hypoxic pulmonary hypertension

Transcription

1 Br. J. Pharmacol. (1992), 17, '." Macmillan Press Ltd, 1992 Reduced relaxant potency of nitroprusside on pulmonary artery preparations taken from rats during the development of hypoxic pulmonary hypertension 'Janet C. Wanstall, 2Ian E. Hughes & Stella R. O'Donnell Pulmonary Pharmacology Group, Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland 472, Australia 1 Relaxant responses to nitroprusside were examined on U46619-contracted pulmonary artery ring preparations from rats exposed to hypoxia, in chambers containing 1% oxygen, for 1, 3, or 14 days, or for 14 days followed by 12 days in room air. rats were housed in room air. 2 After 3 days of hypoxia (but not 1 day), rats had elevated pulmonary artery pressure, right ventricular hypertrophy and polycythemia. After 14 days of hypoxia there was, in addition, hypertrophy of the pulmonary artery. In rats returned to room air for 12 days after 14 days of hypoxia, there was still some right ventricular and vascular hypertrophy but no increase in pulmonary artery pressure or polycythemia. 3 The potency (neg log EC5) of nitroprusside on pulmonary arteries taken from rats after 3 or 14 days of hypoxia was significantly less than on preparations from control rats (3 and 11 fold, respectively). This was not seen after I day of hypoxia or after 14 days of hypoxia followed by 12 days in room air. Removal of the endothelium from the preparations had no effect on the potency of nitroprusside in control or hypoxic rats (14 days). 4 In preparations from hypoxic, but not control, rats (14 days), the maximum response to nitroprusside was > 1% (177% reversal of the U46619 contraction) in the absence, but not in the presence, of the endothelium, indicating that pulmonary arteries from hypoxic rats had inherent tone which could be counteracted by a relaxing factor from the endothelium. 5 Exposure of rats to hypoxia (14 days) did not affect the potency of nitroprusside on aorta or trachea. 6 It is concluded that exposure of rats to hypoxia results in reversible desensitization of the vascular smooth muscle of pulmonary artery to nitroprusside. The time course of this desensitization suggests that it is probably associated with the elevated pulmonary artery pressure or maintained hypoxaemia rather than with the vascular hypertrophy. 7 It is postulated that the increase in pulmonary artery pressure and/or the maintained hypoxaemia may cause chronic release of nitric oxide from the pulmonary vascular endothelium or smooth muscle resulting in desensitization of soluble guanylate cyclase to the action of nitroprusside. Keywords: ; pulmonary hypertension; nitroprusside; rat pulmonary artery; endothelium Introduction Exposure of rats to chronic hypoxia leads to the development of pulmonary hypertension (Herget et al., 1978; Reid, 1979; Kay, 198) and to changes in the reactivity of pulmonary blood vessels to various vasoactive agents (Emery et al., 1981; Lowen et al., 1987; Barer et al., 1989; Wanstall & O'Donnell, 1992). One particular change in reactivity is a 5-1 fold reduction in the potency of the vasodilator drug, nitroprusside. This was seen on pulmonary arteries taken from rats exposed to hypoxia for 14 days (Wanstall & O'Donnell, 1992). In these rats, the characteristic pathophysiological features of hypoxic pulmonary hypertension were already well developed (Wanstall & O'Donnell, 1992). Hence it was not known whether the loss of vascular reactivity to nitroprusside coincided with the rise in pulmonary artery pressure, the onset of vascular hypertrophy and/or the maintained arterial hypoxaemia. The aim of the present study was to compare the time course of the decline in pulmonary vascular reactivity to I Author for correspondence. 2 On study leave from The Department of Pharmacology, University of Leeds, U.K. nitroprusside with that of the appearance of the various pathophysiological features of hypoxic pulmonary hypertension by studying rats exposed to hypoxia for different periods of time. We have also investigated whether the reactivity of pulmonary arteries to nitroprusside reverted to normal if, after exposure to hypoxic conditions, rats were allowed to recover (for 12 days) in room air. To obtain information on the mechanism(s) underlying the reduction in the potency of nitroprusside, we have investigated whether the exposure of rats to hypoxia affected (1) the responses of pulmonary artery to the spasmogen used to contract the preparations, (2) the influence, if any, of the endothelium on responses to nitroprusside, and (3) the sensitivity of smooth muscle in other tissues to nitroprusside. The thromboxane analogue, U46619, was selected as the contractile spasmogen (in contrast to noradrenaline used in our previous study, Wanstall & O'Donnell, 1992), and data have been obtained on pulmonary artery preparations with and without endothelium, and on tissues other than pulmonary artery, viz. a non-pulmonary blood vessel, aorta, and non-vascular tissue from the airways, trachea. A preliminary account of some of these data was presented to a meeting of the Australasian Society of Clinical and Experimental Pharmacologists (Wanstall et al., 199).

2 48 J.C. WANSTALL et al. Methods Exposure of rats to hypoxia Male Wistar rats were exposed to chronic hypoxia for 1, 3 and 14 days in chambers containing 1-11% oxygen (Wanstall & O'Donnell, 1992). rats were housed in room air (21% oxygen) for the same periods of time. An additional group of rats was exposed to hypoxia for 14 days and subsequently allowed to recover in room air for 12 days. All rats were the same age (8-9 weeks old) at the completion of the period of exposure to hypoxia. The hypoxic chamber was continuously flushed with a mixture of nitrogen and compressed air, at a flow rate of min-'. The ratio of nitrogen to air was adjusted so that the oxygen component of the gas in the chamber was 1-11% (measured with a Datex Normocap Gas Monitor). Trays of soda lime were included in the chamber to maintain the CO2 content below.5%. The rats were removed from the chamber for a maximum of 15 min each day, when the chamber was cleaned and food and drinking water were replenished. On the day of the experiment, the rats were anaesthetized with pentobarbitone (9 mg kg-', i.p.), were artifically ventilated via a tracheal cannula (6 breaths min '), and the pulmonary artery pressure (PAP) was recorded via a hypodermic needle inserted into the pulmonary artery through the right ventricle (Wanstall & O'Donnell, 199). Mean PAP was calculated as diastolic PAP + 1/3 [systolic - diastolic PAP]. A blood sample was removed for measurement of the haematocrit. After removal of the pulmonary artery (and, in some experiments, the aorta and trachea) the heart and lungs were removed and weighed for the determination of the ratio of the weight of right ventricle to the weight of left ventricle plus septum (RV/LV + S), and the ratio of lung wet to dry weight, as described previously (Wanstall & O'Donnell, 199). Increases in PAP, RV/LV + S, haematocrit and lung wet/dry weight were taken as indications of pulmonary hypertension, right ventricular hypertrophy, polycythemia and lung oedema respectively. Blood vessel preparations Single ring preparations (3 mm in length) of main pulmonary artery and ventral aorta were set up in physiological salt solution (PSS), at 37C, around 2 stainless steel wires in a vertical organ bath. In one series of experiments on pulmonary artery, the endothelium was removed by gently rubbing the luminal surface of the preparations with small forceps. In the remaining preparations care was taken not to damage the endothelium. Force in the circular muscle was recorded isometrically with a Statham Universal Transducer (UC3 + UCL) attached to a micrometer (Mitutoyo, Tokyo, Japan), as described previously (Wanstall & O'Donnell, 1988). The composition of the PSS was (mm): NaCl 118, KCl 5.9, CaCl2 1.5, MgSO4.72, NaHCO3 25, glucose 11.7, ascorbic acid 1.14 (95% 2/5% C2; ph 7.4). Pulmonary artery preparations were set up under resting forces of 1 mn (control) or 2 mn (hypoxic), and aortic preparations (control and hypoxic) under a resting force of 65 mn. These resting forces were selected in order to reflect the circumferential wall tensions corresponding to the different pressures in these arteries in vivo, and were determined, in separate experiments, from passive length/tension studies and the Laplace equation, as described in detail elsewhere (Wanstall & O'Donnell, 199). At the completion of the experiment, the distance between the 2 horizontal wires (with the preparation under the selected resting force, see above) was measured with the micrometer holding the transducer, and the wet weight of the preparation was determined. The cross sectional area of the preparation, in the plane perpendicular to the direction of the applied force, was then calculated as described by Wanstall & O'Donnell (1988), from the formula: cross-sectional area= w (hd)-' where h = distance between the wires plus the diameters of the wires (mm), w = wet weight (mg) and d = density = 1.6 mg mm-3. Since all the preparations were the same length (3 mm, see above), differences in cross-sectional area between vessels reflected differences in vessel wall thickness. Thus an increase in cross-sectional area indicated the development of vascular hypertrophy. Tracheal preparations Segments of trachea, 4 cartilage rings wide, were taken from the bronchial end of the trachea and set up, with the cartilage cut, in PSS under a resting force of 1 mn. Changes in force in the smooth muscle were measured isometrically. Experimental protocols Blood vessel preparations were allowed to equilibrate for 1 h. They were then contracted with.1 AiM noradrenaline and, when the contraction was stable, acetylcholine (ACh 1 SAM) was added. A relaxant response to ACh indicated the presence of a functional endothelium. After washing with PSS, a contraction to potassium-depolarizing PSS (in which 8 mm NaCl was replaced with 8 mm KCI) was obtained. The preparations were then washed, allowed to relax and a cumulative concentration-response (contraction) curve to U46619 was then determined. The preparations were again washed and allowed to relax. They were then submaximally contracted with 1 nm U46619 (approximate EC7 on pulmonary artery and EC9 on aorta), and a cumulative concentrationresponse (relaxation) curve to nitroprusside was determined. Tracheal preparations were allowed to equilibrate for 1 h and were then contracted with 1!M carbachol. This was followed, after washout with PSS, by a contraction to potassium-depolarizing PSS. The preparations were then washed, allowed to relax, and a cumulative concentrationresponse (contraction) curve to carbachol was obtained. After wash-out, the preparations were submaximally contracted with 1 IAM carbachol (EC7-8) and a cumulative concentration-response (relaxation) curve to nitroprusside was determined. Analysis of data Contractile responses to U46619 on pulmonary artery and aorta were determined as force (mn) and expressed as stress (mn mm-2) by dividing by the cross-sectional area of the preparations. Contractile responses to carbachol on trachea were expressed as force (mn). Relaxant responses to nitroprusside were expressed as 'percentage reversal' of the contraction induced by either 1 nm U46619 (pulmonary artery and aorta) or 1 gm carbachol (trachea). The potency of U46619 and nitroprusside was expressed as the negative log EC5 (where EC5 is the concentration producing 5% of the maximum contraction or relaxation to U46619 or nitroprusside, respectively). Preliminary experiments The pulmonary artery preparations from control (normotensive) and hypoxic (pulmonary hypertensive) rats were set up at different resting forces, in order to try to mimic in vivo wall tensions (see above). Thus concentration-response curves to nitroprusside were also obtained on preparations from control rats set up at a resting force of 2 mn, i.e. the resting force used in experiments on pulmonary arteries from hypoxic rats. These curves, in which the nitroprusside negative log EC5 was 8. ±.7 and maximum relaxation was 96 ± 1.1 % (n = 4), were superimposable on curves obtained on control preparations set at a resting force of 1 mn (control values in Table 3). Thus the use of a higher resting force for the preparations from hypoxic rats, compared with

3 NITROPRUSSIDE ON PULMONARY ARTERY FROM HYPOXIC RATS 49 control rats, was not responsible for the differences in reactivity to nitroprusside reported in the Results section for the different groups of rats. Drugs and solutions Acetylcholine chloride (ACh, Sigma); carbamylcholine (carbachol, Sigma); (-)-noradrenaline acid tartrate (Sigma); sodium nitroprusside (Sigma); U46619 ((1,5,5)-hydroxy-1 1a, 9a-(epoxymethano) prosta 5Z, 13E-dienoic acid; Upjohn). Solutions of drugs were prepared as follows: ACh (I mm), carbachol (1O mm) and nitroprusside (1O mm) in deionised water; noradrenaline (1 mm) in 1 mm HCl; U46619 (1 mm) in absolute ethanol. Dilutions were made in PSS and kept on ice during the course of an experiment. Statistical analyses Mean values are quoted together with their standard errors (s.e.mean). The significance of differences between mean values has been assessed by Student's t test, except for percentage values, which were assessed by Mann Whitney U-test. Results Effects of hypoxia on the rats All control rats housed in room air gained weight. Rats exposed to hypoxia for I or 3 days lost weight. Rats exposed to hypoxia for 14 days gained weight but this gain was significantly less than that in the corresponding control rats (Table 1). After 3 days exposure to hypoxia (but not I day) rats had significantly elevated pulmonary artery pressures, right ventricular hypertrophy and polycythemia (Table 1). When the time of exposure to hypoxia was increased to 14 days, these changes were more pronounced and there was, in addition, pulmonary vascular hypertrophy (Table 1). In rats exposed to hypoxia for 14 days and then allowed to recover in room air for 12 days, pulmonary artery pressure was not significantly different from controls and there was no polycythemia, but there was still significant right ventricular and pulmonary vascular hypertrophy (Table 1). There was no lung oedema in the hypoxic rats (Table 1), and all the pulmonary artery preparations relaxed equally well in response to ACh (33-49% reversal of a noradrenaline-induced contraction), indicating the presence of a functional pulmonary vascular endothelium in all rats irrespective of treatment. Effects of exposure of rats to hypoxia on responses of isolated tissue preparations The potency of U44619 in contracting pulmonary arteries was the same on preparations from control and hypoxic rats (Table 2). The magnitude of the submaximal contraction to 1O nm U46619 was also the same on all preparations, with one exception, viz. on endothelium-denuded preparations taken from rats exposed to hypoxia for 14 days this contraction was significantly less than in the corresponding controls (Table 2). The potency of nitroprusside, when compared with controls, was significantly less on pulmonary arteries taken from rats exposed to 3 or 14 days of hypoxia (3 and 11 fold less potent, respectively), but not on those from rats exposed to only 1 day of hypoxia (Table 3; Figure 1). The decrease in the potency of nitroprusside was apparently reversible in that it was not seen on preparations from rats allowed to recover in room air for 12 days after exposure to 14 days of hypoxia (Table 3; Figure 1). The reduction in potency associated with 14 days of hypoxia was also seen in preparations without endothelium, since removal of the endothelium did not affect the potency of nitroprusside in preparations from either the control or the hypoxic rats (Table 3). However in the endothelium-denuded preparations from the hypoxic rats, in contrast to matching preparations from control rats, there was greater than 1% reversal of the U46619-induced contractions (Figure 2). This was not seen in any of the other groups of rats studied (Table 3). In contrast to the findings on pulmonary artery, there was no change in the potency or maximum relaxation to nitroprusside on preparations of aorta or trachea taken from the rats exposed to hypoxia for 14 days (Table 4; Figure 3). It was noted that both the potency and maximum relaxation to nitroprusside on trachea were less than on the vascular preparations (Table 4). Discussion In the present study, nitroprusside was less potent in relaxing isolated preparations of pulmonary artery from rats made pulmonary hypertensive by exposure to hypoxia for 14 days, than in preparations from control rats, i.e. the tissues were desensitized to nitroprusside. The preparations were contracted with the thromboxane analogue, U46619, and the data confirmed a previous finding on preparations contracted with noradrenaline (Wanstall & O'Donnell, 1992). Thus the influence of hypoxic pulmonary hypertension on pulmonary vascular reactivity to nitroprusside is not restricted to vessels contracted by noradrenaline. Furthermore the desensitization Table 1 Effects of exposure of rats to 1% oxygen (hypoxia) or room air (control) for different periods of time Initial weight (g) Change in weight (g)a Mean PAP (mmhg) RV/(LV + S)b Haematocrit (%) Lung wet/dry weight Cross-sectional area of pulmonary artery preparations (mm2)c Values are means ± s.e.mean I day (n = 4) (n = 4) 287 ± 1 I ± 1.34 ±.1 44± ±.7.71 ± ± 7-35 ± 3"' 14±2.37 ±.1 47 ± ±.3.6 ±.6 3 days (n = 4) (n = 4) 229 ± 7 28 ± 3 1 ± 2.33 ±.1 43 ± ±.4.67 ±.6 24 ± 1-26± 1"' 19 ± 2*.42 ±.1 ** 52 ± 3* 4.92 ±.5.69 ±.4 14 days 14 days plus 12 days recovery (n = 9) (n = 8) (n =4) (n =4) 198 ± 1 96± ± 2.34 ±.1 43 ± ±.4.66 ±.3 194± ± 5"' 26 ± 2 '.64 ±.5*** 57 ± 2"' 4.68 ± ±.7"' 183 ± ±9 14 ± 2.34 ±.1 45± ±.3.65 ±.4 186± ± 7* 18±2.45 ±.2** 44± ±.3.88 ±.8* 'Positive values indicate weight gain and negative values indicate weight loss during period of exposure to hypoxia or room air. bweight of right ventricle + weight of left ventricle plus septum. cfor definition see Methods. 'Value significantly different from corresponding control value: '.5>P>.1; ".1>P>.1; ***P<.1 (Student's t test).

4 41 J.C. WANSTALL et al. Table 2 U46619: potency (negative log EC5) and submaximal contraction (to 1 nm) on pulmonary artery preparations from rats exposed to 1% 2 (hypoxia) or room air (control) for different times Time of exposure 1 day 3 days 14 days 14 days ( + 12 days recovery in room air) 14 days (preparations without endothelium) Neg log EC ± ± ± ± ± ± ± ± ± ±.14 1 nm contractiona (mn mm-2) 24.2 ± ± ± ± ± ± ± ± ± ± 1.** Values are mean ± s.e.mean (numbers of preparations from different rats in parentheses). asubmaximal contractions to U44169 (1 nm) used for nitroprusside concentration-response (relaxation) curves. **Value significantly lower than control value.1 > P>.1. (Student's t test). Table 3 Nitroprusside: potency (negative log EC5) and maximum relaxation on pulmonary artery preparations from rats exposed to 1% 2 (hypoxia) or room air (control) for different times Time of exposure 1 day 3 days 14 days 14 days (+ 12 days recovery in room air) 14 days (preparations without endothelium) Neg log EC ± ± ± ± ± ± ±.12* 7. ±.14*** 7.92 ± ±.4*** Max relaxation (%)a 14± 1 14±3 99 ± 1 11 ± 1 14± 1 98 ± 1 89 ± 3 86±1 11 ± ± 12t Values are mean ± s.e.mean (numbers of preparations from different rats in parentheses) amaximum relaxation expressed as a percentage of the U44619 (1 nm)-induced contraction. *Value significantly lower than corresponding control value: *.5> P>.1; ***P<.1 (Student's t test). tvalue significantly greater than corresponding control value: P <.5 (Mann Whitney U test). to nitroprusside in rats exposed to hypoxia was not a nonspecific effect on either blood vessels or lung tissues in general, since the potency of nitroprusside on preparations of aorta and trachea from the same animals was not affected. The desensitization was not the indirect result of an effect of the hypoxic treatment on responses to the spasmogen, since the potency of U46619 and the magnitude of the contraction to 1 nm U46619 (the concentration used in the nitroprusside experiments) were unchanged. The reduction in the potency of nitroprusside on pulmonary arteries was not an acute response to hypoxia since it was not apparent after the first 24 h of exposure. However, it was seen in preparations from rats after 3 days of hypoxia and appeared to be progressive, in that the potency of the drug was even less after an exposure time of 14 days. The potency was not reduced in preparations taken from rats that were returned to room air for 12 days after 14 days exposure to hypoxia, indicating that the desensitization to nitroprusside was completely reversible. The time-course for the desensitization to nitroprusside was compared with that for the various pathophysiological changes associated with hypoxic pulmonary hypertension in an attempt to determine which of these changes might account for the desensitization. Desensitization appeared to parallel the increase in pulmonary artery pressure and the development of polycythemia, in that each of these three changes occurred in rats exposed to hypoxia for 3 or 14 days, but not in the hypoxic rats that had been returned to room air for 12 days. In contrast, desensitization to nitroprusside did not coincide with vascular hypertrophy; vascular hypertrophy was seen after 14, but not after 3 days, of hypoxia, and persisted up to 12 days after hypoxic rats had been returned to room air. These observations suggest that the desensitization to nitroprusside may result either from the elevated pulmonary artery pressure (although not from the vascular hypertrophy arising from this) or from the maintained hypoxaemia (which gives rise to the polycythemia). Further experiments over a wider range of exposure and recovery times will be required to substantiate this suggestion. The results of the present study showed that pulmonary artery pressure reverted to normal if rats exposed to hypoxia for 14 days were returned to room air for 12 days (see above). This was in contrast to findings in rats exposed to hypoxia for 3-4 weeks, where pulmonary artery pressure was still elevated even after 6 weeks in room air (Herget et al., 1978; Kay, 198). The recovery period in room air required for reversal of the increase in pulmonary artery pressure may therefore depend on the duration of the initial hypoxic exposure. The same may apply to the desensitization to nitroprusside, if, as postulated above, desensitization is associated with the increase in pulmonary artery pressure. We have not yet explored this possibility. In seeking to provide an explanation for the reduction in the potency of nitroprusside on pulmonary arteries from hypoxic rats, the possible role of the endothelium was investigated. Although nitroprusside is not an endothelium-dependent vasodilator (Shirasaki & Su, 1985), there is evidence

5 NITROPRUSSIDE ON PULMONARY ARTERY FROM HYPOXIC RATS 411 C Co so C C) a) 4- C a 11 1C A * 4~4 A1- b ( D - -o 4X D...\._... \ T\ c l C.) -o Co C-) CD CD 16 6U) ) Or 5k N S 1l k, \ * _ -9-7 log [Nitroprussidel M -5 Figure 2 Preparations without endothelium. Mean concentrationresponse (relaxation) curves (n = 4) to nitroprusside on U46619 (1 nm)-contracted preparations of pulmonary artery taken from rats housed in room air (control; ) or 1% oxygen (hypoxia; ) for 14 days. Responses are expressed as percentage reversal of the U induced contraction. Points are means with s.e. mean, when not smaller than the size of the symbols, shown by a vertical bar. The asterisk indicates that the maximum relaxation was significantly greater than the corresponding value in the control preparations (P<.5, Mann Whitney U test). CT) CD co ) ) 1 c C - To d \ O * \.I * \.... * - * ",\V V\ V\..~ ~~~ A\I V \" _ log [Nitroprusside] M Figure 1 Mean concentration-response (relaxation) curves for nitroprusside on U46619 (1 nm)-contracted preparations of pulmonary artery taken from rats housed in room air (control; closed symbols) or 1% oxygen (hypoxia; open symbols) for (a) 1 day (b) 3 days (c) 14 days and (d) 14 days, followed by 12 days recovery in room air. Responses are expressed as percentage reversal of the U induced contraction. Points are means with s.e. mean shown by vertical bars except when smaller than the size of the symbols. For numbers of observations refer to Table 3. that in some, but not all, vessell types, relaxant responses to this drug are less when the endothelium is present than when it is absent (Shirasaki & Su, 1985; Shirasaki et al., 1986; Pohl & Busse, 1987). Shirasaki et al. (1986) suggested that this could be due to the release by nitroprusside of a contractile substance from the endothelium, which would presumably act as a functional antagonist of nitroprusside. In contrast, Pohl & Busse (1987) postulated that continual basal release of nitric oxide (EDRF) from the endothelium might either compete with the nitroprusside for soluble guanylate cyclase or interfere with the relaxant response at a site distal to guanylate cyclase activation. In the present study the potency of nitroprusside on pulmonary arteries was the same whether or not the endothelium had been removed, i.e. in vitro, the endothelium did not modulate responses to nitroprusside on this vessel type. Furthermore, in vessels from rats exposed to hypoxia for 14 days, the potency of nitroprusside was reduced by the same amount in preparations with and without endothelium. These findings are in contrast to the results of experiments on systemic vessels (aorta and mesenteric artery) taken from rats with systemic hypertension where the endothelium did modulate responses to nitroprusside, and where a reduction in the potency of nitroprusside was seen in the hypertensive rats, but only on preparations with an intact endothelium (Shirasaki et al., 1986). Thus it appears that desensitization of systemic vessels to nitroprusside in systemic hypertension involves a change in the influence of the endothelium on responses of the smooth muscle (Shirasaki et al., 1986), whereas that in pulmonary arteries in pulmonary hypertension reflects a change in the responsiveness of the smooth muscle itself. The removal of the endothelium from vessels from 14 days hypoxic rats did result in a dramatic increase in the maximum relaxant response to nitroprusside such that reversal of the contraction to U46619 was greater than 1%. This provides evidence that the smooth muscle of pulmonary arteries from 14 day hypoxic rats had significant inherent tone, which may, in turn, explain the reduction in the size of the contraction to U46619 that was also seen on these preparations. This evidence for inherent tone was not seen in matching preparations with intact endothelium, suggesting that, when present, the endothelium may continuously release a relaxing factor that can effectively counteract (and therefore conceal) the inherent tone in the smooth muscle. It was not the purpose of this study to establish the nature of the relaxing factor involved, but one could predict that it might be either nitric oxide (Palmer et al., 1987) or endotheliumderived hyperpolarizing factor (Chen et al., 1988).

6 412 J.C. WANSTALL et al. Table 4 Nitroprusside: potency (negative log EC5) and maximum relaxation on aortic and tracheal preparations from rats exposed to 1% 2 (hypoxia) or room air (control) for 14 days Neg log EC5 Maximum relaxation (%)c Aortaa 7.73 ± ±.2 12 ± 2 15 ± 3 Tracheab 6.11 ±.1 6. ± ± 2 58 ± 15 Values are mean ± s.e.mean (numbers of preparations from different rats in parentheses). aaortic preparations submaximally contracted with 1 nm U btracheal preparations submaximally contracted with 1 JAM carbachol. cmaximum relaxation expressed as a percentage of the U or carbachol-induced contraction. c o O ab -' o\f 9. -.@ > To *:.:. >1. g.** > log [Nitroprusside] M Figure 3 Aorta and trachea. Mean concentration-response (relaxation) curves for nitroprusside on preparations of (a) aorta and (b) trachea taken from rats housed in room air (control; closed symbols) or 1% oxygen (hypoxia; open symbols) for 14 days. The aortic preparations were contracted with 1 nm U46619 (contractions were (mn mm-2): controls 36. ± 3.7 (n = 5); hypoxia 36.1 ± 3.7 (n = 4)). The tracheal preparations were contracted with 1 pm carbachol (contractions were (mn): control 7.4 ±.88 (n = 4); hypoxia 11. ± 3.1 (n = 4)). Responses are expressed as percentage reversal of the induced contractions. Points are means, with s.e. mean shown by vertical bars except when smaller than the size of the symbols. The mechanisms underlying the two functional changes in the smooth muscle of pulmonary arteries from hypoxic rats reported in this study, viz. the desensitization of the smooth muscle to the relaxant effects of nitroprusside and the development of inherent tone, remain to be established. It is possible that depolarization of the smooth muscle cell membrane, which has been described by Suzuki & Twarog (1982) in main pulmonary arteries from hypoxic rats, could explain the inherent tone. Explanations for the reduced sensitivity to nitroprusside remain speculative. It is possible that during prolonged exposure of rats to hypoxia the resultant increased pulmonary artery pressure and/or the maintained hypoxaemia could stimulate or augment the release of nitric oxide in vivo. This might then downregulate or desensitize the soluble guanylate cyclase on which both nitric oxide and nitroprusside act, as suggested previously (Wanstall & O'Donnell, 1992). The source of the nitric oxide could be either the endothelium (Palmer et al., 1987) or the smooth muscle (Mollace et al., 1991). Alternatively the hypoxaemia might inhibit hydrogen peroxide-dependent activation of guanylate cyclase. The latter is a novel, oxygen-dependent mechanism for vascular smooth muscle relaxation proposed by Burke-Wolin & Wolin (1989). In conclusion, this study has shown that in rats with hypoxic pulmonary hypertension there is a reduction in the relaxant potency of nitroprusside on isolated pulmonary artery preparations. This desensitization to nitroprusside is reversible and appears to be due to an alteration in the responsiveness of the pulmonary vascular smooth muscle and not to an alteration in the influence, in vitro, of the endothelium on responses of the underlying smooth muscle. It is unlikely that this functional change reflects the structural changes to the pulmonary artery that occur in hypoxic pulmonary hypertension, since the time course did not coincide with the development and reversal of vascular hypertrophy. It is more likely to be associated with the rise in pulmonary artery pressure or the maintained hypoxaemia, both of which followed the same time course as the changes in the functional response to nitroprusside. It is postulated that the increase in pressure and/or the maintained hypoxaemia could give rise, in vivo, to sustained release of nitric oxide, which might desensitize soluble guanylate cyclase in the smooth muscle thereby reducing responsiveness to nitroprusside. This research was supported by the National Health and Medical Research Council of Australia. J.C.W. is an NH&MRC Research Scientist. Financial assistance to I.E.H. from the Royal Society of London is gratefully acknowledged. We thank Agatha Gambino for excellent technical assistance. References BARER, G.R., CAI, Y., RUSSELL, P.C. & EMERY, C. (1989). Reactivity and site of vasomotion in pulmonary vessels of chronically hypoxic rats: relation to structural changes. Am. Rev. Resp. Dis., 14, BURKE-WOLIN, T. & WOLIN, M.S. (1989). H22 and cgmp may function as an 2 sensor in the pulmonary artery. J. Appl. Physiol., 66, CHEN, G., SUZUKI, H. & WESTON, A.H. (1988). 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