Minutes Of Hypoxia Research Articles

Long-term Facilitation of Ventilation in Humans During NREM Sleep

The purpose of this study was to determine whether episodic hypoxic exposure would elicit long term facilitation (LTF) of ventilation (V(I)) in sleeping humans. Twenty subjects gave written informed consent. Of these, six subjects were unable to maintain stable stage 2 sleep or deeper for a majority of the experiment and their data were excluded from the analysis. On night 1 after subjects had reached stable sleep (stage 2 or deeper), the subjects breathed room air for 5 minutes, followed by 3 minutes of hypoxia (F(I)O2 = 8%). This sequence was repeated 10 times, and the breathing pattern was observed for a further 60 minutes. Subjects returned to the laboratory for a second visit, which served as a sham night. Instrumentation and study time were the same as on night 1, but subjects breathed room air only. Airflow, tidal volume (V(T)), end tidal O2 and CO2, and estimation of arterial O2 saturation (%) were measured. Seven of the subjects had long-term facilitation (LTF), which was manifested as a significant increase in V(I) that persisted for up to 40 minutes following the last hypoxic exposure. In the other seven subjects, no substantial increase in V(I) was found. We could not explain this difference based on body size (BMI), gender, level of hypoxemia, or magnitude of the hyperpnea during hypoxia. The difference between the two groups was that the LTF group consisted of habitual snorers, and that the NLTF were not inspiratory-flow-limited during the experiment.

Dopamine Depresses Minute Ventilation in Patients With Heart Failure

Low-dose dopamine is frequently used in patients in the intensive care setting. Dopamine may inhibit chemoreceptor afferents and hence decrease chemoreflex sensitivity to hypoxia. In a double-blind, randomized, crossover study, we determined the effects of dopamine (5 microg x kg(-1) x min(-1)) and placebo infusion on oxygen saturation, minute ventilation, and sympathetic nerve activity during normoxia and 5 minutes of hypoxia in 10 normal young subjects. We further investigated the effects of dopamine and placebo on minute ventilation during normoxic breathing in 8 patients with severe heart failure and in 8 age-matched control subjects. Dopamine did not decrease minute ventilation during normoxia in normal subjects. During hypoxia, minute ventilation was 12.9+/-1.3 L/min on dopamine and 15.8+/-1.5 L/min on placebo (P<0.0001). Oxygen saturation during hypoxia was lower with dopamine (78+/-3%) than placebo (84+/-2%; P<0.0001). Sympathetic nerve activity during hypoxia was not enhanced with dopamine despite the lower O2 saturation. Subjects were able to maintain a voluntary apnea to a lower oxygen saturation on dopamine than on placebo (P<0.05). In heart failure patients breathing room air, but not in age-matched control subjects, dopamine decreased minute ventilation despite decreased oxygen saturation and increased PETCO2 during dopamine (all P< or =0.02). Dopamine inhibits chemoreflex responses during hypoxic breathing in normal humans, preferentially affecting the ventilatory response more than the sympathetic response. Dopamine also depresses ventilation in normoxic heart failure patients breathing room air. Ventilatory inhibition by low-dose dopamine may adversely influence outcome in hypoxic patients, especially in patients with heart failure.

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture

Hypoxia-Mediated Modulation of Vascular Function—Implications for Organ Preservation and Thrombogenesis: Roger S. Mitchell Lecture

Ventilatory responses to sustained eucapnic hypoxia in healthy males during wakefulness and NREM sleep.

The effects of sustained eucapnic hypoxia (SEH, 20 minutes SaO2, approximately 80%) on ventilation and supraglottic airflow resistance (Rua) plus genioglossal (gg) and diaphragmatic (di) electromyograms (EMGs) were compared during wakefulness and nonrapid eye movement (NREM) sleep in six healthy normal male subjects. Early augmentation of ventilation was followed by decline or roll-off in both states. The augmentation of ventilation was less in sleep than wakefulness (e.g., after 5 minutes hypoxia, 140% and 167% of baseline, respectively, p < 0.05). This appeared to be due to three factors: 1) sleep-related increases in Rua [the ventilatory responses to SEH (sleep vs. awake) were inversely related to changes in Rua (sleep vs. awake) (p < 0.05)], 2. reduced central neural drive (inspiratory phasic EMG di after 5 minutes SEH, 111% and 121% of baseline, p < 0.05), and 3) failure to increase respiratory frequency during SEH sleep. There was also a nonsignificant trend to a biphasic response in EMG gg and a small increase in Rua during SEH.

The Ventilatory and Arousal Responses to Prolonged Hypoxia in Preterm Infants are Sleep State Dependent. 1575

Recently, we have shown that during the response to a more prolonged (20 min) hypoxia in preterm infants (Lemke, et al, Pediatr. Res., 39:389A, 1996), ventilation falls after initial hyperventilation and remains depressed between 5 and 20 min. To test the hypothesis that the response is influenced by the infant's sleep state and the presence or absence of arousal we studied 14 healthy preterm infants [BW 1.6±0.1 kg (mean±SE), SW 2.0±0.2 kg, GA 31±1 wk, PNA 27±4 d]. Ventilation was measured using a flow-through system. Sleep states were defined using EEG, EOG, and body movements. Arousal was defined using eye opening, body movements, agitation, and crying. The experimental procedure consisted in giving the infants 21% O2 for 2 min, 15% O2 for 20 min, followed by 2 min of recovery in 21% O2. Fourteen experiments were conducted in quiet sleep and 7 in REM sleep. Arousal was observed in 71% of experiments in quiet sleep (10 out of 14) and 0% in REM sleep (0 out of 7; p<0.01). In quiet sleep, minute ventilation remained above baseline at 5(6.7±2.5%), 10 (7.4±2.8%), 15 (3.4±2.1%), and 20(6.3±2.7%) minutes of hypoxia. On the contrary, in REM sleep minute ventilation decreased below baseline at 5 (-4.2±2.3), 10(-4.5±5.0), 15 (-7.4±2.3%), and 20 (-7.9±3.1%) minutes of hypoxia (p<0.001 quiet vs REM for all time points). During quiet sleep minute ventilation at 10, 15, and 20 min of hypoxia was significantly higher in infants who exhibited arousal (12.6±3.1%, 8.6±2.3%, and 12.0±3.1% respectively) than in infants who did not (-5.6±4.9%,-8.2±3.5%, and -4.8±4.4% respectively; p<0.001). The difference in minute ventilation at each time point was primarily related to a decrease in frequency with no significant change in VT. These observations suggest 1) preterm infant's ventilatory response to prolonged hypoxia is sleep state dependent, 2) arousal is more frequent during quiet sleep than in to REM sleep. We speculate that sleep state determines ventilatory and arousal responses to prolonged hypoxia in preterm infants which in turn may determine the infant's predisposition to SIDS.Supported by the Children's Hospital of Winnipeg Research Foundation.

Differential Effects of Short-term Hypoxia and Hypercapnia on N -Methyl- d -Aspartate–Induced Cerebral Vasodilatation in Piglets

Recent studies in piglets show that either asphyxia or global cerebral ischemia, which combines effects of hypoxia and hypercapnia, transiently attenuates N-methyl-D-aspartate (NMDA)-induced pial arteriolar dilation. The purpose of this study was to determine individually the effects of hypoxic hypoxia and normoxic hypercapnia on NMDA-dependent cerebrovascular reactivity. In addition, we examined mechanisms involved in reduced cerebral vascular dilation to NMDA. In anesthetized piglets, we examined pial arteriolar diameters using a cranial window and intravital microscopy. Arteriolar responses to topically applied NMDA were determined under control conditions and after arterial hypoxia or arterial hypercapnia. In addition, arteriolar responses to NMDA were examined in animals given indomethacin (10 mg/kg IV) or superoxide dismutase (100 U/mL, topical application) before hypoxia. Under control conditions, application of NMDA produced a dose-related dilation of pial arterioles (eg, 9 +/- 1% to 10(-5), 15 +/- 2% to 5 x 10(-5), and 28 +/- 5% to 10(-4) mol/L NMDA above baseline, respectively, in the hypoxic group; n = 6, P < .05). After transient exposure to 15 minutes of hypoxic hypoxia, arteriolar responses to NMDA were reduced at 30 minutes and at 60 minutes (10(-4) mol/L NMDA dilated by 12 +/- 5% and 18 +/- 5%, respectively; n = 6, P < .05). Five minutes of hypoxic hypoxia also reduced dilatation to NMDA. Indomethacin or superoxide dismutase preserved arteriolar responses to NMDA after 15 minutes of hypoxia. Pial arteriolar responses to NMDA remained unimpaired during and after hypercapnia. Short-term severe hypoxic hypoxia and reventilation impair the NMDA-induced dilatation of pial arterioles. Respiratory acidosis alone does not modify pial arteriolar reactivity to NMDA. The reduced responsiveness of the cerebral blood vessels to NMDA caused by hypoxia appears to be due to action of oxygen radicals.

Effect of theophylline on ventilatory roll-off during hypoxia in goats

The increase in pulmonary ventilation (V̇ e) during the first minutes of hypoxia is not sustained as after several minutes V̇ e decreases or “rolls-off” toward control levels. We hypothesized that intravenous infusion of theophylline, by blocking the central inhibitory effects on breathing of adenosine, would attenuate the hypoxic V̇ e roll-off. Twelve unanesthetized adult goats were exposed for 20 min to a 12% O 2-88% N 2 gas mixture. In some studies, theophylline was infused intravenously (IV) for 20 min before and during the hypoxia. The highest infusion rate of 6.0–8.0 mg/min was sufficient to totally prevent the arterial hypertension and bradycardia that occurred with IV infusion of 4 mg·min −1 of adenosine. Nine of the 12 goats demonstrated V̇ e roll-off without the theophylline infusion. In goats that demonstrated V̇ e roll-off without theophylline, a significant ( P < 0.05) V̇ e roll-off was observed even at the highest theophylline infusion rate. We therefore conclude that the V̇ e roll-off during hypoxia is not primarily or critically mediated by adenosine in awake, adult goats.

Influence of hypoxia and hypoxia-reoxygenation on endothelial P-selectin expression.

P-selectin is an endothelial adhesion molecule involved in the initial step of the neutrophil recruitment. We investigated the effect of hypoxia (95% N2, 5% CO2) and of hypoxia-reoxygenation (95% air, 5% CO2) on the expression of P-selectin by human umbilical vein endothelial cells (HUVEC). P-selectin expression was detected by immunolabelling and quantified by flow cytometric analysis. Our data indicate that hypoxia induces an increase in P-selectin expression with a maximum reached after 90 minutes. A hypoxic exposure of 90 minutes results in a highly significant increase compared to normoxia (p < 0.001, n = 13). Furthermore, when a reoxygenation period follows 90 minutes of hypoxia, the initially elevated levels of P-selectin are dramatically enhanced with a maximum obtained after 60 minutes of reoxygenation.

Cerebral and ventilatory depression during hypoxia in anaesthetized newborn guinea‐pigs

The effects of hypoxia on ventilation and cerebral activity were studied in urethane-anaesthetized newborn guinea-pigs. Ventilation was measured by a pneumotachograph, and cerebral activity by a cerebral function monitor (CFM). All animals were subjected to either 9% O2 or 6% O2 in N2 for 10 minutes or until apnoea occurred. Hypoxia produced a biphasic response in ventilation, that is, an increase followed by a decrease. The initial increase was attributed to the elevation of the respiratory rate, whereas the tidal volume showed a pure decline. The respiratory rate reached its peak at 3 minutes of hypoxia (170 +/- 12% during 9% O2 and 169 +/- 12% during 6% O2). Cerebral activity during both 9 and 6% O2 breathing showed a small increase followed by a decrease. In the group subjected to 9% O2 the maximum CFM activity increased to 114 +/- 8% of the control level and the minimum activity increased to 113 +/- 7%, while in the group subjected to 6% O2 the maximum CFM activity increased to 104 +/- 5% and the minimum CFM activity to 101 +/- 3%. The depression of CFM activity was more pronounced with 6% O2 than with 9% O2. Regression analysis showed a linear correlation between ventilation and cerebral activity during both 9 and 6% O2 breathing. The results suggest that hypoxic ventilatory depression may be the consequence of cerebral depression produced by acute severe hypoxia.

Angiotensin II Contributes to Cerebral Vasodilatation During Hypoxia in the Rabbit

and Purpose Hypoxia increases cerebral blood flow (CBF). Hypoxia also exerts a major influence on the renin-angiotensin system. In addition to the circulating renin-angiotensin system, a local renin-angiotensin system appears to be present in the brain, and angiotensin II receptors have been identified in cerebral blood vessels. In this study we tested the hypothesis that endogenous angiotensin II attenuates dilatation of the cerebral vessels during hypoxia. Pentobarbital-anesthetized rabbits were prepared for measurement of blood flow (microspheres) and assigned to one of two groups: in group 1 (n = 11), rabbits were subjected to 30 minutes of stable hypoxia (PaO2 = 34 +/- 1 mm Hg, mean +/- SD) followed by 15 minutes of reoxygenation (PaO2 = 177 to 200 mm Hg). Blood flow was measured four times: under control conditions, after 15 and 30 minutes of hypoxia, and after 15 minutes of reoxygenation. This was a control group to characterize changes in CBF during hypoxia. In group 2 (n = 11), blood flow was measured as in the previous group except that an infusion of the angiotensin II receptor antagonist saralasin (1 microgram.kg-1.min-1 IV) was started with the onset of hypoxia and continued through reoxygenation to the end of the experiment. The goal of this group was to examine whether endogenous activation of receptors for angiotensin II influences increases in CBF during hypoxia. In a separate series of experiments we examined the influence of the angiotensin-converting enzyme (ACE) inhibitor captopril on the hypoxic response. Thus, in one group of rabbits we measured CBF in the same manner as in group 1 (n = 13). In another group of rabbits we also measured blood flow as in group 1 except that rabbits received 10 mg/kg of the ACE inhibitor captopril before the control measurement (n = 11). We tested for significant differences between groups using two-way ANOVA. Under control conditions, CBF was similar in all groups and averaged 53 +/- 15 mL.min-1.100 g-1. During hypoxia, CBF increased to a greater extent in the absence versus the presence of saralasin (95 +/- 31 and 104 +/- 30 mL.min-1.100 g-1 versus 72 +/- 24 and 71 +/- 25 mL.min-1.100 g-1, respectively; P = .003). Increase in CBF during hypoxia was also significantly greater in the animals that did not receive captopril versus those that were treated with captopril (100 +/- 24 and 89 +/- 16 mL.min-1.100 g-1 versus 72 +/- 16 and 73 +/- 17 mL.min-1.100 g-1). To rule out the possibility that saralasin produced non-specific attenuation of cerebral vasodilatation, we tested the influence of hypercapnia on CBF in the absence and presence of saralasin. During normocapnia, CBF values were not significantly different in the absence and presence of saralasin (57 +/- 17 and 64 +/- 6 mL.min-1.100 g-1, respectively; P > .05). Hypercapnia increased CBF similarly in the absence and presence of saralasin (81 +/- 22 and 91 +/- 19 mL.min-1.100 g-1; PaCO2 = 61 +/- 2 and 60 +/- 2 mm Hg, respectively; P > .05). Because the ACE inhibitor captopril and the angiotensin II receptor blocker saralasin attenuated increased in CBF during hypoxia, the findings suggest that endogenous release of angiotensin II contributes to the increase in CBF during hypoxia.

Antiarrhythmic agent, MS-551, protects against pinacidil + hypoxia-induced ventricular fibrillation in Langendorff-perfused rabbit isolated heart.

We studied the electrophysiologic and antifibrillatory effects of the class III agent MS-551 in a rabbit isolated heart model in which ventricular fibrillation (VF) occurs reproducibly under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener, pinacidil. Ten minutes after MS-551 or vehicle administration, addition of pinacidil (1.25 microM) to the buffer was followed by a 12-min hypoxic period and 40-min reoxygenation. At a low concentration of MS-551 (1.0 microM), VF occurred in 5 of 6 hearts, the same incidence as in the control group (5 of 6). In contrast 0 of 6 hearts treated with 15 microM MS-551 developed VF (p < 0.05 vs. vehicle). Ventricular effective refractory period (VERP) was determined in a separate group of isolated hearts (n = 13). Pinacidil alone, during normoxic perfusion, decreased VERP 48 +/- 11% (p < 0.05) 15 min after exposure. Five minutes of hypoxia alone also decreased VERP (57 +/- 8%, p < 0.05). Under normoxic conditions, MS-551 increased ERP 31 +/- 10% (p < 0.05 vs. baseline). VERP prolongation by MS-551 was reduced in the presence of pinacidil but remained 22 +/- 6% (p < 0.05) above baseline. The results suggest that VERP shortening owing to pinacidil-mediated ATP-dependent K+ channel opening is associated with development of VF in isolated heart. MS-551 attenuates the pinacidil-mediated decrease in VERP and prevents pinacidil+hypoxia-reoxygenation-induced VF. Because pinacidil and hypoxia open myocardial KATP channels, putatively decreasing VERP, MS-551 may exert its antifibrillatory effect through partial blockade of KATP channels.

Metabolic alterations accompany ionic disturbances and cellular swelling during a hypoxic insult to the retina: an in vitro study.

To study the ionic, metabolic, and morphologic derangements that occur following brain injury we utilized a retina in vitro model of hypoxia. Retinas were dissected into oxygenated (95% O2, 5% CO2) Ames medium, a physiologic solution resembling cerebrospinal fluid, and randomly assigned to either experimental hypoxic conditions (95% N2, 5% CO2) or control conditions. All retinas were incubated and maintained at 37 degrees C. Changes in extracellular K+ and lactate concentration, intracellular incorporation of 45Ca and 14C-leucine, uptake of glucose using [14C]-2-deoxy-D-glucose (2DG), and cell size were determined at 10, 20, 30, and 60 minute time intervals. The results show that compared to control retinas hypoxia produced: (1) an early increase in extracellular concentration of K+ and lactate, (2) a delayed increase in the intracellular incorporation of 45Ca, (3) an early onset of cellular swelling, and (4) a decrease in the intracellular incorporation of 14C-leucine, and (5) increased glucose utilization. All of the results were statistically significant (p < 0.05) and exhibited a dose response relationship with the exception of intracellular incorporation of 45Ca which did not become significantly different until 30 minutes post-hypoxia. A 16% increase in cell size was noted after 10 minutes of hypoxia. Increased hypoxic cell size persisted for 30 minutes but after 60 minutes the control retinas appeared enlarged as well. Our results suggest that ionic, metabolic, and morphologic derangements can be demonstrated utilizing an in vitro model of hypoxia which are similar to those seen following in vivo traumatic brain injury. With use of this model the mechanisms behind these ionic-metabolic relationships can be addressed at the molecular level.

Hypoxic Pulmonary Hypertension: Changes in Platelet Size and Number

Circulating platelets have been implicated in the hypoxic pulmonary pressor response. This study was undertaken to assess the effects of acute hypoxia-induced pulmonary hypertension on platelet volume and number across the pulmonary circulation in anesthetized newborn lambs. Seven animals were instrumented for measurement of pulmonary vascular resistance. All measurements were made during normoxia and after 5 and 30 minutes of hypoxia (10 to 12% oxygen breathing). Hypoxia caused a doubling of the pulmonary vascular resistance. During hypoxic vasoconstriction, platelet volume decreased rapidly while traversing the lung but was not affected on return to the lung after traversing the systemic circulation. Platelet numbers were unchanged on leaving the lung but were decreased on entering the lung during hypoxia. Our data are consistent with the release of platelet contents in the lung during hypoxic pulmonary hypertension.

Delineation of the influence of propionylcarnitine on the accumulation of long-chain acylcarnitines and electrophysiologic derangements evoked by hypoxia in canine myocardium

To investigate the potential influence on one analogue of carnitine on the electrophysiologic derangements elicited by myocardial ischemia and subsequent reperfusion, we evaluated whether increasing concentrations of propionylcarnitine would interact with carnitine acyltransferase I and thereby decrease the accumulation of long-chain acylcarnitines during hypoxia in isolated adult canine myocytes. Propionylcarnitine (1-100 microM) did not alter the sixfold reversible increase in long-chain acylcarnitines elicited by 10 minutes of hypoxia. Likewise, propionylcarnitine did not alter the reversal of the accumulation of long-chain acylcarnitines associated with reoxygenation of hypoxic myocytes. To assess whether analogues of carnitine could influence the development or reversal of the electrophysiologic derangements induced by hypoxia in adult canine epicardial tissue, selected concentrations of propionylcarnitine (1 microM to 10 mM) were administered prior to and during 15 minutes of hypoxic perfusion at 35 degrees C followed by 5-20 minutes of reoxygenation. Continuous intracellular transmembrane action potentials were recorded with glass microelectrodes. Administration of propionylcarnitine prior to and during hypoxia did not alter the electrophysiologic derangements elicited by hypoxia or subsequent reoxygenation. Therefore, propionylcarnitine does not influence the activity of carnitine acyltransferase I and does not alter the accumulation of long-chain acylcarnitines during hypoxia. Although propionylcarnitine may protect ischemic myocardium by enhancing the recovery of contractile function during reperfusion, propionylcarnitine does not attenuate any of the electrophysiologic alterations observed during hypoxia or subsequent reoxygenation in isolated tissue.

Increased Lactate Appearance and Reduced Clearance During Hypoxia in Dogs

In order to assess the effects of severe hypoxia on whole body glucose and lactate kinetics, nine experiments were performed on anesthetized, ventilated mongrel dogs. [U-13C]glucose and [1-14C]lactate (n = 5), or [6-14C]glucose and [U-13C]lactate (n = 4) were infused using the primed-continuous infusion method. Cardiac output was measured by thermodilution. After a control period with 21% O2, inspired O2 was reduced for 90 minutes. Three of the experiments resulted in unstable hemodynamics and lactate levels, and are excluded from the mean data. Arterial PO2 fell from a control level of 106.8 +/- 11.9 to 24.2 +/- 3.5 mmHg during the last 45 minutes of hypoxia, and O2 transport fell to 52% of normoxic values. Arterial lactate concentration and the rate of appearance increased by 428% and 182%, respectively, from control to hypoxia. The metabolic clearance rate for lactate fell by 34%. Arterial glucose levels did not change significantly with hypoxia, but the rate of glucose disappearance rose by 70%, and the rate of glucose conversion to lactate increased 3-fold. It is concluded that acute severe hypoxia in anesthetized dogs causes 1) a large increase in arterial lactate levels, but no significant change in glycemia, 2) a large increase in the rate of lactate disappearance and only a small increase in the rate of glucose disappearance and 3) a fall in the metabolic clearance rate of lactate.

ウサギ新生仔脳室上衣下層の低酸素負荷による超微形態学的変化に関する研究

Fetal and neonatal hypoxia is suggested as a noticeable risk factor related to the occurrence of subependymal hemorrhages. To investigate the cause of newborn subependymal hemorrhages, a 0 day old newborn rabbit was exposed to either 5 or 10 minutes hypoxia by N2 box. After exposure, the brain was observed by optical and electric microscope using horseradish peroxidase (HRP) as the protein tracer to study the change in the capillary permeability. The result obtained were as follows: 1) In the newborn rabbit blood gas analysis, PO2 (mmHg) decreased more in the 5 and 10 min hypoxia groups than the control group which used the N2 box. pH and BE changes were similar to PO2 (mmHg). On the other hand, PCO2 (mmHg) increased proportionately. These results indicated that this hypoxic method changed blood gas and caused acidosis. 2) In the optical microscopic examination, the 0 day old newborn rabbit brain was found to have a thin subependymal layer. We could not find obvious subependymal hemorrhage using optical microscope. 3) In the electric microscopic examination, HRP was found in the cavity of the brain capillary at the subependymal layer and was slightly incorporated into the pinocytic vesicle of the lumen on the control. Since the tight junction filled the roll out, HRP was not found outside the capillary. 4) The 5 min hypoxia group caused astrocyte foot swelling, edema and slight opening of the tight opening of the tight junction. HRP was passed through the tight junction, but not outside the capillary. 5) The 10 min hypoxia group caused destruction of astrocyte and edema around the capillary. The tight junction was more opened than in the 5 min hypoxia group and HRP leaked out of the capillary through the tight junction. From the findings mentioned above, it is suggested that our hypoxic model increased permeability of the capillary in subependymal layers and that this change might be the first ultrastructural change before the onset of subependymal hemorrhages.

Direct measurement of changes in intracellular calcium transients during hypoxia, ischemia, and reperfusion of the intact mammalian heart.

In studies of ischemia and reperfusion, a major experimental problem has been the inability to measure intracellular ionized calcium ([Ca2+]i) in the intact heart. We have developed a new approach in which the bioluminescent calcium indicator aequorin is used to measure [Ca2+]i in the isolated, coronary-perfused ferret heart. Aequorin is loaded into subepicardial myocytes of the left ventricle, and the signals are recorded simultaneously along with isovolumic left ventricular (LV) pressure at a constant pacing rate. This system shows 1) no attenuation or change of time course of LV pressure development or coronary perfusion pressure after aequorin loading; 2) consistent responses to physiological interventions and drugs; 3) individual aequorin and pressure signals that do not require signal averaging for analysis; and 4) [Ca2+]i levels comparable with those reported in tissue or isolated myocyte cell preparations. During 5 minutes of hypoxia, diastolic [Ca2+]i and LV diastolic pressure increased while the systolic values of both [Ca2+]i and pressure decreased. The peak-to-peak systolic [Ca2+]i versus LV isovolumic pressure relation remained close to the control curve. In contrast, during 3 minutes of global ischemia, LV systolic and diastolic pressures fell rapidly, while [Ca2+]i increased substantially. The [Ca2+]i versus pressure relations for both systole and diastole shifted to the right, indicating desensitization of the contractile apparatus to [Ca2+]i. These results provide evidence that different primary mechanisms determine the systolic and diastolic responses to acute hypoxia versus ischemia. During hypoxia, changes in [Ca2+]i handling probably play a major role, while during ischemia, changes in the Ca2+ sensitivity of the myofilaments appear to be of primary importance in the modulation of contractile dysfunction.

Acute effects of hypoxia and phosphate on two populations of heart mitochondria.

Initial Polytron treatment with subsequent exposure to the bacterial proteinase Nagarse has been shown to result in the isolation of two distinct populations of cardiac mitochondria, subsarcolemmal and interfibrillar mitochondria, respectively. Although these populations have been shown to possess distinct biochemical properties, few studies have been reported which document the potential differences in their response to pathological insult. We therefore examined the effect of acute hypoxia with or without reoxygenation as well as treatment with phosphate on oxidative phosphorylation on both groups of mitochondria. Freshly-isolated interfibrillar mitochondria (IFM) exhibited significantly higher respiratory values, with the exception of the ADP:O ratios, than subsarcolemmal mitochondria (SLM). With pyruvate-malate as respiratory substrate, 40 minutes hypoxia alone produced no effect on SLM whereas a stimulation in respiration was seen in IFM. A 40-minute reoxygenation period depressed the oxidative phosphorylation rate in SLM whereas it was stimulated in IFM. These treatments did not produce any effect in either population when succinate was the substrate of choice. Because of the latter observation, the possibility that increased lability of complex I of the electron transport chain accounted for the differences associated with NAD-linked substrates was studied by assessing NADH oxidation of sonicated mitochondria following the treatments. SLM exhibited enhanced permeability to exogenous NADH as well as increased sensitivity to sonication following either hypoxia or hypoxia/reoxygenation compared to IFM. Compared to hypoxia/reoxygenation, increasing concentrations of phosphate (5-15 mM) produced a marked depression in oxidative phosphorylation of SLM whereas IFM were relatively resistant. The toxic effects of phosphate were much more evident with pyruvate-malate as substrates; with succinate, oxidative phosphorylation of IFM was not depressed by phosphate whereas only a slight depression was observed with SLM. The latter population similarly exhibited reduced NADH oxidation following phosphate treatment whereas IFM were unaffected. Our studies show a differential sensitivity of two mitochondrial populations to hypoxia/reoxygenation, and, more markedly to phosphate. Since these effects were much less pronounced with succinate-linked respiration and since they were associated with defective NADH oxidation in SLM, it is suggested that the differences between the two populations may be accounted for by the increased lability of complex I of SLM due to hypoxia/reoxygenation or phosphate.

Progressive hypoxia until brain electrical silence: a useful model for studying protective interventions

Anesthetized spontaneously breathing rats, fitted with epicortical electrodes and catheters for sampling arterial, venous, and cerebral venous blood, were exposed to standardized progressive hypoxia. Three minutes of hypoxia sequentially caused hyperpnea, hypopnea, apnea, and cessation of electrocorticogram "spiking," of synchronization, and of background in electroencephalogram (EEG). Blood data and cerebral blood flow and metabolism were measured throughout and at "insults," i.e., at apnea and cessation events, to clarify their interdependence. Arterial and brain venous PO2 fell linearly with inspired oxygen (final value of 2% at 280 s). Hyperpnea induced arterial alkalosis; subsequent hypopnea led to near-normal PCO2 and pH when EEG ceased. Hypercapnia was more pronounced in cerebral than in systemic venous blood; time courses of pH changes were similar. Sagittal sinus blood pressure and outflow were linearly related and resembled the time course of local cerebral blood flow. Blood flow increased by 25% at apnea and only 60% at EEG silence. Cerebral metabolic rate of O2 rose during the hyperpnea phase and fell exponentially thereafter. Cerebral glucose uptake and lactate release increased within the first 3 min but fell abruptly when cortico-electric spiking ceased. Time courses of cerebral O2 consumption and spike rate were linearly related; both showed inverse linear relations to cerebral perfusion. The hypoxic insults were well defined by blood data; critical PO2 values were lower than previously assumed. This model is proving to be a useful, controlled method by which mechanisms of cerebral hypoxia tolerance may be studied in vivo.

Time course of adaptation to hypoxia in newborn rats

In newborn rats after a few minutes of hypoxia, ventilation is similar to the normoxic value. Nevertheless, after a few days in hypoxia, newborn rats have a sustained hyperventilation. In this study we examined the time course of the newborn rat's adaptation to hypoxia. Measurements of body size, hematocrit, lung and heart mass, and breathing pattern have been performed on newborn rats exposed to hypoxia (10% O2) for different time intervals from 4 to 60 h (hypoxic, H), and on same-age rats growing in air (controls, C). Ventilation measured by flow plethysmography was increased in H rats above the C value from about 8 h; this was due to a higher breathing rate and, from 24 h, also to a larger tidal volume. During the early hours of hypoxia, oxygen consumption measured manometrically was about 50% of C, while after 3 days in hypoxia it was almost like the C value. These observations indicate that the lack of sustained hyperventilation, characteristic of the newborn's acute exposure to hypoxia, is an immediate but transient phenomenon that is resolved after a few hours, and suggest a tight link between metabolic and ventilatory hypoxic responses. Body weight of H rats was less than in C, owing to an immediate decrease below the prehypoxic value. Dry heart and lung weight changed in proportion with the rest of the body during the first 36-48 h of hypoxia, then they increased disproportionately more. Hence, these temporal changes suggest that the large heart and lung weight-body weight ratios of the chronic hypoxic animals result from their smaller body mass and the stimulated growth of cardiac and pulmonary tissues.