Ion exchange activity in pulmonary artery smooth muscle cells: the response to hypoxia

The purposes of this study were to determine 1) the presence of the major ion transport activities that regulate cytoplasmic pH (pH(c)) in cat pulmonary artery smooth muscle cells, i.e., Na+/H+ and the Na+-dependent and -independent Cl-/HCO3- exchange, 2) whether pH(c) changes in cells from small (SPAs) and large (LPAs) pulmonary arteries during hypoxia, and 3) whether changes in pH(c) are due to changes in the balance of exchange activities. Exchange activities as defined by physiological maneuvers rather than molecular identity were ascertained with fluorescence microscopy to document changes in the ratio of the pH(c) indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Steady-state pH(c) was higher in LPA than in SPA normoxic smooth muscle cells. SPAs and LPAs possessed all three transport activities; in HCO3- containing normoxic solutions, Cl-/HCO3- exchange rather than Na+/H+ exchange set the level of pH(c); in HCO3- containing hypoxic solutions, pH(c) increased in SPA and decreased in LPA cells; altering the baseline pH(c) of a cell type to that of the other did not change the direction of the pH(c) response during hypoxia. The absence of Na+ prevented hypoxia-induced alkalinization in SPA cells; in both cell types, inhibiting the Cl-/HCO3- exchange activities reversed the normal direction of pH(c) changes during hypoxia.     

Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response. RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde. The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35%. The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation. On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension. RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters. Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05). Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate. The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates. In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability. Deformability was measured with the same filtration technique. There was no difference in the deformability of hypoxic compared with normoxic RBCs. We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.     

Strength of pulmonary vascular response to regional alveolar hypoxia.

Regional alveolar hypoxia in the lung induces regional pulmonary vasoconstriction which diverts blood flow from the hypoxic area. However, the predominant determinant of the distribution of perfusion in the normal erect lung is gravity so that more perfusion occurs at the base than at the apex. To determine the strength of the regional alveolar hypoxic response in diverting flow with or against the gravity gradient a divided tracheal cannula was placed in anesthetized dogs and unilateral alveolar hypoxia created by venilating one lung with nitrogen while ventilating the other lung with oxygen to preserve normal systemic oxygentation. Scintigrams of the distribution of perfusion obtained with intravenous 13-N and the MGH positron camera revealed a 34 and 32 per cent decrease in perfusion to the hypoxic lung in the supine and erect positions and a 26 per cent decrease in the decubitus position with the hypoxic lung dependent (P equal to 0.94 from supine shift), indicating nearly equal vasoconstriction with shift of perfusion away from the hypoxic lung in all positions. Analysis of regional shifts in perfusion revealed an equal vasoconstrictor response from apex to base in the supine position but a greater response in the lower lung zones in the erect position where perfusion was also greatest.     

Atrial natriuretic factor attenuates the pulmonary pressor response to hypoxia

The influence of endogenous and exogenous atrial natriuretic factor (ANF) on pulmonary hemodynamics was investigated in anesthetized pigs during both normoxia and hypoxia. Continuous hypoxic ventilation with 11% O2 was associated with a uniform but transient increase of plasma immunoreactive (ir) ANF that peaked at 15 min. Plasma irANF was inversely related to pulmonary arterial pressure (Ppa; r = -0.66, P less than 0.01) and pulmonary vascular resistance (PVR; r = -0.56, P less than 0.05) at 30 min of hypoxia in 14 animals; no such relationship was found during normoxia. ANF infusion after 60 min of hypoxia in seven pigs reduced the 156 +/- 20% increase in PVR to 124 +/- 18% (P less than 0.01) at 0.01 microgram.kg-1.min-1 and to 101 +/- 15% (P less than 0.001) at 0.05 microgram.kg-1.min-1. Cardiac output (CO) and systemic arterial pressure (Psa) remained unchanged, whereas mean Ppa decreased from 25.5 +/- 1.5 to 20.5 +/- 15 mmHg (P less than 0.001) and plasma irANF increased two- to nine-fold. ANF infused at 0.1 microgram.kg-1.min-1 (resulting in a 50-fold plasma irANF increase) decreased Psa (-14%) and reduced CO (-10%); systemic vascular resistance (SVR) was not changed, nor was a further decrease in PVR induced. No change in PVR or SVR occurred in normoxic animals at any ANF infusion rate. These results suggest that ANF may act as an endogenous pulmonary vasodilator that could modulate the pulmonary pressor response to hypoxia.     

Chronic hypoxia opposes pregnancy-induced increase in uterine artery vasodilator response to flow

We tested the hypotheses that pregnancy increases the uterine artery (UA) vasodilator response to flow and that this increase is impaired under conditions of chronic hypoxia (30 days, simulated elevation 3,960 m). UA were isolated from 24 normoxic or chronically hypoxic midpregnant guinea pigs and studied with the use of pressure myography. Normoxic pregnancy increased UA flow vasodilator response and protected against a rise in wall shear stress (WSS). Chronic hypoxia opposed these effects, prompting vasoconstriction at high flow and increasing WSS above levels seen in normoxic pregnant UA. The nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA) eliminated the pregnancy-associated increase in flow vasodilation in normoxic UA, suggesting that increased nitric oxide production was responsible. The considerable residual vasodilation after nitric oxide synthase and cyclooxygenase inhibition implicated endothelial-derived hyperpolarizing factor (EDHF) as an additional contributor to flow vasodilation. l-NNA increased flow vasodilation in UA from chronically hypoxic animals, suggesting that chronic hypoxia may have lowered EDHF or elevated peroxynitrite production. In conclusion, flow is an important physiological vasodilator for the acute and more chronic UA dimensional changes required to increase uteroplacental blood flow during normal pregnancy. Chronic hypoxia may be a mechanism that opposes the pregnancy-associated rise in UA flow vasodilation, thereby increasing the incidence of preeclampsia and intrauterine growth restriction at a high altitude.     

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O2) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O2 levels respectively, p less than 0.05, compared to 21% O2 exposure values). Release of 3H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 microM for 5 min) or PGH2 (4 microM for 2 min) immediately after exposure. Endothelium exposed to 0% O2, but not to 10% O2, produced significantly less prostacyclin after addition of either arachidonic acid (25 +/- 5% of 21% O2 exposure values, n = 6, p less than 0.01) or PGH2 (31 +/- 3% of 21% O2 exposure values, n = 6, p less than 0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O2 level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O2 exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.     

Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium

Exposure of cultured bovine pulmonary artery endothelial cells to varying levels of hypoxia (10% or 0% O₂) for 4 hours resulted in a significant dose-dependent inhibition in endothelial prostacyclin synthesis (51% and 98%, at the 10% and 0% O₂ levels respectively, p <0.05, compared to 21% O₂ exposure values). Release of ³H-arachidonic acid from cellular pools was not altered by hypoxia. Some of the cells were incubated with arachidonic acid (20 μM for 5 min) or PGH₂ (4 μM for 2 min) immediately after exposure. Endothelium exposed to 0% O₂, but not to 10% O₂, produced significantly less prostacyclin after addition of either arachidonic acid (25 ± 5% of 21% O₂ exposure values, n=6, p <0.01) or PGH₂ (31 ± 3% of 21% O₂ exposure values, n=6, p <0.05). These results suggest that hypoxia inhibits cyclooxygenase at the 10% O₂ level and both cyclooxygenase and prostacyclin synthetase enzymes at the 0% O₂ exposure levels. Exposure of aortic endothelial cells resulted in a 44% inhibition of prostacyclin at the 0% exposure level. No significant alteration in prostacyclin production was found in pulmonary vascular smooth muscle cells exposed to hypoxia. These data suggest that the increased prostacyclin production reported in lungs exposed to hypoxia is not due to a direct effect of hypoxia on the main prostacyclin producing cells of the pulmonary circulation.     

Role of the gap junctions in the contractile response to agonists in pulmonary artery from two rat models of pulmonary hypertension

Background

Pulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH.

Methods

Intrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine.

Results

Cx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with ^37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with ⁴⁰Gap27 (a specific blocker of Cx 40) or ^37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by ⁴⁰Gap27 or ^37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following ^37-43Gap27 incubation.

Conclusion

Altogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.     

Dihydroceramide-based response to hypoxia

To understand the mechanisms of ceramide-based responses to hypoxia, we performed a mass spectrometry-based survey of ceramide species elicited by a wide range of hypoxic conditions (0.2-5% oxygen). We describe a rapid, time-dependent, marked up-regulation of dihydroceramides (DHCs) in mammalian cells and in the lungs of hypoxic rats. The increase affected all DHC species and was proportional with the depth and duration of hypoxia, ranging from 2- (1 h) to 10-fold (24 h), with complete return to normal after 1 h of reoxygenation at the expense of increased ceramides. We demonstrate that a DHC-based response to hypoxia occurs in a hypoxia-inducible factor-independent fashion and is catalyzed by the DHC desaturase (DEGS) in the de novo ceramide pathway. Both the impact of hypoxia on DHC molecular species and its inhibitory effect on cell proliferation were reproduced by knockdown of DEGS1 or DEGS2 by siRNA during normoxia. Conversely, overexpression of DEGS1 or DEGS2 attenuated the DHC accumulation and increased cell proliferation during hypoxia. Based on the amplitude and kinetics of DHC accumulation, the enzymatic desaturation of DHCs fulfills the criteria of an oxygen sensor across physiological hypoxic conditions, regulating the balance between biologically active components of ceramide metabolism.     

Developmental differences in pulmonary eNOS expression in response to chronic hypoxia in the rat.

Chronic hypoxia (CH) increases pulmonary endothelial nitric oxide synthase (eNOS) protein levels in adult rats but decreases eNOS protein levels in neonatal pigs. We hypothesized that this differing response to CH is due to developmental rather than species differences. Adult and neonatal rats were placed in either hypobaric hypoxia or normoxia for 2 wk. At that time, body weight, hematocrit, plasma nitrite/nitrate (NOx(-)), and right ventricular and total ventricular heart weights were measured. Percent pulmonary arterial wall area of 20-50 and 51-100 microm arteries were also determined. Total lung protein extracts were assayed for eNOS levels by using immunoblot analysis. Compared with their respective normoxic controls, both adult and neonatal hypoxic groups demonstrated significantly decreased body weight, elevated hematocrit, and elevated right ventricular-to-total ventricular weight ratios. Both adult and neonatal hypoxic groups also demonstrated significantly larger percent pulmonary arterial wall area compared with their respective normoxic controls. Hypoxic adult pulmonary eNOS protein and plasma NOx(-) were significantly greater than levels found in normoxic adults. In contrast, hypoxic neonatal pulmonary eNOS protein and plasma NOx(-) were significantly less compared with normoxic neonates. We conclude that there is a developmental difference in eNOS expression and nitric oxide production in response to CH.