and enhanced production of the vasodilator carbon monoxide (CO). Thus we hypothesized that a hypoxia-induced increase in HO activity is responsible for decreased vasoconstrictor responsiveness observed in conscious CH rats. CH (4 wk at 0.5 atm) and control rats were renal denervated and instrumented for the measurement of renal blood flow (RBF) and blood pressure. First, renal vasoconstrictor responses to graded intravenous infusion of phenylephrine (PE) were assessed in conscious rats. CH rats demonstrated significantly diminished renal vasoconstrictor responses to PE compared with control responses that persisted even with acute restoration of normoxia. In additional experiments, CH rats exhibited increased renal vascular resistance and decreased RBF in response to the HO inhibitor zinc protoporphyrin IX (11 micromol/kg iv), whereas renal hemodynamics were unaffected by the inhibitor in control animals. Furthermore, we demonstrated greater HO enzyme activity in renal tissue from CH rats compared with controls. These data suggest that enhanced HO activity contributes a tonic vasodilatory influence in the renal vasculature of CH rats that may be responsible for the diminished sensitivity to vasoconstrictor agonists observed under these conditions.     

A morphometric study of the carotid body in chronically hypoxic rats

We performed morphometric studies of carotid body in acutely and chronically hypoxic rats (inspired PO2 = 70 Torr, at sea level). Acute exposure was for the duration of about 10 min, and chronic exposure lasted for 28 days. We confirmed that the total volume of the organ increased by severalfold. At the light-microscopy level we found an enlargement of the volume density of the blood sinuses from 14 to 31% due to chronic hypoxia. The morphometric hematocrit increased from 39 to 70% paralleling changes in the conventionally measured venous hematocrit. These data do not show any specific plasma skimming in the carotid body blood vessels. With the electron microscope we found that the mean average volume of type I cells increased from 320 micron3 in controls to 1,120 micron3 in the chronically hypoxic rats without hyperplasia, whereas type II cells had increased in number without alteration in size. Qualitative observations revealed that the normal appearance of clusters of ovoid type I cells interspersed by capillaries had been transformed into a pattern of individual cells forming plates between expanded blood vessels with a large increase of contact area between the cells and vessels. Type II cells appeared to have proliferated without changes in individual size to cover the enlarged periphery of type I cells. The observed structural changes in the carotid body parenchyma and vasculature appear to be physiologically adaptive and provide further support for the idea that various elements in the organ are particularly sensitive to hypoxia.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.     

Mitochondrial contacts of cardiomyocytes

The myocardium of the left and right ventricles in mature rabbits has been studied electron microscopically. The material is fixed by means of vital perfusion and/or by immersion in 2.5% glutaraldehyde with cacodylate buffer 0.05-0.1 M, pH 7.4 and treated in 1% OsO4 with the same buffer. For revealing intercellular contacts and inter-mitochondrial gaps, colloid lanthanum is applied. In order the colloid particles penetrate into cytoplasm, the model of ischemic myocardium is used. The myocardial infarction is produced by ligation of the coronary artery. The inter-mitochondrial interactions in cardiomyocytes are various and can be performed not only via hyaloplasm, but immediately by means of direct specific inter-mitochondrial contacts (IC). The IC are limited areas of maximal bringing together of the external membranes of the adjoining mitochondria. These areas are characterized by an increase electron density both of the contacting mitochondrial membranes and of the contents in the intermembranous spaces. A close topographic connection is revealed between mitochondria and cytolemma in the zone of the gap intercellular junction of the intercalated disc, where the mitochondrial nexus complex is formed. The IC can evidently ensure the metabolic and adhesive connection between separate mitochondria.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Metalloproteinase inhibition by Batimastat attenuates pulmonary hypertension in chronically hypoxic rats

Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 +/- 3 mmHg in hypoxic controls, 24 +/- 1 mmHg in Batimastat-treated hypoxic rats, and 16 +/- 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.     

Temporal increase in the reactivity of pulmonary vasculature to substance P in chronically hypoxic rats

We previously demonstrated that the pulmonary vascular response to substance P (SP) increased in chronically hypoxic rats. This study explored the temporal increase in reactivity of the pulmonary vascular response to SP and its underlying mechanisms. First, young female Wistar rats were exposed to sea level (SL) or simulated high altitude (HA) for 15 h/day for 3 days, 1 wk, 2 wk, and 4 wk. Lungs were isolated and perfused with 4% bovine serum albumin in Krebs-Henseleit buffer solution. SP (1.5 x 10(-4) M) induced significant increases in pulmonary arterial pressure (P(pa)), venous pressure (P(v)), capillary pressure (P(c)), arterial resistance (R(a)), and filtration coefficient (K(fc)) in SL lungs. Increases in P(pa) and R(a) were significantly augmented in HA lungs, with a temporal increase trend peaking at 2 wk of HA exposure. The selective neurokinin (NK) type 1 (NK1) receptor antagonist SR-14033 significantly attenuated SP-induced increases in P(pa), P(v), P(c), R(a), and K(fc) in SL lungs. In lungs exposed to HA for 2 wk, SR-14033 suppressed the effect of SP on P(pa). Also, chronic hypoxia induced significant increases in NK1 receptors and NK1 receptor mRNA, with a temporal trend. We conclude that chronic hypoxia temporally augments SP-induced vascular responses, which are closely associated with increases in NK1 receptors and gene expression.     

Dopamine and ventilatory effects of hypoxia and almitrine in chronically hypoxic rats

We hypothesized that the temporary blunted ventilatory response to hypoxia seen in chronically hypoxic rats could be related to the increased amount of dopamine found in their carotid bodies. Rats, kept 2-3 wk in 10% O2, showed reduced nonisocapnic ventilatory responses to 21-12% inspiratory O2 fraction compared with control rats. Stimulus-response curves to almitrine, which simulates the action of hypoxia on the carotid body, were also depressed in chronically hypoxic rats. Responses to hypoxia and almitrine were significantly correlated in the two groups of rats. Dopamine depressed ventilation during normoxia, hypoxia, and almitrine stimulation in both groups, an action abolished by the dopamine-2 antagonist domperidone. Domperidone slightly increased responses to hypoxia and almitrine in control rats but had a greater enhancing effect in chronically hypoxic rats, such that there was no longer a difference between the responses of the two groups.     

Absence of MK801-induced inspiratory prolongation in chronically hypoxic rats.

N-methyl-D-aspartate (NMDA) receptors play important roles in the neural control of respiration. We hypothesized that the brainstem circuit for respiratory control is modulated in response to chronic hypoxia during postnatal maturation, and the modulation may involve changes in the neurotransmission mediated by the NMDA receptors for inspiratory termination. Electrophysiological studies were performed on anesthetized, vagotomized, paralyzed and ventilated rats. Phrenic nerve activity was recorded in normoxic control and chronically hypoxic (CH) rats maintained in normobaric hypoxia (10% O2) for 4-5 weeks from birth. In normoxic rats, the NMDA receptor antagonist, dizocilpine (MK801, i.p.) irreversibly increased inspiratory time (Ti) by 53% and decreased expiratory time (Te) by 29%. However, MK801 did not change the Ti, Te, respiratory rate and peak phrenic nerve activity in CH rats. Results suggest that brainstem mechanisms underlying inspiratory termination mediated by NMDA receptors are modulated by early chronic hypoxia.     

Conditioned medium from hypoxic cells protects cardiomyocytes against ischemia

The hypothesis of the present study is that cardiomyocytes subjected to prolonged ischemia, may release survival factors that will protect new cardiac cells from ischemic stress. We exposed neonatal rat cardiomyocyte primary cultures to hypoxia, collected the supernatant, treated intact cardiac cells by this posthypoxic supernatant, and exposed them to hypoxia. The results show cardioprotection of the treated cells compared with the untreated ones. We named the collected posthypoxic supernatant "conditioned medium" (CM), which acts in a dose-dependent manner to protect new cardiac cells from hypoxia: 100 or 75% of CM diluted in phosphate-buffered saline (PBS) protected cells as if they were not exposed to hypoxia (P < 0.001). When CM was removed from the cells before hypoxia, protection was not observed. CM also protected skeletal muscle cultures from hypoxia, but not cardiac cells against H(2)O(2)-induced cell damage. Finally, CM treatment protected the isolated heart in Langendorff set-up against ischemia. Smaller infarct size (9.9 ± 4.4% vs. 28.3 ± 8.5%, P < 0.05), better Rate Pressure Product (67 ± 11% vs. 48.6 ± 13.4%, P < 0.05) and better rate of contraction and relaxation were observed following ischemia and reperfusion (1341 ± 399 mmHg/s vs. 951 ± 349 mmHg/s, P < 0.05 and 1053 ± 347 mmHg/s vs. 736 ± 314 mmHg/s, P < 0.05). To conclude, there are factors that are released from the heart cells subjected to ischemia/hypoxia that protects cardiomyocytes from ischemic stress.