Modulation of total ceramide and constituent ceramide species in the acutely and chronically hypoxic mouse heart at different ages

Ceramide has been implicated in regulatory processes vital for cell survival under different stressors, most notably hypoxia. Little has been done to investigate the contributions of the different ceramide species to the regulation of cell survival. This study aims to highlight the patterns of variation in total ceramide and its species in the growing and hypoxic mouse heart. Mus musculus mice were placed in a hypoxic environment at birth. Control animals remained in room air. The hearts were extracted at different time points: 1 day, 1 week, 4 weeks, and 8 weeks. The total ceramide content and the amounts of component species were assayed by a modified diacylglycerol kinase assay and high-performance liquid chromatography-tandem mass spectroscopy, respectively. Data was collected from both ventricles in hypoxic and control conditions. There was significant polycythemia in the hypoxic versus control animals with a nearly twofold increase in hematocrit levels. Hypoxic right ventricle (RV) mass significantly increased over that of controls at different age groups. When ceramide content was compared in the hypoxic versus control animals, there was a significant increase at day 1 and a significant decrease at week 4 in the left ventricle, whereas a significant decrease was found in the RV at 1 week, 4 weeks, and 8 weeks. There was also a differential involvement of the RV with regard to levels of N-palmitoyl-D-erythro-sphingosine (C16-Cer) and its synthetic precursor dihydro-N-palmitoyl-D-erythro-sphinganine (DHC-16-Cer). The decrease in C16-Cer observed in both hypoxic and control RV's over time was paralleled by a significant increase in DHC-16-Cer in hypoxic (142.1+/-15.0 pmol; p<0.05) but not control (52.8+/-4.0 pmol) RV's suggesting a role for DHC-16-Cer in the RV adaptive response to hypoxia. Another species, N-arachidoyl-D-erythro-sphingosine (C20-Cer), was specifically and significantly decreased in the hypoxic RV. These studies support the presence of distinct roles for different ceramide species and their precursors. A better assessment of cyanotic congenital heart disease in light of the mechanism and timing of cardiomyocyte death, will lead to punctual interventions and even novel cardioprotective strategies.     

Exercise delays the hypoxic thermal response in rats.

Exercise exacerbates acute mountain sickness. In infants and small mammals, hypoxia elicits a decrease in body temperature (Tb) [hypoxic thermal response (HTR)], which may protect against hypoxic tissue damage. We postulated that exercise would counteract the HTR and promote hypoxic tissue damage. Tb was measured by telemetry in rats (n = 28) exercising or sedentary in either normoxia or hypoxia (10% O2, 24 h) at 25 degrees C ambient temperature (Ta). After 24 h of normoxia, rats walked at 10 m/min on a treadmill (30 min exercise, 30 min rest) for 6 h followed by 18 h of rest in either hypoxia or normoxia. Exercising normoxic rats increased Tb ( degrees C) vs. baseline (39.68 +/- 0.99 vs. 38.90 +/- 0.95, mean +/- SD, P < 0.05) and vs. sedentary normoxic rats (38.0 +/- 0.09, P < 0.05). Sedentary hypoxic rats decreased Tb (36.15 +/- 0.97 vs. 38.0 +/- 0.36, P < 0.05) whereas Tb was maintained in the exercising hypoxic rats during the initial 6 h of exercise (37.61 +/- 0.55 vs. 37.72 +/- 1.25, not significant). After exercise, Tb in hypoxic rats reached a nadir similar to that in sedentary hypoxic rats (35.05 +/- 1.69 vs. 35.03 +/- 1.32, respectively). Tb reached its nadir significantly later in exercising hypoxic vs. sedentary hypoxic rats (10.51 +/- 1.61 vs. 5.36 +/- 1.83 h, respectively; P = 0.002). Significantly greater histopathological damage and water contents were observed in brain and lungs in the exercising hypoxic vs. sedentary hypoxic and normoxic rats. Thus exercise early in hypoxia delays but does not prevent the HTR. Counteracting the HTR early in hypoxia by exercise exacerbates brain and lung damage and edema in the absence of ischemia.     

Pulmonary metabolic function in the awake lamb: effect of development and hypoxia

The effect of postnatal development and acute alveolar hypoxia on pulmonary metabolic function was studied in conscious newborn lambs. Measurements of the ability of the lungs of these animals to metabolize [3H]benzoyl-L-phenyl-alanyl-L-alanyl-L-proline ([3H]BPAP; a synthetic substrate for angiotensin-converting enzyme, ACE) and to remove 5-hydroxy-[14C]tryptamine (5-[14C]HT) were made by modified indicator-dilution techniques during normoxic and hypoxic (fraction of inspired O2 = 0.10) conditions at 1 day, 1 wk, and 1 mo of age. Six additional sheep (8-23 wk old) were studied acutely as "adult" controls. BPAP metabolism in the 1-day-old group was 48 +/- 3% and increased slowly to 57 +/- 1% (P less than 0.05) at 1 mo of age and to 79 +/- 3% (P less than 0.01) by 23 wk of age. Pulmonary 5-[14C]HT removal was adultlike at birth (69 +/- 2%). Alveolar hypoxia significantly decreased BPAP only in the 1-day-old group (41 +/- 3%; P less than 0.05) and had no significant effect on 5-[14C]HT removal over the range of ages studied. These data demonstrate a selective and gradual postnatal development of pulmonary ACE which could be due to alterations in either the affinity or maximal capacity of pulmonary ACE, or increased endothelial cell surface area secondary to rapid growth of small blood vessels in this period. Alveolar hypoxia does not appear to closely regulate either ACE activity or 5-HT removal in conscious lambs greater than 1 day old when trace amounts of substrate are used.     

L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.

Previously, our laboratory found that pulmonary hypertension developed and lung nitric oxide (NO) production was reduced when piglets were exposed to chronic hypoxia (Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung Cell Mol Physiol 274: L517-L526, 1998). The purposes of this study were to determine whether L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10% O(2) (chronic hypoxia) for 10-12 days. Lung NO production was assessed in isolated lungs from both groups by measuring the perfusate accumulation of nitrites and nitrates (collectively termed NO(-)(x)) before and after addition of L-arginine (10(-2) M) to the perfusate. The rate of perfusate NO(-)(x) accumulation increased by 220% (from 0.8 +/- 0.4 to 2.5 +/- 0.5 nmol/min, P < 0.05) after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 +/- 0. 8 before vs. 3.3 +/- 0.4 nmol/min after L-arginine) in control lungs. In the second series of studies, L-arginine uptake was evaluated by measuring the perfusate concentration of L-[(3)H]arginine at fixed time intervals. The perfusate concentration of L-[(3)H]arginine at each time point was less (P < 0.05) in control than in chronic hypoxic lungs. Thus L-arginine uptake was impaired and may underlie in part the reduction in lung NO production that occurs when piglets are exposed to 10-12 days of chronic hypoxia. Moreover, these findings in isolated lungs lead to the possibility that L-arginine supplementation might increase in vivo lung NO production in piglets with chronic hypoxia-induced pulmonary hypertension.     

Polycythemia and vascular remodeling in chronic hypoxic pulmonary hypertension in guinea pigs.

Chronic hypoxia increases pulmonary arterial pressure (PAP) as a result of vasoconstriction, polycythemia, and vascular remodeling with medial thickening. To determine whether preventing the polycythemia with repeated bleeding would diminish the pulmonary hypertension and remodeling, we compared hemodynamic and histological profiles in hypoxic bled (HB, n = 6) and hypoxic polycythemic guinea pigs (H, n = 6). After 10 days in hypoxia (10% O2), PAP was increased from 10 +/- 1 (SE) mmHg in room air controls (RA, n = 5) to 20 +/- 1 mmHg in H (P less than 0.05) but was lower in HB (15 +/- 1 mmHg, P less than 0.05 vs. H). Cardiac output and pulmonary artery vasoreactivity did not differ among groups. Total pulmonary vascular resistance increased from 0.072 +/- 0.011 mmHg.ml-1.min in RA to 0.131 mmHg.ml-1.min in H but was significantly lower in HB (0.109 +/- 0.006 mmHg.ml-1.min). Hematocrit increased with hypoxia (57 +/- 3% in H vs. 42 +/- 1% in RA, P less than 0.05), and bleeding prevented the increase (46 +/- 4% in HB, P less than 0.05 vs. H only). The proportion of thick-walled peripheral pulmonary vessels (53.2 +/- 2.9% in HB and 50.6 +/- 4.8% in H vs. 31.6 +/- 2.6% in RA, P less than 0.05) and the percent medial thickness of pulmonary arteries adjacent to alveolar ducts (7.2 +/- 0.6% in HB and 7.0 +/- 0.4% in H vs. 5.2 +/- 0.4% in RA, P less than 0.05) increased to a similar degree in both hypoxic groups. A similar tendency was present in larger bronchiolar vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gender differences in the endocrine and metabolic responses to hypoxic exercise.

This study tested the hypothesis that women would have blunted physiological responses to acute hypoxic exercise compared with men. Fourteen women taking oral contraceptives (28 +/- 0.9 yr of age) and 15 men (30 +/- 1.0 yr of age) with similar peak O(2) consumption (VO(2 peak)) values (56 +/- 1.1 vs. 57 +/- 0.8 ml x kg fat-free mass(-1) x min(-1)) were studied under hypoxic (H; fraction of inspired oxygen = 13%) vs. normoxic (fraction of inspired oxygen = 20.93%) conditions. Cardiopulmonary, metabolic, and neuroendocrine measures were taken before, during, and 30 min after three 5-min consecutive workloads at 30, 45, and 60% VO(2 peak). In women compared with men, glucose levels were greater during recovery from H (P < 0.05) and lactate levels were lower at 45% VO(2 peak), 60% VO(2 peak), and up to 20 min of recovery (P < 0.05), regardless of trial (P < 0.0001). Although the women had greater baseline levels of cortisol and growth hormone (P < 0.0001), gender did not affect these hormones during H or exercise. Catecholamine responses to H were also similar between genders. Thus the endocrine response to hypoxia per se was not blunted in women as we had hypothesized. Other mechanisms must be at play to cause the gender differences in metabolic substrates in response to hypoxia.     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH.     

Chronic hypercapnia inhibits hypoxic pulmonary vascular remodeling

Chronic hypercapnia is commonly found in patients with severe hypoxic lung disease and is associated with a greater elevation of pulmonary arterial pressure than that due to hypoxia alone. We hypothesized that hypercapnia worsens hypoxic pulmonary hypertension by augmenting pulmonary vascular remodeling and hypoxic pulmonary vasoconstriction (HPV). Rats were exposed to chronic hypoxia [inspiratory O(2) fraction (FI(O(2))) = 0.10], chronic hypercapnia (inspiratory CO(2) fraction = 0.10), hypoxia-hypercapnia (FI(O(2)) = 0.10, inspiratory CO(2) fraction = 0.10), or room air. After 1 and 3 wk of exposure, muscularization of resistance blood vessels and hypoxia-induced hematocrit elevation were significantly inhibited in hypoxia-hypercapnia compared with hypoxia alone (P < 0.001, ANOVA). Right ventricular hypertrophy was reduced in hypoxia-hypercapnia compared with hypoxia at 3 wk (P < 0.001, ANOVA). In isolated, ventilated, blood-perfused lungs, basal pulmonary arterial pressure after 1 wk of exposure to hypoxia (20.1 +/- 1.8 mmHg) was significantly (P < 0.01, ANOVA) elevated compared with control conditions (12.1 +/- 0.1 mmHg) but was not altered in hypoxia-hypercapnia (13.5 +/- 0.9 mmHg) or hypercapnia (11.8 +/- 1.3 mmHg). HPV (FI(O(2)) = 0.03) was attenuated in hypoxia, hypoxia-hypercapnia, and hypercapnia compared with control (P < 0.05, ANOVA). Addition of N(omega)-nitro-L-arginine methyl ester (10(-4) M), which augmented HPV in control, hypoxia, and hypercapnia, significantly reduced HPV in hypoxia-hypercapnia. Chronic hypoxia caused impaired endothelium-dependent relaxation in isolated pulmonary arteries, but coexistent hypercapnia partially protected against this effect. These findings suggest that coexistent hypercapnia inhibits hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy, reduces HPV, and protects against hypoxia-induced impairment of endothelial function.     

PO2 modulation of paraquat-induced microvascular injury in isolated dog lungs

We determined the effects of paraquat (PQ) concentrations ranging from 10(-3) to 10(-2) M and three levels of venous PO2 [hypoxia (41 +/- 3 Torr), normoxia (147 +/- 8 Torr), and hyperoxia (444 +/- 17 Torr)] in the presence of 4 x 10(-3) M PQ on microvascular permeability in isolated blood-perfused dog lungs. Capillary filtration coefficient (Kf,c) increased and isogravimetric capillary pressure (Pc,i) decreased 3 h after perfusion with 10(-2) M PQ (n = 7) and 5 h after perfusion with 4 x 10(-3) M PQ (n = 6) but not with 10(-3) M PQ (n = 4). In hyperoxic lungs perfused with 4 x 10(-3) M PQ, Kf,c increased to nine times the base-line value 5 h after PQ [0.15 +/- 0.01 to 1.35 +/- 0.25 (SE) ml.min-1.cmH2O-1.100 g-1]. Pc,i significantly decreased from a base-line value of 9.4 +/- 0.2 to 7.1 +/- 0.4 cmH2O at 3 h. In hypoxic lungs perfused with 4 x 10(-3) M PQ (n = 5), Pc,i and Kf,c changes were not significantly different from those in normoxic lungs treated with PQ. Thus both hyperoxia and an increased dose of PQ shortened the latent period and increased the severity of the PQ-induced microvascular permeability lesion, but hypoxia failed to prevent the PQ damage.     

Hypoxia Differentially Reduces GABAA Receptor Density During Embryonic Chick Optic Lobe Development

It has been demonstrated that the CNS is severely affected by hypoxic-ischemic insults during the prenatal-perinatal period, including imbalance in excitatory and inhibitory neurotransmitter release. Using a previously developed model of acute normobaric hypoxic hypoxia on chick embryos, we studied alterations observed both on [3H]GABA binding saturation parameters and on lactate concentration on successive embryonic days (ED). While maximal density of GABA binding sites (Bmax) from the low-affinity site was significantly reduced in an age-dependent manner, earlier stages of development (ED12 and 16) proving more vulnerable (ED12: control = 5.48 +/- 0.20, hypoxia = 3.90 +/- 0.39 pmol/mg prot, P < .05; ED16: control = 3.89 +/- 0.26, hypoxia = 2.80 +/- 0.28 pmol/mg prot, P < .05), ligand affinity (Kd) values and kinetic constants of the high-affinity site remained unaltered. Not unlikely, a physiological hypoxic state prevailing from ED17 up to hatching time rendered the whole embryo less sensitive to an externally induced hypoxic state (ED17: control = 2.93 +/- 0.06, hypoxia = 2.38 +/- 0.04 pmol/mg prot, P < .05; ED18: control = 2.97 +/- 0.12, hypoxia = 2.87 +/- 0.27 pmol/mg prot). Lactate levels in chick optic lobe homogenates were constant during development. The increase observed after hypoxic treatment compared to control value was significant at all stages studied, but increased percentage changes proved similar, indicating that all days of development equally perceive externally induced hypoxia. In conclusion, the present work demonstrates that after normobaric hypoxic hypoxia at different embryonic days, the embryo senses the externally induced hypoxic state as from ED12, but the GABA(A) receptor is differentially affected. It may be speculated that a different subunit composition of GABA(A) receptor is assembled in order to build a more stable receptor capable of resisting the physiological hypoxic state observed during the last few days before hatching.     

Role of leukotriene C4 in pulmonary hypertension: platelet-activating factor vs. hypoxia

The aim of this study was to determine whether leukotriene C4 (LTC4) is a mediator of hypoxic pulmonary vasoconstriction. We hypothesized that similar increases in LTC4, detected in the lung parenchyma and pulmonary vascular compartment during cyclooxygenase blockade with indomethacin (INDO), would be observed during an equal increase in pulmonary arterial pressure caused by acute alveolar hypoxia (HYP, 100% N2) or platelet-activating factor (PAF, 10 micrograms into the pulmonary artery). Rat lungs were perfused at constant flow in vitro with an albumin-Krebs-Henseleit solution. Mean pulmonary arterial pressure (n = 6 per group) increased from a base line of 10.9 +/- 1.2 to 15.8 +/- 2.1 (HYP + INDO) and 15.5 +/- 1.9 (SE) Torr (PAF + INDO). LTC4 levels increased only in response to PAF + INDO; perfusate levels increased from 0.4 +/- 0.07 to 5.3 +/- 1.1 ng/40 ml, and lung parenchymal levels increased from 1.9 +/- 0.07 to 22.8 +/- 5.3 ng/lung. Diethylcarbamazine (lipoxygenase inhibitor) reduced PAF-induced lung parenchymal levels of LTC4 by 68% and pulmonary hypertension by 63%. We conclude that 1) LTC4 is not a mediator of hypoxic pulmonary vasoconstriction and 2) intravascular PAF is a potent stimulus for LTC4 production in the lung parenchyma.     

Perinatal history of hypoxia leads to lower vascular pressures and hyporeactivity to angiotensin II in isolated lungs of adult rats

The most dramatic changes in pulmonary circulation occur at the time of birth. We hypothesized that some of the effects of perinatal hypoxia on pulmonary vessels are permanent. We studied the consequences of perinatal exposure to hypoxia (12 % O2 one week before and one week after birth) in isolated lungs of adult male rats (approximately 12 weeks old) perfused with homologous blood. Perfusion pressure-flow relationship was tilted towards lower pressures in the perinatally hypoxic as compared to the control, perinatally normoxic rats. A non-linear, distensible vessel model analysis revealed that this was due to increased vascular distensibility in perinatally hypoxic rats (4.1 +/- 0.6 %/mm Hg vs. 2.3 +/- 0.4 %/mm Hg in controls, P = 0.03). Vascular occlusion techniques showed that lungs of the perinatally hypoxic rats had lower pressures at both the pre-capillary and post-capillary level. To assess its role, basal vascular tone was eliminated by a high dose of sodium nitroprusside (20 microM). This reduced perfusion pressures only in the lungs of rats born in hypoxia, indicating that perinatal hypoxia leads to a permanent increase in the basal tone of the pulmonary vessels. Pulmonary vasoconstrictor reactivity to angiotensin II (0.1-0.5 microg) was reduced in rats with the history of perinatal-hypoxia. These data show that perinatal hypoxia has permanent effects on the pulmonary circulation that may be beneficial and perhaps serve to offset the previously described adverse consequences.     

Synergistic effects of ANP and sildenafil on cGMP levels and amelioration of acute hypoxic pulmonary hypertension

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/ kg) or vehicle and exposed to acute hypoxia with and without ANP (10(-8)-10(-5) M ). Sildenafil decreased systemic blood pressure (103 +/- 10 vs. 87 +/- 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% +/- 9.4% in sildenafil-treated rats vs. 117.2% +/- 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r(2) = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.     

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide

In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.     

Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator

The mechanism of hypoxia-induced pulmonary vasoconstriction remains unknown. To explore the possible dependence of the hypoxic response on voltage-activated calcium (Ca2+) channels, the effects of BAY K 8644 (BAY), a voltage-dependent Ca2+ channel potentiator, were observed on the pulmonary vascular response to hypoxia of both the intact anesthetized dog and the perfused isolated rat lung. In six rat lungs given BAY (1 X 10(-6)M), hypoxia increased mean pulmonary arterial pressure (Ppa) to 30.5 +/- 1.7 (SEM) Torr compared with 14.8 +/- 1.2 Torr for six untreated rat lungs (P less than 0.01). After nifedipine, the maximum Ppa during hypoxia fell 14.1 +/- 2.4 Torr from the previous hypoxic challenge in the BAY-stimulated rats (P less than 0.01). BAY (1.2 X 10(-7) mol/kg) given during normoxia in seven dogs increased pulmonary vascular resistance 2.5 +/- 0.3 to 5.0 +/- 1.2 Torr X 1(-1) X min (P less than 0.05), and systemic vascular resistance 55 +/- 4.9 to 126 +/- 20.7 Torr X 1(-1) X min (P less than 0.05). Systemic mean arterial pressure rose 68 Torr, whereas Ppa remained unchanged. Administration of BAY during hypoxia produced an increase in Ppa: 28 +/- 1.5 to 33 +/- 1.9 Torr (P less than 0.05). Thus BAY, a Ca2+ channel potentiator, enhances the hypoxic pulmonary response in vitro and in vivo. This, together with the effect of nifedipine on BAY potentiation, suggests that increased Ca2+ channel activity may be important in the mechanism of hypoxic pulmonary vasoconstriction.     

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.     

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.     

Attenuation of chronic hypoxic pulmonary hypertension by simvastatin

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to improve multiple normal endothelial cell functions and inhibit vascular wall cell proliferation. We hypothesized that one such agent, simvastatin, would attenuate chronic hypoxic pulmonary hypertension. Male adult Sprague-Dawley rats were exposed (14 days) to normoxia (N), normoxia plus once-a-day administered simvastatin (20 mg/kg ip) (NS), hypoxia (10% inspired O2 fraction) (H), or hypoxia plus simvastatin (HS). Mean pulmonary artery pressure, measured in anesthetized, ventilated rats with an open-chest method, was reduced from 25 +/- 2 mmHg in H to 18 +/- 1 in HS (P < 0.001) but did not reach normoxic values (12 +/- 1 mmHg). Similarly, right ventricular/left ventricular plus interventricular septal weight was reduced from 0.53 +/- 0.02 in the H group to 0.36 +/- 0.02 in the HS group (P < 0.001). The increased hematocrit in H (0.65 +/- 0.02) was prevented by simvastatin treatment (0.51 +/- 0.01, P < 0.001). Hematocrit was similar in N versus NS. Alveolar vessel muscularization and medial thickening of vessels 50-200 microM in diameter induced by hypoxia were also significantly attenuated in the HS animals. Lung endothelial nitric oxide synthase (eNOS) protein expression in the HS group was less than H (P < 0.01) but was similar in N versus NS. We conclude that simvastatin treatment potently attenuates chronic hypoxic pulmonary hypertension and polycythemia in rats and inhibits vascular remodeling. Enhancement of lung eNOS expression does not appear to be involved in mediating this effect.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.