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Effect of acidosis on α1- and α2-adrenoceptor-mediated vasoconstrictor responses in isolated arteries
443
European Journal of Pharmacology, 135 (1987) 443-447
Elsevier EJP 173SC Short communication
Effect of acidosis on am- and a2-adrenoceptor-mediated vasoconstrictor responses in isolated arteries l a i n C. M e d g e t t , Peter E. H i c k s a n d S a l o m o n Z. L a n g e r * Department of Biology, Laboratoires d'Etudes et de Recherches Synth~labo (L.E.R.S.), 58, rue de la Glaci~re, 75013 Paris, France
Received 19 January 1987, accepted 27 January 1987
We have investigated the effect of reducing the pH (from 7.5 to 7.0 by addition of HC1) on vasoconstrictor responses to noradrenaline in cat middle cerebral artery (in which responses are mediated almost entirely by a2-adrenoceptors ) and in rabbit pulmonary artery (in which responses are mediated by al-adrenoceptors ). In the cerebral artery, a reduction in pH caused a pronounced inhibition of the responses to noradrenaline, and the antagonistic effect of idazoxan (100 nM) was increased 10-fold. In contrast, in the pulmonary artery, a reduction in pH had no effect on the responses to noradrenaline and the antagonistic effect of prazosin (100 nM) was not altered. We conclude that acidosis selectively reduces the vasoconstriction mediated by a2-adrenoceptors in vitro. Cerebral artery (middle); Pulmonary artery; Acidosis; a-Adrenoceptor subtypes; (Cat, Rabbit)
1. Introduction
The difference between a~- and a2-adrenoce ptor-mediated vasoconstrictor responses with regard to their dependence on arterial p H was first investigated by McGrath and coworkers (McG r a t h et al., 1982). It was reported that pressor responses to catecholamines in pithed rats were decreased under acidotic conditions: the antagonistic effect of prazosin on these pressor responses remained constant as the p H was lowered whereas the antagonist effects of rauwolscine increased. These results led to the suggestion that acidosis favours responses mediated by the a2-subtype. However more recent results (Korstanje et al., 1985) obtained with a wide range of synthetic
* To whom all correspondence should be addressed: Department of Biology, L.E.R.S.-Synthrlabo, 58, rue de la Glaci~re, 75013 Paris, France.
agonists that have varying degrees of selectivity for cq- or a2-adrenoceptors have failed to provide clear-cut support for this proposal. In the present study we have attempted to clarify the effect of acidosis on a-adrenoceptormediated vasoconstriction by examining the effects of a reduction in p H on responses to noradrenaline in isolated arterial preparations in which only one of the two a-adrenoceptor subtypes is present. We have chosen the cat middle cerebral artery, a vessel in which responses to noradrenaline are mediated by a2-adrenoceptors (although a very small al-adrenoceptor-mediated component is also present; Medgett and Langer, 1983), and the rabbit pulmonary artery, which contains only a~-adrenoceptors (see e.g. Shepperson and Langer, 1981). In addition to avoiding the complications of a mixed population of receptors in the tissues and the problem of agonist selectivity which occur in vivo, the use of isolated vessels makes it possible to maintain equilibrium conditions in the biophase.
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
444 2. Materials and methods
were to be assessed. N o more than three successive concentration-response curves were obtained with each tissue and the effects of no more than two antagonist concentrations were assessed in either preparation. The pH of the Krebs solution was reduced from 7.5 to 7.0 by dropwise addition of HC1 according to the method of Edvinsson and Sercombe (1976). This method allows the pH to be monitored continuously and maintained within 0.1 log unit of pH 7.0. The Krebs solution had the following composition (in mM): NaC1 118, KC1 4.7, N a H C O 3 25, N a H 2 P O 4 1.0, MgC12 1.2, CaC12 2.6, glucose 11.1, ascorbic acid 0.1. Cocaine (4/tM) and propranolol (1 ~M) were present routinely. The following drugs were used: cocaine hydrochloride (Cooprration Pharmaceutique Fran~aise), Paris, France; 1noradrenaline bitartrate (Sigma, St. Louis, MO, USA): prazosin hydrochloride (Pfizer, Groton, CT, USA); d,l-propranolol hydrochloride (Sigma, St. Louis, MO, USA); idazoxan hydrochloride (Reckitt and Colman, Hull, England). The salts were dissolved in distilled water, which in the case of noradrenaline contained 50 t t g / m l ascorbic acid to prevent oxidation. Statistical analysis was performed using Student's t-test for paired or unpaired data, as appropriate.
Both middle cerebral arteries were removed under pentobarbitone anaesthesia from cats of either sex weighing 2-3 kg. The arteries were prepared for perfusion and superfusion with Krebs solution according to the method of Medgett and Langer (1983). After passage through the lumen the perfusate was allowed to superfuse the extraluminal surface of the vessel. The bathing solution was maintained at 37 ° C and was gassed with a mixture of 5% CO 2 in O 2. Vasoconstriction was measured as an increase in perfusion pressure at constant flow (5 ml/min). Non-cumulative concentration-response curves to noradrenaline were obtained (see Medgett and Langer, 1983). Rabbits (CEGAV) of either sex weighing 2-3 kg were killed by a blow on the head and the main pulmonary artery was rapidly removed and cut into 3 mm wide spiral strips which were then mounted, under a resting tension of 2 g, in 5 ml organ baths containing Krebs solution. Cumulative concentration-response curves to noradrenaline were obtained. A period of equilibration of at least 20 min was allowed for both cerebral arteries and pulmonary arteries when a-adrenoceptor antagonists were used or when the effects of a reduction in pH on the responses to noradrenaline
150
100
(B)
(A)
% max
(C)
i% max
=•3rd
% max
st
60
li0.zo-o]/
2nd
o.'~1
o.1
1
lO
0.~1
,/
1 JJM
I
0.1
1
10
100
O.Ol o.1
lO
lOO jim
Fig. 1. Effectsof reduction in pH on responses to noradrenaline in cat middle cerebral artery. The points represent the mean, and the vertical bars show the S.E.M. of at least 5 experiments. Responses are expressed as % of the maximum response to noradrenaline in the control concentration-response curve. (A) Effect of reduction in pH from 7.5 to 7.0 on responses to noradrenaline. (B) Effect of idazoxan on responses to noradrenaline at pH 7.5. (C) Effect of idazoxan on responses to noradrenaline at pH 7.0.
445 150~
% max
(A)
% max
(B)
Y/
3rd
100
% max
[PRZl 0 0.01
(C)
[PRZ] 00.01
0.1 u.M
0.1 .u.M
50
I
0.1
1
10
100
0.1
1
10
100
1000
0.1
1
10
100
[NA] ,uM
Fig. 2. Effects of reduction in p H on responses to noradrenaline in rabbit pulmonary artery. The points represent the mean and the vertical bars show the S.E.M. of 3 or 4 experiments. Responses are expressed as % of the m a x i m u m response to noradrenaline in the control concentration-response curve. (A) Effect of reduction in p H from 7.5 to 7.0 on responses to noradrenaline. (B) Effect of prazosin on responses to noradrenaline at p H 7.5. (C) Effect of prazosin on responses to noradrenaline at p H 7.0.
3. Results
In cat middle cerebral arteries, a reduction in p H from 7.5 to 7.0 caused a marked inhibition of responses to noradrenaline: the concentration-response curve was shifted to the right and the maximum response was halved. The responses were completely restored when the tissues were returned to pH 7.5 (fig. 1A). At p H 7.5 the a 2adrenoceptor antagonist idazoxan produced concentration-dependent, parallel rightward shifts of the noradrenaline concentration-response curve (fig. 1B). At pH 7.0, the magnitude of the rightward shifts was approximately 10-fold greater (compare rightward shifts produced by 1 and 10 /~M idazoxan in fig. 1B with those produced by 0.1 and 1 # M idazoxan in fig. 1C). At this p H it was not possible to overcome the antagonist effect of idazoxan. The aa-adrenoceptor antagonist prazosin (0.3 #M) produced a small (less than 3-fold) but significant rightward shift in the noradrenaline curve at pH 7.5. When the pH was reduced to 7.0, the shift was smaller and did not reach statistical significance (results not shown).
In rabbit pulmonary artery strips, reducing the pH from 7.5 to 7.0 had no effect on responses to noradrenaline or on the antagonistic activity of prazosin (fig. 2). Idazoxan, in concentrations below 1 /~M, produced no antagonism at either pH (results not shown).
4. Discussion
The main finding of the present study was that, in an isolated artery in which smooth muscle a-adrenoceptors are of the et2-type (cat middle cerebral artery), contractile responses to noradrenaline are markedly inhibited under acidotic conditions whereas in an isolated artery in which the receptors are of the at-type (rabbit pulmonary artery) the responses to noradrenaline are unaffected by a reduction in pH. These results are in agreement with those obtained in previous studies. Thus, in ring segments of cat middle cerebral artery, although in this case noradrenaline was about 10 times less potent than in the present study, Edvinsson and Sercombe (1976) showed
446 that reducing p H from 7.4 to 7.0 caused a similar inhibition of the responses. In strips of rabbit aorta, a vessel which would be expected to behave similarly to the pulmonary artery, a reduction in p H from 7.6 to 7.1 did not alter the sensitivity to noradrenaline (Williamson and Moore, 1960). In the present study, the effectiveness of idazoxan as an antagonist of the responses of cat middle cerebral artery to noradrenaline was greatly increased at an acidotic p H whereas the effectiveness of prazosin in rabbit pulmonary artery was not altered. F r o m these in vitro data it would seem clear that acidosis causes a reduction in vasoconstrictor responses mediated by a2-adrenoceptors but does not affect responses mediated by al-adrenoceptors. Korstanje et al. (1985) have proposed that acidosis might decrease the effectiveness of a-adrenoceptor agonists dependent on extracellular calcium. Although we have not addressed this proposal directly, it is relevant to note that the responses to noradrenaline obtained in cat middle cerebral arteries at a calcium ion concentration of 2.6 raM, were about twice as great as those obtained in the study of Medgett and Langer (1983) in which the calcium ion concentration was 1.3 mM. Thus, like a reduction in pH, a reduced concentration of calcium ions in the Krebs solution is associated similarly with a marked inhibition of the responses to noradrenaline in the cerebral artery. It may also be noted that, in rat tail artery, which contains a mixed population of a 1- and a2-adrenoceptors on smooth muscle cells (Medgett and Langer, 1984), the response to noradrenaline is less sensitive to a reduction in calcium ion concentration (Medgett and Rajanayagam, 1984) than in cat middle cerebral artery. Reducing the p H by 0.5 log unit also produces a much smaller inhibition of the responses in rat tail artery (Medgett and Young, in preparation) than in cat middle cerebral artery. Notwithstanding the above conclusions, it may be argued that a reduction in p H will inhibit responses mediated by ct2- but not al-adrenoce ptors simply because there is a much smaller receptor reserve for the a2-subtype. In fact, Ruffolo and Zeid (1986) have recently reported that dog saphenous vein has a 4-fold larger receptor reserve
for smooth muscle a 1- compared to a2-adrenoce ptots and have also suggested that this difference might exist in other vessels. Although we cannot rule out such an explanation for our results, it should be pointed out that the marked increase in the antagonistic effectiveness of idazoxan at a low p H is not consistent with a reduction in receptor reserve, but points instead to a specific relationship between p H and a2-adrenoceptors. The nature of this relationship is presently unclear and requires further investigation. Some speculative comments may be made concerning the reasons why the available in vivo data (see Introduction for references) are not always consistent with the present results. In all the studies carried out in the pithed rat, respiratory acidosis was induced by changing ventilation and hence p C O 2. In the present study, as in that of Edvinsson and Sercombe (1976), acidotic conditions were obtained by the addition of HC1. Tobian et al. (1959) found that the inhibition of noradrenalineinduced contraction in rat aorta was less pronounced with Krebs solutions in which the p H was reduced b y - a d d i t i o n of hydrogen ions ('metabolic' type of acidosis) than with solutions in which the p H was reduced by gassing with a high C O 2 mixture ('respiratory' type of acidosis). As noted by Tobian et al. (1959) the high CO 2 tension in respiratory acidosis probably leads to a very rapid penetration of lipid soluble CO 2 into smooth muscle cells while in metabolic acidosis the excess hydrogen ions, not being so lipid soluble, penetrate more slowly. Hence there is probably more intracellular and membrane acidosis in respiratory acidosis than in metabolic acidosis. Considerable evidence has accumulated to show that, with some exceptions, the pressor responses in the pithed rat that are mediated by activation of smooth muscle a2-adrenoceptors are more dependent on extracellular calcium than are responses mediated by the al-subtype (see Korstanje et al., 1985), which preferentially utilise intraceUular stores of calcium to initiate the contractile process. Thus it can be proposed that respiratory acidosis, which causes acidosis both intraceUularly and extracellularly, will not distinguish so markedly as metabolic acidosis be-
447 t w e e n r e s p o n s e s m e d i a t e d b y a 2- a n d a l - a d r e n o c e p t o r s . I f t h i s is i n d e e d t h e case, it is n o t s u r p r i s i n g t h a t K o r s t a n j e e t al. ( 1 9 8 5 ) f a i l e d t o o b t a i n any correlation between the effect of respiratory acidosis on a wide variety of a-adrenoceptor a g o n i s t s , r e g a r d l e s s o f t h e i r s e l e c t i v i t y f o r a 1- o r a2-adrenoceptors or their dependence on extracelular calcium. It can be concluded from the present study that acidosis selectively reduces the vasoconstriction m e d i a t e d b y a 2 - a d r e n o c e p t o r s i n v i t r o , w h i l e it fails t o m o d i f y t h e a ~ - a d r e n o c e p t o r - m e d i a t e d vasoconstriction.
Acknowledgements The authors would like to thank Catherine Grandet for expert technical assistance, and Miss Anny Sebbah for preparing the manuscript.
References Edvinsson, L. and R. Sercombe, 1976, Influence of pH and pCO 2 on a-receptor mediated contraction in brain vessels, Acta Physiol. Scand. 97, 325. Korstanje, C., M.J. Mathy, K. Van Charldorp, A. De Jonge and P.A. Van Zwieten, 1985, Influence of respiratory acidosis or alkalosis on pressor responses mediated by a 1- and
a2-adrenoceptors in pithed normotensive rats, NaunynSchmiedeb. Arch. Pharmacol. 330, 187. McGrath, J.C., N.A. Flavahan and C.E. McKean, 1982, a 1and a2-adrenoceptor-mediated pressor and chronotropic effects in the rat and rabbit, J. Cardiovasc. Pharmacol. 4, S101. Medgett, I.C. and S.Z. Langer, 1983, Characterization of smooth muscle a-adrenoceptors and of responses to electrical stimulation in the cat isolated perfused middle cerebral artery, Naunyn-Schmiedeb. Arch. Pharmacol. 323, 24. Medgett, I.C. and S.Z. Langer, 1984, Heterogeneity of smooth muscle a-adrenoceptors in rat tail artery in vitro, J. Pharmacol. Exp. Ther. 229, 823. Medgett, I.C. and M.A.S. Rajanayagam, 1984, Effects of reduced calcium ion concentration and of diltiazem on vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in rat isolated tail artery, Br. J. Pharmacol. 83, 889. Ruffolo, R.R. and R.Z. Zeid, 1986, Relationship between a-adrenoceptor occupancy and response for the al-adrenoceptor agonist, cirazoline, and the a2-adrenoceptor agonist, BHT 933, in canine saphenous vein, J. Pharmacol. Exp. Ther. 235, 636. Shepperson, N.B. and S.Z. Langer, 1981, The effects of the 2-amino-tetrahydronaphtalene derivative M7, a selective a2-adrenoceptor agonist in vitro, Naunyn-Schmiedeb. Arch. Pharmacol. 318, 10. Tobian, L., S. Martin and W. Eilers, 1959, Effect of pH on norepinephrine-induced contractions of isolated arterial smooth muscle, Am. J. Physiol. 196, 998. Williamson, A.W.R. and F.D. Moore, 1960, Norepinephrine sensitivity of isolated rabbit aorta strips in solutions of varying pH and electrolyte content, Am. J. Physiol. 198, 1157.
European Journal of Pharmacology, 135 (1987) 443-447
Elsevier EJP 173SC Short communication
Effect of acidosis on am- and a2-adrenoceptor-mediated vasoconstrictor responses in isolated arteries l a i n C. M e d g e t t , Peter E. H i c k s a n d S a l o m o n Z. L a n g e r * Department of Biology, Laboratoires d'Etudes et de Recherches Synth~labo (L.E.R.S.), 58, rue de la Glaci~re, 75013 Paris, France
Received 19 January 1987, accepted 27 January 1987
We have investigated the effect of reducing the pH (from 7.5 to 7.0 by addition of HC1) on vasoconstrictor responses to noradrenaline in cat middle cerebral artery (in which responses are mediated almost entirely by a2-adrenoceptors ) and in rabbit pulmonary artery (in which responses are mediated by al-adrenoceptors ). In the cerebral artery, a reduction in pH caused a pronounced inhibition of the responses to noradrenaline, and the antagonistic effect of idazoxan (100 nM) was increased 10-fold. In contrast, in the pulmonary artery, a reduction in pH had no effect on the responses to noradrenaline and the antagonistic effect of prazosin (100 nM) was not altered. We conclude that acidosis selectively reduces the vasoconstriction mediated by a2-adrenoceptors in vitro. Cerebral artery (middle); Pulmonary artery; Acidosis; a-Adrenoceptor subtypes; (Cat, Rabbit)
1. Introduction
The difference between a~- and a2-adrenoce ptor-mediated vasoconstrictor responses with regard to their dependence on arterial p H was first investigated by McGrath and coworkers (McG r a t h et al., 1982). It was reported that pressor responses to catecholamines in pithed rats were decreased under acidotic conditions: the antagonistic effect of prazosin on these pressor responses remained constant as the p H was lowered whereas the antagonist effects of rauwolscine increased. These results led to the suggestion that acidosis favours responses mediated by the a2-subtype. However more recent results (Korstanje et al., 1985) obtained with a wide range of synthetic
* To whom all correspondence should be addressed: Department of Biology, L.E.R.S.-Synthrlabo, 58, rue de la Glaci~re, 75013 Paris, France.
agonists that have varying degrees of selectivity for cq- or a2-adrenoceptors have failed to provide clear-cut support for this proposal. In the present study we have attempted to clarify the effect of acidosis on a-adrenoceptormediated vasoconstriction by examining the effects of a reduction in p H on responses to noradrenaline in isolated arterial preparations in which only one of the two a-adrenoceptor subtypes is present. We have chosen the cat middle cerebral artery, a vessel in which responses to noradrenaline are mediated by a2-adrenoceptors (although a very small al-adrenoceptor-mediated component is also present; Medgett and Langer, 1983), and the rabbit pulmonary artery, which contains only a~-adrenoceptors (see e.g. Shepperson and Langer, 1981). In addition to avoiding the complications of a mixed population of receptors in the tissues and the problem of agonist selectivity which occur in vivo, the use of isolated vessels makes it possible to maintain equilibrium conditions in the biophase.
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
444 2. Materials and methods
were to be assessed. N o more than three successive concentration-response curves were obtained with each tissue and the effects of no more than two antagonist concentrations were assessed in either preparation. The pH of the Krebs solution was reduced from 7.5 to 7.0 by dropwise addition of HC1 according to the method of Edvinsson and Sercombe (1976). This method allows the pH to be monitored continuously and maintained within 0.1 log unit of pH 7.0. The Krebs solution had the following composition (in mM): NaC1 118, KC1 4.7, N a H C O 3 25, N a H 2 P O 4 1.0, MgC12 1.2, CaC12 2.6, glucose 11.1, ascorbic acid 0.1. Cocaine (4/tM) and propranolol (1 ~M) were present routinely. The following drugs were used: cocaine hydrochloride (Cooprration Pharmaceutique Fran~aise), Paris, France; 1noradrenaline bitartrate (Sigma, St. Louis, MO, USA): prazosin hydrochloride (Pfizer, Groton, CT, USA); d,l-propranolol hydrochloride (Sigma, St. Louis, MO, USA); idazoxan hydrochloride (Reckitt and Colman, Hull, England). The salts were dissolved in distilled water, which in the case of noradrenaline contained 50 t t g / m l ascorbic acid to prevent oxidation. Statistical analysis was performed using Student's t-test for paired or unpaired data, as appropriate.
Both middle cerebral arteries were removed under pentobarbitone anaesthesia from cats of either sex weighing 2-3 kg. The arteries were prepared for perfusion and superfusion with Krebs solution according to the method of Medgett and Langer (1983). After passage through the lumen the perfusate was allowed to superfuse the extraluminal surface of the vessel. The bathing solution was maintained at 37 ° C and was gassed with a mixture of 5% CO 2 in O 2. Vasoconstriction was measured as an increase in perfusion pressure at constant flow (5 ml/min). Non-cumulative concentration-response curves to noradrenaline were obtained (see Medgett and Langer, 1983). Rabbits (CEGAV) of either sex weighing 2-3 kg were killed by a blow on the head and the main pulmonary artery was rapidly removed and cut into 3 mm wide spiral strips which were then mounted, under a resting tension of 2 g, in 5 ml organ baths containing Krebs solution. Cumulative concentration-response curves to noradrenaline were obtained. A period of equilibration of at least 20 min was allowed for both cerebral arteries and pulmonary arteries when a-adrenoceptor antagonists were used or when the effects of a reduction in pH on the responses to noradrenaline
150
100
(B)
(A)
% max
(C)
i% max
=•3rd
% max
st
60
li0.zo-o]/
2nd
o.'~1
o.1
1
lO
0.~1
,/
1 JJM
I
0.1
1
10
100
O.Ol o.1
lO
lOO jim
Fig. 1. Effectsof reduction in pH on responses to noradrenaline in cat middle cerebral artery. The points represent the mean, and the vertical bars show the S.E.M. of at least 5 experiments. Responses are expressed as % of the maximum response to noradrenaline in the control concentration-response curve. (A) Effect of reduction in pH from 7.5 to 7.0 on responses to noradrenaline. (B) Effect of idazoxan on responses to noradrenaline at pH 7.5. (C) Effect of idazoxan on responses to noradrenaline at pH 7.0.
445 150~
% max
(A)
% max
(B)
Y/
3rd
100
% max
[PRZl 0 0.01
(C)
[PRZ] 00.01
0.1 u.M
0.1 .u.M
50
I
0.1
1
10
100
0.1
1
10
100
1000
0.1
1
10
100
[NA] ,uM
Fig. 2. Effects of reduction in p H on responses to noradrenaline in rabbit pulmonary artery. The points represent the mean and the vertical bars show the S.E.M. of 3 or 4 experiments. Responses are expressed as % of the m a x i m u m response to noradrenaline in the control concentration-response curve. (A) Effect of reduction in p H from 7.5 to 7.0 on responses to noradrenaline. (B) Effect of prazosin on responses to noradrenaline at p H 7.5. (C) Effect of prazosin on responses to noradrenaline at p H 7.0.
3. Results
In cat middle cerebral arteries, a reduction in p H from 7.5 to 7.0 caused a marked inhibition of responses to noradrenaline: the concentration-response curve was shifted to the right and the maximum response was halved. The responses were completely restored when the tissues were returned to pH 7.5 (fig. 1A). At p H 7.5 the a 2adrenoceptor antagonist idazoxan produced concentration-dependent, parallel rightward shifts of the noradrenaline concentration-response curve (fig. 1B). At pH 7.0, the magnitude of the rightward shifts was approximately 10-fold greater (compare rightward shifts produced by 1 and 10 /~M idazoxan in fig. 1B with those produced by 0.1 and 1 # M idazoxan in fig. 1C). At this p H it was not possible to overcome the antagonist effect of idazoxan. The aa-adrenoceptor antagonist prazosin (0.3 #M) produced a small (less than 3-fold) but significant rightward shift in the noradrenaline curve at pH 7.5. When the pH was reduced to 7.0, the shift was smaller and did not reach statistical significance (results not shown).
In rabbit pulmonary artery strips, reducing the pH from 7.5 to 7.0 had no effect on responses to noradrenaline or on the antagonistic activity of prazosin (fig. 2). Idazoxan, in concentrations below 1 /~M, produced no antagonism at either pH (results not shown).
4. Discussion
The main finding of the present study was that, in an isolated artery in which smooth muscle a-adrenoceptors are of the et2-type (cat middle cerebral artery), contractile responses to noradrenaline are markedly inhibited under acidotic conditions whereas in an isolated artery in which the receptors are of the at-type (rabbit pulmonary artery) the responses to noradrenaline are unaffected by a reduction in pH. These results are in agreement with those obtained in previous studies. Thus, in ring segments of cat middle cerebral artery, although in this case noradrenaline was about 10 times less potent than in the present study, Edvinsson and Sercombe (1976) showed
446 that reducing p H from 7.4 to 7.0 caused a similar inhibition of the responses. In strips of rabbit aorta, a vessel which would be expected to behave similarly to the pulmonary artery, a reduction in p H from 7.6 to 7.1 did not alter the sensitivity to noradrenaline (Williamson and Moore, 1960). In the present study, the effectiveness of idazoxan as an antagonist of the responses of cat middle cerebral artery to noradrenaline was greatly increased at an acidotic p H whereas the effectiveness of prazosin in rabbit pulmonary artery was not altered. F r o m these in vitro data it would seem clear that acidosis causes a reduction in vasoconstrictor responses mediated by a2-adrenoceptors but does not affect responses mediated by al-adrenoceptors. Korstanje et al. (1985) have proposed that acidosis might decrease the effectiveness of a-adrenoceptor agonists dependent on extracellular calcium. Although we have not addressed this proposal directly, it is relevant to note that the responses to noradrenaline obtained in cat middle cerebral arteries at a calcium ion concentration of 2.6 raM, were about twice as great as those obtained in the study of Medgett and Langer (1983) in which the calcium ion concentration was 1.3 mM. Thus, like a reduction in pH, a reduced concentration of calcium ions in the Krebs solution is associated similarly with a marked inhibition of the responses to noradrenaline in the cerebral artery. It may also be noted that, in rat tail artery, which contains a mixed population of a 1- and a2-adrenoceptors on smooth muscle cells (Medgett and Langer, 1984), the response to noradrenaline is less sensitive to a reduction in calcium ion concentration (Medgett and Rajanayagam, 1984) than in cat middle cerebral artery. Reducing the p H by 0.5 log unit also produces a much smaller inhibition of the responses in rat tail artery (Medgett and Young, in preparation) than in cat middle cerebral artery. Notwithstanding the above conclusions, it may be argued that a reduction in p H will inhibit responses mediated by ct2- but not al-adrenoce ptors simply because there is a much smaller receptor reserve for the a2-subtype. In fact, Ruffolo and Zeid (1986) have recently reported that dog saphenous vein has a 4-fold larger receptor reserve
for smooth muscle a 1- compared to a2-adrenoce ptots and have also suggested that this difference might exist in other vessels. Although we cannot rule out such an explanation for our results, it should be pointed out that the marked increase in the antagonistic effectiveness of idazoxan at a low p H is not consistent with a reduction in receptor reserve, but points instead to a specific relationship between p H and a2-adrenoceptors. The nature of this relationship is presently unclear and requires further investigation. Some speculative comments may be made concerning the reasons why the available in vivo data (see Introduction for references) are not always consistent with the present results. In all the studies carried out in the pithed rat, respiratory acidosis was induced by changing ventilation and hence p C O 2. In the present study, as in that of Edvinsson and Sercombe (1976), acidotic conditions were obtained by the addition of HC1. Tobian et al. (1959) found that the inhibition of noradrenalineinduced contraction in rat aorta was less pronounced with Krebs solutions in which the p H was reduced b y - a d d i t i o n of hydrogen ions ('metabolic' type of acidosis) than with solutions in which the p H was reduced by gassing with a high C O 2 mixture ('respiratory' type of acidosis). As noted by Tobian et al. (1959) the high CO 2 tension in respiratory acidosis probably leads to a very rapid penetration of lipid soluble CO 2 into smooth muscle cells while in metabolic acidosis the excess hydrogen ions, not being so lipid soluble, penetrate more slowly. Hence there is probably more intracellular and membrane acidosis in respiratory acidosis than in metabolic acidosis. Considerable evidence has accumulated to show that, with some exceptions, the pressor responses in the pithed rat that are mediated by activation of smooth muscle a2-adrenoceptors are more dependent on extracellular calcium than are responses mediated by the al-subtype (see Korstanje et al., 1985), which preferentially utilise intraceUular stores of calcium to initiate the contractile process. Thus it can be proposed that respiratory acidosis, which causes acidosis both intraceUularly and extracellularly, will not distinguish so markedly as metabolic acidosis be-
447 t w e e n r e s p o n s e s m e d i a t e d b y a 2- a n d a l - a d r e n o c e p t o r s . I f t h i s is i n d e e d t h e case, it is n o t s u r p r i s i n g t h a t K o r s t a n j e e t al. ( 1 9 8 5 ) f a i l e d t o o b t a i n any correlation between the effect of respiratory acidosis on a wide variety of a-adrenoceptor a g o n i s t s , r e g a r d l e s s o f t h e i r s e l e c t i v i t y f o r a 1- o r a2-adrenoceptors or their dependence on extracelular calcium. It can be concluded from the present study that acidosis selectively reduces the vasoconstriction m e d i a t e d b y a 2 - a d r e n o c e p t o r s i n v i t r o , w h i l e it fails t o m o d i f y t h e a ~ - a d r e n o c e p t o r - m e d i a t e d vasoconstriction.
Acknowledgements The authors would like to thank Catherine Grandet for expert technical assistance, and Miss Anny Sebbah for preparing the manuscript.
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