Effects of NOS inhibition on the cardiopulmonary system and brain microvascular markers after intermittent hypoxia in rats.

We previously demonstrated that rats subjected to intermittent hypoxia (IH) by exposure to 10% O(2) for 4 h daily for 56 days in a normobaric chamber, developed pulmonary hypertension, right ventricular hypertrophy and wall-thickening in pulmonary arterioles, compared with normoxic (N) controls. These changes were greater in rats subjected to continuous hypoxia (CH breathing 10% O(2) for 56 days). Cerebral angiogenesis was demonstrated by immunostaining with glucose transporter 1 (GLUT1) antibody, in viable vessels, in CH and to a lesser degree in IH. In this study, adult Wistar rats were subjected to the same hypoxic regimes and given the nitric oxide synthase (NOS) inhibitor N(6)-nitro-L-arginine methyl ester (L-NAME) in drinking water (NLN, IHLN and CHLN regimes) to induce hypertension. There was significant systemic hypertension in NLN and IHLN rats, compared with N and IH, but surprisingly not in CHLN compared with CH. Hematocrit rose in all hypoxic groups (up to 79% in CHLN). There was no significant pulmonary hypertension in IHLN versus NLN rats, although there was asymmetric wall thickening in pulmonary arterioles. Cerebral GLUT1 immunoreactivity increased with L-NAME, with or without hypoxia, especially in CHLN rats, but conspicuously there was no evidence of angiogenesis in brains of IHLN compared with NLN rats. NOS blockade may attenuate the cerebral and pulmonary vascular changes of IH while augmenting cerebral angiogenesis in continuous hypoxia. However, whether cerebral effects are due to systemic hypertension or changes in cerebral nitric oxide production needs to be evaluated.

Effect of alveolar pressure on pulmonary artery pressure in chronically hypoxic rats.

The effect on pulmonary artery pressure of a rise in alveolar pressure differed in chronically hypoxic rats (10% O2 for 3-5 weeks) compared with control rats. Chronically hypoxic rats have newly muscularised walls in arterioles in the alveolar region. Isolated lungs of chronically hypoxic and control rats were perfused with blood under conditions in which alveolar pressure was greater than left atrial pressure during both normoxia and hypoxia. Alveolar pressure was the effective downstream pressure. Pressure-flow lines were measured at low and high alveolar pressure (5 and 15 mmHg). During normoxia pressure-flow lines of chronically hypoxic rats had a steeper slope (higher resistance) and greater extrapolated intercept on the pressure axis (effective downstream pressure) than control rats. In both groups of rats the change from low to high alveolar pressure during normoxia caused an approximately parallel shift in the pressure-flow line similar to the change in alveolar pressure. During hypoxia, which led to an increase in slope and intercept in both groups of rats, the effect of a rise in alveolar pressure differed in chronically hypoxic from control rats. In control rats there was a small parallel shift in the pressure-flow line that was much less than the increase in alveolar pressure; in chronically hypoxic rats there was a large parallel shift in the pressure-flow line that was greater than the increase in alveolar pressure. Thus in chronically hypoxic rats hypoxic vasoconstriction probably occurred mainly in muscular alveolar vessels, whereas in control rats it probably occurred upstream in extra-alveolar vessels. At constant blood flow the relation between pulmonary artery pressure and alveolar pressure was measured while alveolar pressure was reduced from approximately 15 mmHg to zero during both normoxia and hypoxia. In control and chronically hypoxic rats the slope of this line was less than 1. At an alveolar pressure of 2-3 mmHg there was an inflection point below which the line was nearly horizontal in control but negative in chronically hypoxic rats. During hypoxia the inflection point increased in control but not in chronically hypoxic rats, whereas the preinflection slope became negative. Apart from a rise in pulmonary artery pressure at all values of alveolar pressure, which occurred in both groups of rats, there was no change in the form of the curve in chronically hypoxic rats during hypoxia. These results also suggest constriction of extra-alveolar vessels in control rats and alveolar vessels in chronically hypoxic rats during hypoxia.

Quantitation of isoprenaline-induced changes in the ventricular myocardium.

Small doses of isoprenaline sulphate given intermittently produce a characteristic cardiopathy consisting of subendocardial scarring and myocardial hypertrophy. A morphometric technique was successfully applied to the quantitation of these changes. This technique improves the use of the isoprenaline model for the study of cardiac necrosis as statistical analysis can be applied and objective comparisons made. No hypertrophy was seen in the absence of myocardial necrosis which suggests that it is at least in part compensatory.

In vitro responses of lung arteries to acute hypoxia after NO synthase blockade or chronic hypoxia.

Responses to hypoxia of lung arteries (200-350 microns) from control (C) and chronically hypoxic (CH) rats were compared in a myograph before and after blockade of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME). After precontraction with prostaglandin F2 alpha (PGF2 alpha), hypoxia caused a four-phase tension change: brief dilation, transient contraction, prolonged dilation, and slow contraction (we studied the first three phases). In CH rats, the first dilation and first contraction were significantly reduced. After L-NAME, the first dilation was reduced in C rats and abolished in CH rats; thus the first phase is attributable to NO release and is affected by chronic hypoxia. The first contractile phase was significantly reduced by L-NAME in C but not in CH rats, where it was small. Thus NO synthase inhibition inhibits hypoxic constriction in isolated vessels, whereas it enhances hypoxic constriction in perfused lungs. The third dilator phase was unaffected by chronic hypoxia; it was increased after L-NAME in CH rats. Thus, in vitro, responses to hypoxia are complex; there is a balance between two dilator and two constrictor processes.

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.

Ligustrazine is a vasodilator of human pulmonary and bronchial arteries.

We have investigated the dilator effect of ligustrazine, the semisynthetic principle of a traditional Chinese herbal remedy, on human pulmonary and bronchial arteries in vitro. Ligustrazine caused a concentration-dependent relaxation of human small pulmonary arteries, which was independent of endothelium. Although ligustrazine was equally potent in inducing dilatation of pulmonary and bronchial arteries, it was about 10 times more potent in relaxing small pulmonary arteries (300-500 microns i.d.) compared with lobar pulmonary arteries (7-8 mm i.d.). By contrast, the relaxant responses of small and lobar pulmonary arteries to sodium nitroprusside was not significantly different. Ligustrazine was equally potent in relaxing prostaglandin F2 alpha- or 5-hydroxytryptamine-precontracted pulmonary arteries, suggesting that it is not a prostaglandin F2 alpha or 5-hydroxytryptamine antagonist. Preincubating the vessels with propranolol (1 microM) or indomethacin (10 microM) had no significant effect on the ligustrazine-induced vasodilatation. However, ligustrazine caused concentration-dependent inhibition of calcium-evoked contraction when applied to rat aorta in calcium-free K(+)-depolarizing medium. We conclude that ligustrazine is a dilator of human pulmonary and bronchial arteries, which is endothelium-independent and that ligustrazine preferentially relaxes pulmonary resistance vessels rather than large conduit pulmonary arteries.

Vasoreactions to acute hypoxia, whole lungs and isolated vessels compared: modulation by NO.

We aimed to explain diverse pulmonary vascular responses to hypoxia in different preparations and their modulation by NO. In rats we compared isolated perfused lungs (IPL), small vessels in vitro (PRVs) and in vivo preparations. In IPL and in vivo, acute and chronic nitric oxide synthase (NOS) blockade with L-NAME left normoxic pulmonary artery pressure unchanged but enhanced hypoxic vasoconstriction, hypoxia-induced pulmonary vasoconstriction (HPV). PRVs in vitro, precontracted with PGF(2alpha), showed four tension changes in acute hypoxia: dilatation, contraction, dilatation, contraction. Acute and chronic NOS blockade reduced the first two phases. In non-precontracted PRVs (from other laboratories), NOS inhibition enhanced HPV as in vivo and IPL; attenuation of HPV seems associated with precontraction. Thus reduced NOS activity does not cause pulmonary hypertension but exaggerates HPV. In IPL, prolonged severe hypoxia caused biphasic vasoconstriction separated by dilatation; the time course resembled that seen in PRVs. We suggest that the sequence of events during hypoxia in PRVs can be detected in whole lung preparations.

Hypoxia and the pulmonary circulation: a brief review.

Hypoxia constricts small pulmonary arteries. Local hypoxia regulates blood flow/ventilation ratios, while general hypoxia elevates pulmonary artery pressure (Ppa). There is a continuum of responses from flow reduction to Ppa elevation dependent on the proportion of lung involved. Stimulus-response curves to hypoxia show the effect is maximal within the physiological range and resemble that for the carotid body. In widespread lung disease so much of the lung becomes hypoxic, that blood flow/ventilation matching fails, hypoxaemia and pulmonary hypertension follow. In chronic hypoxia structural changes take place which maintain a high pressure even when hypoxia is removed; small arterioles become muscularized and there is right ventricular hypertrophy and polycythaemia. Animal models of hypoxic pulmonary hypertension have brought some understanding of the growth processes involved and shown that several drugs will prevent these changes. The reactivity of the restructured pulmonary vessels in chronic hypoxia is altered.

Some observatoons on the carotid bodies of the New Zealand strain of genetically hypertensive rats.

We report preliminary studies of the carotid bodies in the New Zealand strain of hypertensive rats. Female animals have a higher blood pressure than males of the same colony, but in both sexes mean arterial pressure is elevated significantly when compared to normal animals. The carotid bodies are enlarged in both the hypertensive and normotensive animals and there is no correlation between carotid body size and arterial pressure. The only structural abnormality detected in the hypertensive carotid bodies was a gross thickening of the intimal layer of the arterioles. The content of dopamine in the organs was similar in normotensives and hypertensives but the noradrenaline levels were some 50% lower in the hypotensives. These results are discussed and compared with data available for SHR animals.

Experimental prevention of hypoxic pulmonary hypertension in animals by drugs.

The rat model of chronic hypoxic pulmonary hypertension has been extensively studied and shows many of the features seen in man with chronic pulmonary hypertension. The development and reversibility of these changes by various treatments and by drugs is discussed. The experimental model may provide valuable clues as to the mechanisms involved in the aetiology of pulmonary hypertension in man.

Effect of ligustrazine on pulmonary vascular changes induced by chronic hypoxia in rats.

1. Acute and chronic effects on the pulmonary circulation of ligustrazine, a chemically identified and synthesized principle of a Chinese herb, were studied in rats. It dilated lung vessels and reversed hypoxic pulmonary vasoconstriction. 2. In rats kept 2 weeks in 10% O2 in a normobaric chamber and simultaneously treated with ligustrazine, right ventricular hypertrophy and muscularization of pulmonary arterioles were attenuated compared with saline-treated rats. Pulmonary artery pressure, measured in isolated lungs perfused at a constant flow rate, was also less in ligustrazine-treated rats. 3. In isolated blood-perfused lungs of chronically hypoxic and control rats, the relation between pressure and flow was measured during normoxia (ventilation with air plus 5% CO2), hypoxia (2% O2 plus 5% CO2) and after ligustrazine during continued hypoxia. Alveolar pressure was always greater than left atrial pressure; thus flow was determined by the pulmonary artery minus alveolar pressure difference. 4. Pressure/flow lines were measured during normoxia in four groups of rats: (1) control, saline-treated; (2) control, ligustrazine-treated; (3) chronically hypoxic, saline-treated; (4) chronically hypoxic, ligustrazine-treated. Both chronically hypoxic groups had steeper lines (higher resistance) than the control groups, which were similar in all respects. However, in chronically hypoxic rats, the extrapolated intercept of the line on the pressure axis, probably attributable to small newly muscularized arterioles in a state of tone, was much increased in the saline-treated group but did not differ from controls in the ligustrazine-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary vasodilator and vasoconstrictor actions of carbon dioxide.

1. Three preparations were used to study the actions of CO(2) on the pulmonary vasculature: isolated rat and cat lungs perfused at a constant flow rate with homologous blood and a lobe of cat lung perfused at a constant flow rate in vivo. In all three changes in pulmonary artery pressure (P(Pa)) reflected changes in pulmonary vascular resistance.2. In the isolated rat lung CO(2) caused vasodilatation when vascular tone was high. The lung was first ventilated with N(2) causing a rise in P(Pa). CO(2) caused vasodilatation during hypoxia whether the initial blood CO(2) level was low or normal and in spite of a fall in blood pH which usually augments the constrictor action of N(2).3. In the rat lung ventilation with CO(2) from the control state usually caused weak vasoconstriction. Reducing blood pH with acids also caused weak vasoconstriction while alkali caused vasodilatation.4. The over-all effect of CO(2) on the pulmonary vasculature depends on a balance between a vasoconstrictor action probably caused by carbonic acid and vasodilatation caused by some other property of the molecule. The dilator action is powerful in the isolated rat lung.5. By contrast, in both cat lung preparations, no direct evidence for a vasodilator action of CO(2) was obtained. Ventilation with CO(2) when vascular tone was raised by hypoxia, drugs or acids caused further vasoconstriction. From the control state CO(2) caused strong vasoconstriction.6. Indirect evidence from other work suggests that a pulmonary vasodilator action of CO(2) exists in the cat but is usually masked by the strong vasoconstrictor action of carbonic acid. In life the dilator mechanism may be important when pH changes caused by CO(2) are minimized by renal compensation.

Respiratory stimulation by almitrine during acute or chronic hypoxia/hypercapnia in rats.

In anaesthetized rats studied in a body plethysmograph, 1-(4'6-diallylamino-2'-triazinyl)-4(bis parafluorobenzydryl) piperazine (almitrine) caused large increases in ventilation (VE) which were abolished by cutting the IXth and Xth cranial nerves. Maximal effects followed infusions of 0.5-1 mg X kg-1; the solvent had no action. When the rats breathed 10% O2 or 10% O2 + 5% CO2, gas mixtures which increased VE, almitrine caused further increases in VE and improved blood gas tensions. Chronically hypoxic (4 weeks in 10% O2) and chronically hypoxic and hypercapnic rats (4 weeks in 10% O2 + 4% CO2), kept in an environmental chamber, were similarly studied. While they breathed, under anaesthesia, the gas mixture to which they had been chronically exposed, infusions of almitrine caused further large increases in VE and improved blood gas tensions. Thus almitrine stimulates VE, probably through peripheral chemoreceptors, when these are already strongly stimulated. This drug increased both tidal volume and frequency and thus increased alveolar ventilation. No convulsant action was observed. It caused a fall in blood pressure which lasted 5-10 min and was seen with urethane but not pentobarbitone anaesthesia; it was not due to the solvent.

Contribution of polycythaemia to pulmonary hypertension in simulated high altitude in rats.

A rat model was used to assess the viscosity factor in pulmonary hypertension of high altitude. Rats exposed to 10% O2 for three weeks developed increased pulmonary vascular resistance (p.v.r.) and polycythaemia; the haematocrit (Hct) was 50-60%, values similar to those in normal men at high altitudes. The contribution of high Hct to the increased p.v.r. was assessed in isolated perfused lungs of chronically hypoxic rats perfused with their own high Hct blood, or normal Hct blood from control rats. Pressure/flow relationships were measured over a wide range and the slope (P/Q) of this relationship and its extrapolated intercept on the pressure axis were increased by high Hct blood. A return to low Hct blood did not restore initial conditions although a second perfusion with high Hct blood again increased p.v.r. and intercept. Lack of reversibility was attributed to changes with time in blood or lung. In a second experiment designed to eliminate time changes, chronically hypoxic or litter-mate control rats were each perfused with only one blood, their own or each other's and P/Q relations were rapidly measured. The P/Q slope and pressure intercept increased progressively in the following groups: control rats perfused with their own blood (Hct 34%), control rats perfused with chronically hypoxic blood (Hct 56%), chronically hypoxic rats perfused with control blood (Hct 35%) and chronically hypoxic rats perfused with chronically hypoxic blood (Hct 53%). To exclude factors in chronically hypoxic blood other than high Hct which might increase p.v.r., control rats were perfused with blood of different Hct obtained by centrifugation. High Hct again increased p.v.r. There was a significant relationship in all rats between pulmonary artery pressure (Ppa), which takes into account both P/Q slope, intercept and Hct. There was substantial batch variation which may reflect sensitivity to hypoxia. In chronically hypoxic rats with high Hct blood, Ppa varied from 29-47 mmHg; with low Hct blood the range was 26-38 mmHg. Comparable values for control rats were 21-29 and 17-20 mmHg. We conclude that the polycythaemic blood of chronic hypoxia contributes substantially to pulmonary hypertension. Where it is excessive, it may prejudice tissue blood flow.

The enlarged carotid body of the chronically hypoxic and chronically hypoxic and hypercapnic rat: a morphometric analysis.

Rats were subjected to chronic hypoxia (10% O2) or hypoxia and hypercapnia (10% O2 + 4% CO2) for 3-4 weeks and their carotid bodies (twenty-three from twenty rats) were compared with those of litter-mate controls. Both chronic exposures, which simulated high altitude or chronic lung disease, caused a 4-10-fold increase in carotid body volume. The larger increases were attributed to higher fixation-perfusion pressures. The organs were fixed by perfusion with glutaraldehyde. Semi-thin (1 micron) sections for light microscopy and ultra-thin sections for electron microscopy were cut at regular intervals and were examined by stereological techniques to determine the nature of the enlargement. The proportion occupied by blood vessels was much increased in both chronic hypoxia and hypoxia plus hypercapnia; the endothelium appeared stretched with conspicuous fenestrations. There were increased numbers of endothelial cells which suggested new growth as well as stretching of endothelium and the mean transectional area of the vessels was increased. The mean surface area of blood vessels per unit area of carotid body was unaltered but the total surface area of blood vessels in the whole carotid body was greatly increased. Both the Type 1 cell nucleus and cytoplasm were increased in size. The proportion nucleus/cytoplasm was unaltered in hypoxia but reduced in hypoxia plus hypercapnia. There were fewer Type 1 cell nuclei per unit area but the estimated total number of Type 1 cell nuclei per carotid body was increased 2-4-fold; this was interpreted as Type 1 cell hyperplasia. Some of the dense-cored vesicles in Type 1 cells were enlarged with eccentric dense cores but their number per unit area of cytoplasm was decreased. Their mean size was not significantly altered. However, the total number of vesicles per carotid body was presumed to be increased because their decreased density in the cell was offset by a greater increase in total Type 1 cell volume. The harmonic and arithmetic mean distances between endothelium and the boundary of glomus tissue were significantly reduced. The harmonic mean distance is an indication of the diffusion distance for gases to and from blood and glomus tissue. The arithmetic mean distance is a measure of the amount of tissue in between. The significance of the vascular enlargement and hyperplasia and the Type 1 cell hyperplasia cannot be assessed at present. We do not know if enlargement is associated with the same, greater, or lesser activity of the organ for a given stimulus.