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Studies on the mechanism of potentiation of arteriolar constriction by dexamethasone in vivo
MICROVASCULAR
Studies
RESEARCH
9,
317-323(1975)
on the Mechanism of Potentiation Constriction by Dexamethasone
of Arteriolar in Viva
A. C. DARKE Department
of Physiology,
University
of Manitoba,
Winnipeg, Canada R3E 0 W3
Received August 19, 1974
The effectof dexamethasoneon the responsesof rat mesentericarteriolesin viva to epinephrineand vasopressinhas beeninvestigated.Superfusionof the rat mesoappendix with a solution containing dexamethasonesodium phosphate(0.2mg/ml) resultedin a potentiation of the arteriolar constriction producedby topically applied epinephrine. Vasopressin-inducedarteriolar constrictions were potentiated by dexamethasonein five of elevenpreparations.Theseresults demonstratethat the potentiation of the effectsof vasoconstrictoragentson vascularsmooth muscleby dexamethasoneis not restricted to agents acting on adrenergic receptors. Any general theory of the mechanismwhereby glucocorticoids potentiate arteriolar constriction must therefore explain potentiation of the effects of nonadrenergic agents. INTRODUCTION The mechanism by which adrenal steroids potentiate the activity of vasoconstrictor agents on vascular smooth muscle is not clearly understood. A good deal of study has been made specifically of the potentiation of the vascular effects of the catecholamines by glucocorticoids. Kalsner (1969a and b) has produced extensive evidence to support his view that hydrocortisone and other adrenal steroids enhance vascular reactivity to adrenaline by inhibiting catechol-o-methyl transferase (COMT), resulting in a decreased rate of inactivation of epinephrine within the tissue. Results with norepinephrine provide something of a paradox in that potentiation by hydrocortisone can be demonstrated using isolated arterial preparations in z&o (Fowler and Chou, 1961; Besse and Bass, 1966; Kalsner, 1969a) but not when constrictor activity is measured in terms of the pressor response to norepinephrine in the intact animal (Lecomte et ai., 1959; Besse and Bass, 1966; Margolius et al., 1967). In the experiments of Kalsner (1969a) hydrocortisone potentiation of the constrictor action of norepinephrine was much smaller than that of epinephrine. This was explained by the effectiveness of monamine oxidase as an alternate pathway for norepinephrine inactivation. In contrast to the results obtained using pressor responses to norepinephrine, the constrictor action of both epinephrine and norepinephrine on mesenteric arterioles measured by intravital microscopy has been found to be potentiated by systemic administration of cortisone prednisolone or dexamethasone (Altura, 1966, 1971). Similarly, in the abstract of Baez et al. (1970) superfusion of the rat mesentery with a solution of dexamethasone was reported to potentiate the increase in arteriolar smooth muscle cell thickness produced by topical epinephrine or norepinephrine. However, Copyright 0 1975 by Academic Press, Inc. 317
All rights ofreproduction Printed in Great Britain
in any form
reserved.
318
A. C. DARKE
in a recent article it has been reported that methylprednisolone and hydrocortisone in fact inhibit the action of epinephrine and norepinephrine on mesenteric arterioles (Altura et al., 1974). Using constant-flow perfusion of the hindquarters of the cat or the dog hindpaw Yard and Kadowitz (1972) demonstrated a potentiation by hydrocortisone of the rise in perfusion pressure produced by intra-arterial injections of epinephrine but not norepinephrine. This potentiation was still obtained after previous administration of either a COMT inhibitor or cocaine. These authors concluded that the affects of hydrocortisone were therefore not due to interference with the mechanismsfor uptake or inactivation of epinephrine, and thus proposed that its action was due to an increase in adrenergic receptor affinity for epinephrine. In these experiments aldosterone also potentiated the vascular effectsof epinephrine but, in contrast to the results of Altura (1966, 1971) and Baez et al. (1970), dexamethasonewas without effect. It is apparent that COMT inhibition cannot completely explain the potentiation of catecholamines by glucocorticoids. Moreover, in a preliminary report Baezet al. (1970) have demonstrated that dexamethasonealso potentiates the action of methoxamine, a noncatechol adrenergic amine, on arteriolar smooth muscle. In contrast, in the aortic strip preparation methoxamine contractions are not potentiated by hydrocortisone (Besseand Bass, 1966; Kalsner, 1969).It is also an open question whether the potentiating action of glucocorticoids is actually restricted to agents acting on adrenergic receptors. The only evidence in the literature relevant to this point seem to be the observations of Altura (1966) that after systemic administration of glucocorticoid, arterioles became responsive to serotonin and that chronic treatment of rats with cortisone results in an enhanced constrictor responseof mesenteric arterioles to vasopressin. The present experiments were therefore designed to determine whether acute topical application of the potent glucocorticoid dexamethasonewould potentiate the constriction of single arterioles in the rat mesentery to vasopressin in addition to potentiating the action of a catecholamine (epinephrine).
MATERIALS
AND METHODS
The mesoappendix of 16 female Long-Evans rats (weight range 63-244 g) was prepared for microscopic observation essentially according to the procedures outlined by Zweifach and Metz (1956). Anesthesia was induced with nembutal (30 mg/kg im) plus ether if required and maintained by supplementary doses of nembutal given through a cannulated jugular vein. The trachea and carotid artery were also cannulated routinely. The cecum was exteriorized through a mid-line abdominal incision and the mesoappendix spread over a plexiglass block. Exposed portions of gut were covered with moist cotton wool and thin transparent film. Except for periods of topical drug application the surface of the mesoappendix was superfused with warm (38°C) Ringer’sgelatin solution. This solution was made up fresh on the day of the experiment by a 10 times dilution of a stock solution (90 g NaCl, 4.2 g KCI, 2.4 g CaCI, in I litre) plus gelatin to a concentration of 1%. The pH of the resulting solution was adjusted to 7.2-7.4 with NaHCO,. The rat’s oesophagealtemperature was controlled by means
DEXAMETHASONE
AND ARTERIOLAR
CONSTRICTION
319
of a heated microscope stage-the temperature of the cecum and ileum was separately controlled by placing them on a plexiglass stagethrough which water at 38°C circulated. The microcirculation was viewed on a Leitz Orthoplan microscope equipped with a 32 x UMK objective and 12.5 x oculars. The light source was a 100 W Quartz Iodine lamp. Internal arteriolar diameters were measured with a Watson imageshearing eyepiececalibrated against a stage micrometer. Arterial blood pressure was recorded from the carotid cannula with a Statham P23Gb transducer and displayed on a Beckman RP dynograph. Vasoactive agents were applied to the surface of the mesenteryin volumes of 0.1 ml. Epinephrine was used both as the bitartrate (Sigma) and as the hydrochloride (Parke Davis). Stock solutions of epinephrine in 5% dextrose solution were kept on ice and diluted 10 times with Ringer’s-gelatin solution immediately before warming and topical application to the mesentery. Doses of epinephrine are expressedin terms of the base.Vasopressin was used either as Pitressin (Parke Davis) or as synthetic lysinevasopressin (Sigma) and also diluted in Ringer’s gelatin. Dexamethasone sodium phosphate (Decadron-Merck, Sharp and Dohme) was diluted in Ringer%-gelatin solution to a concentration of 0.2 mg/ml, and this solution was used for superfusion of the mesentery in the sameway as the normal Ringer’s solution. Observations were made of the effect of epinephrine and vasopressin on arterioles of diameter 21-48 pm. Measurements of diameter were made at a site on the arterioles where the best visualization of the internal wall of the arteriole could be made. Usually four or more topical applications of the vasoconstrictor agent were made before, during and again after superfusion of the mesenterywith the dexamethasone-Ringer’sgelatin solution, After each measurementsufficient time (2-3 min) was allowed for full recovery of the preparation and this interval was not increased by more than about 2 min at the time the superfusion solution was changed. For each application of adrenaline or vasopressinthe maximum decreasein diameter was measured.The doses of adrenaline and vasopressin used were chosen so that they produced constrictor responseswell below maximal. The unpaired t test was used for statistical analysis of the results a P value of less than 0.05 being considered significant. RESULTS Superfusion of the mesoappendix with Ringer’s-gelatin solution containing 0.2 mg/ml dexamethasone did not result in a change in systemic arterial blood pressure, nor produce a significant change in mean arteriolar diameter when all the experiments were considered together. In four individual experiments the mean diameter before and after dexamethasone was significantly greater than the mean diameter during dexamethasone superfusion. The mean decrease in diameter produced by dexamethasone in these four experiments was 2.53% (range 0.95-3.81x) of the control diameter. A statistically significant increasein diameter occurred during dexamethasone superfusion in two of the preparations although the percentage changes in diameter were again very small (2.59 and 2.31 %). The results of the five experiments in which the effect of dexamethasone on the constrictor action of epinephrine was tested are shown in Fig. 1. In all cases,except
320
EPINEPHRINE
(p/ml
1
FIG. 1. The effect of dexamethasone on epinephrine-induced arteriolar constriction in five mesoappendix preparations (experiments l-5). Experiment numbers are reference numbers only. Height of bars indicate mean decrease in arteriolar diameter (‘m k 1 SE). Numbers above each bar indicate the number of observations. Numbers below the bars indicate concentration of epinephrine @g/ml).
6.
’IcI
034
ill 6.
C CON-rRol.
I LIEXAMETHASONE 02 mg/ml
7.
6
6
I
002
5 b
‘1 la .008
68. 9. . 10.
WNTROL
.02
5
1% 5
5
~$Y-fy 5-A A 4L
5
6 .02
CONlROL
5
6
DEXAMETHASONE
11. 12. 6
b 13. 6 9.
14.
15.
4 16.
CONlROL
02 mgp
FIG. 2. The effect of dexamethasone on vasopressin-induced arteriolar constriction in eleven mesoappendix preparations (experiments 6-16). Experiment numbers are reference numbers only. AExperiments in which dexamethasone potentiated the effect of vasopressin. B-Experiments in which dexamethasone did not potentiate the effect of vasopressin. Mean decreases in diameter are shown (pm k 1 SE). Numbers adjacent to SE lines indicate the number of observations. Numbers adjacent to the response lines indicate the concentration of vasopressin (units/ml) used in each experiment.
DEXAMETHASONE
AND ARTERIOLAR
CONSTRICTION
321
one, the mean of the decreasesin arteriolar diameter produced by epinephrine before and after dexamethasonewas significantly less than the constriction obtained during dexamethasonesuperfusion. In one experiment (1 in Fig. 1) the responseto 0.2 pg/ml epinephrine was markedly potentiated by dexamethasone but the response to 0.5 pg/ml epinephrine was not significantly potentiated. This may be explained by the fact that the higher dose of epinephrine produced almost complete closure of the arteriole even in the control period before superfusion with dexamethasone. Mesoappendix preparations from 11 rats were tested for the possible potentiation by dexamethasoneof the arteriolar constriction induced by vasopressin. A statistically significant increase in the response to vasopressin during superfusion with dexamethasone was recorded in five experiments (see Fig. 2A). In one experiment (9 in Fig. 2A) two periods of dexamethasone superfusion, each preceded and followed by control periods of Ringers-gelatin superfusion were used. In the first trial there was an increased response to vasopressin but it was not statistically significant, whereas in the second trial the potentiation was statistically significant. For this reason the results of experiment 9 also appear in Fig. 2B which shows the results obtained in those experiments in which the mean of the decreasesin diameter produced by vasopressin before and after dexamethasone superfusion was not significantly different from the mean decreasein diameter produced by vasopressinduring dexamethasonesuperfuaion. DlSCUSSlON While cortisone, hydrocortisone and methylprednisolone topically applied to the mesoappendix do not produce changes in arteriolar diameter (Altura, 1971; Altura et al., 1974) it has been found by Baez (quoted by Altura, 1971) that topical dexamethasone will increase microvascular tone in 20-25 % of mesoappendix preparations. This proportion is similar to that found in the present experiments where a small but statistically significant decreasein arteriolar diameter occurred in 4 of I6 preparations during superfusion of the mesoappendix with Ringer’s-gelatin solution containing 0.2 mg/ml dexamethasone. However, in two of the other preparations there was a small, although statistically significant, increase in diameter during dexamethasone superfusion. The variability of the effect of dexamethasonealone and the very small changesin diameter would make it unlikely that these are changesof relevance to the overall effect of dexamethasone on the arteriolar response to adrenaline and vasopressin. Moreover since the mean change in diameter for all 16 preparations was not statistically significant and systemic blood pressure was unaffected by superfusion of the mesentery with dexamethasone, decreasesin arteriolar diameter produced by a particular vasoconstrictor agent in the presenceand absenceof dexamethasonemay be compared directly. In the caseof epinephrine, the results presented in this paper confirm the findings of Baez et al. (1970) that topically applied dexamethasone potentiates its constrictor action on arterioles in the rat mesoappendix. Systemicadministration of dexamethasone has also been reported to potentiate arteriolar constriction by epinephrine (Altura, 1966, 1971)although it has not been possible to confirm this in this laboratory (Darke, unpublished observations). The results obtained with vasopressindemonstrate that acute topical administration
322
A. C. DARKE
of dexamethasone can potentiate its constrictor action on arterioles in vivo. A significant increase in the vasoconstrictor response to vasopressin was obtained during dexamethasone superfusion in 5 of 1l mesoappendix preparations. In the remaining six experiments a statistically significant increase in the response could not be demonstrated and in one preparation there is a significant decrease in the response. The reason for this variation in the effect of dexamethasone is unclear. It is unrelated to any effect of dexamethasone alone since a statistically significant effect of dexamethasone alone on arteriolar diameter was observed in only three of the vasopressin experiments. Moreover in two of the experiments an increase in diameter during dexamethasone superfusion was associated with nonpotentiation of vasopressin whereas in the other experiment, although the effect of vasopressin was also not potentiated, there was in fact a decrease in diameter during dexamethasone superfusion. The presence or absence of potentiation was also not found to be related to the use of pitressin as opposed to synthetic lysine-vasopressin. Even though potentiation of vasopressin was not found in all preparations the positive results nevertheless mean that any comprehensive explanation of the potentiation of the effect of vasoactive agents by glucocorticoids must involve a mechanism to explain the potentiation of nonadrenergic agents. It is unlikely, as discussed by Altura (197l), that inhibition of COMT by glucocorticoids can completely explain even the potentiation of all adrenergic agents since, as reported by Baez et al. (1970), dexamethasone potentiates the action of methoxamine on arterioles. While it is possible that potentiation of the action of adrenergic agents by glucocorticoids might involve a modification of the affinity of these agents for the a-receptor (Besse and Bass, 1966; Yard and Kadowitz, 1972) it is clear that this cannot be the only site of action of glucocorticoids. Moreover, it appears that it may not be possible to consider all glucocorticoids as having the same effect on the microcirculatory action of vasopressin. Altura et al. (1974) recently found that systemic administration of hydrocortisone and methylprednisole inhibited the actions of vasopressin epinephrine and norepinephrine on mesenteric arterioles, in contrast to the present results with dexamethasone. It is apparent that further experiments are required to elucidate the reason for these differences between individual glucocorticoids with respect to their effect on the actions of vasoactive agents. ACKNOWLEDGMENTS This work was supported by grants from the Manitoba Heart Foundation. I am grateful to Dr. W. D. Dorian, Merck, Sharp and Dohme Ltd., for a generous supply of dexamethasone, and to Dr. P. Gaskell for his helpful criticism of the manuscript. REFERENCES ALTURA, B. M. (1966). Role of glucocorticoids in local regulation of blood flow. Amer. J. Physiol.
211,1393-1397. ALTURA, B. M. (1971). Chemical and humoral regulation of blood flow through the precapillary sphincter. Microvasc. Res. 3, 361-384. ALTURA, B. M., ALTURA, B. T., AND HERSHEY, S. G. (1974). Peripheral vascular action of glucocorti-
coids and protection in circulatory shock (abstract). VIII Touquet, France. BAEZ, S., LQRENZO, M., AND ORKIN, L. R. (1970). Interference hydrocortisone potentiation of responses to epinephrine, single smooth muscle cell in situ. Physiologist 13, (Abstract),
Conference on microcirculation,
Le
by histamine and betahistine HCI with norepinephrine and methoxamine in 140.
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AND ARTERIOLAR
CONSTRICTION
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BESSE,J. C., AND BASS,A. D. (1966). Potentiation by hydrocortisone of responses to catecholamines in vascular smooth muscle. J. Pharmacol. Exp. Thu. 154,224-238. FOWLER,N. O., AND CHOU, H. F. (1961). Potentiation of smooth muscle contraction by adrenal steroids. Circ. Res. 9, 153-156. KALSNER, S. (1969a). Mechanism of hydrocortisone potentiation of responses lo epinephrine and norepinephrine in rabbit aorta. Circ. Res. 24, 383-395. KALSNER,S. (1969b). Steroid potentiation of responses to sympathomimetic amines in aortic strips. Brit. J. Pharmacol. 36, 582-593. LECOMTE, J., GREVISSE, J., AND BEAUMARRIAGE, M. L. (1959). Potentiation par l’hydrocortisone des effets moteurs de l’adrenaline. Arch. Znt. Pharmacodyn. 119,133-141. MARGOLIUS, M. S., REID, P., MOHAMMED,S., AND GAFFNEY,T. E. (1967). Effect of adrenalectomy
and hydrocortisone on responses to norepinephrine and stimulation of the cardioaccelerator nerve. J. Pharmacol. Exp. Ther. 155415421. YARD, A. C., AND KADOWITZ, P. J. (1972). Studies on the mechanism of hydrocortisone potentiation of vasoconstrictor responses to epinephrine in the anesthetised animal. Eur. J. Pharmncol. 20, l-9. ZWEIFACH,B. W., AND METZ, D. B. (1956). Rat mesoappendix procedure for bioassay of humoral substances acting on peripheral blood vessels. Ergeb. Anat. Entwickhmgsgesch. 35,176-239.
Studies
RESEARCH
9,
317-323(1975)
on the Mechanism of Potentiation Constriction by Dexamethasone
of Arteriolar in Viva
A. C. DARKE Department
of Physiology,
University
of Manitoba,
Winnipeg, Canada R3E 0 W3
Received August 19, 1974
The effectof dexamethasoneon the responsesof rat mesentericarteriolesin viva to epinephrineand vasopressinhas beeninvestigated.Superfusionof the rat mesoappendix with a solution containing dexamethasonesodium phosphate(0.2mg/ml) resultedin a potentiation of the arteriolar constriction producedby topically applied epinephrine. Vasopressin-inducedarteriolar constrictions were potentiated by dexamethasonein five of elevenpreparations.Theseresults demonstratethat the potentiation of the effectsof vasoconstrictoragentson vascularsmooth muscleby dexamethasoneis not restricted to agents acting on adrenergic receptors. Any general theory of the mechanismwhereby glucocorticoids potentiate arteriolar constriction must therefore explain potentiation of the effects of nonadrenergic agents. INTRODUCTION The mechanism by which adrenal steroids potentiate the activity of vasoconstrictor agents on vascular smooth muscle is not clearly understood. A good deal of study has been made specifically of the potentiation of the vascular effects of the catecholamines by glucocorticoids. Kalsner (1969a and b) has produced extensive evidence to support his view that hydrocortisone and other adrenal steroids enhance vascular reactivity to adrenaline by inhibiting catechol-o-methyl transferase (COMT), resulting in a decreased rate of inactivation of epinephrine within the tissue. Results with norepinephrine provide something of a paradox in that potentiation by hydrocortisone can be demonstrated using isolated arterial preparations in z&o (Fowler and Chou, 1961; Besse and Bass, 1966; Kalsner, 1969a) but not when constrictor activity is measured in terms of the pressor response to norepinephrine in the intact animal (Lecomte et ai., 1959; Besse and Bass, 1966; Margolius et al., 1967). In the experiments of Kalsner (1969a) hydrocortisone potentiation of the constrictor action of norepinephrine was much smaller than that of epinephrine. This was explained by the effectiveness of monamine oxidase as an alternate pathway for norepinephrine inactivation. In contrast to the results obtained using pressor responses to norepinephrine, the constrictor action of both epinephrine and norepinephrine on mesenteric arterioles measured by intravital microscopy has been found to be potentiated by systemic administration of cortisone prednisolone or dexamethasone (Altura, 1966, 1971). Similarly, in the abstract of Baez et al. (1970) superfusion of the rat mesentery with a solution of dexamethasone was reported to potentiate the increase in arteriolar smooth muscle cell thickness produced by topical epinephrine or norepinephrine. However, Copyright 0 1975 by Academic Press, Inc. 317
All rights ofreproduction Printed in Great Britain
in any form
reserved.
318
A. C. DARKE
in a recent article it has been reported that methylprednisolone and hydrocortisone in fact inhibit the action of epinephrine and norepinephrine on mesenteric arterioles (Altura et al., 1974). Using constant-flow perfusion of the hindquarters of the cat or the dog hindpaw Yard and Kadowitz (1972) demonstrated a potentiation by hydrocortisone of the rise in perfusion pressure produced by intra-arterial injections of epinephrine but not norepinephrine. This potentiation was still obtained after previous administration of either a COMT inhibitor or cocaine. These authors concluded that the affects of hydrocortisone were therefore not due to interference with the mechanismsfor uptake or inactivation of epinephrine, and thus proposed that its action was due to an increase in adrenergic receptor affinity for epinephrine. In these experiments aldosterone also potentiated the vascular effectsof epinephrine but, in contrast to the results of Altura (1966, 1971) and Baez et al. (1970), dexamethasonewas without effect. It is apparent that COMT inhibition cannot completely explain the potentiation of catecholamines by glucocorticoids. Moreover, in a preliminary report Baezet al. (1970) have demonstrated that dexamethasonealso potentiates the action of methoxamine, a noncatechol adrenergic amine, on arteriolar smooth muscle. In contrast, in the aortic strip preparation methoxamine contractions are not potentiated by hydrocortisone (Besseand Bass, 1966; Kalsner, 1969).It is also an open question whether the potentiating action of glucocorticoids is actually restricted to agents acting on adrenergic receptors. The only evidence in the literature relevant to this point seem to be the observations of Altura (1966) that after systemic administration of glucocorticoid, arterioles became responsive to serotonin and that chronic treatment of rats with cortisone results in an enhanced constrictor responseof mesenteric arterioles to vasopressin. The present experiments were therefore designed to determine whether acute topical application of the potent glucocorticoid dexamethasonewould potentiate the constriction of single arterioles in the rat mesentery to vasopressin in addition to potentiating the action of a catecholamine (epinephrine).
MATERIALS
AND METHODS
The mesoappendix of 16 female Long-Evans rats (weight range 63-244 g) was prepared for microscopic observation essentially according to the procedures outlined by Zweifach and Metz (1956). Anesthesia was induced with nembutal (30 mg/kg im) plus ether if required and maintained by supplementary doses of nembutal given through a cannulated jugular vein. The trachea and carotid artery were also cannulated routinely. The cecum was exteriorized through a mid-line abdominal incision and the mesoappendix spread over a plexiglass block. Exposed portions of gut were covered with moist cotton wool and thin transparent film. Except for periods of topical drug application the surface of the mesoappendix was superfused with warm (38°C) Ringer’sgelatin solution. This solution was made up fresh on the day of the experiment by a 10 times dilution of a stock solution (90 g NaCl, 4.2 g KCI, 2.4 g CaCI, in I litre) plus gelatin to a concentration of 1%. The pH of the resulting solution was adjusted to 7.2-7.4 with NaHCO,. The rat’s oesophagealtemperature was controlled by means
DEXAMETHASONE
AND ARTERIOLAR
CONSTRICTION
319
of a heated microscope stage-the temperature of the cecum and ileum was separately controlled by placing them on a plexiglass stagethrough which water at 38°C circulated. The microcirculation was viewed on a Leitz Orthoplan microscope equipped with a 32 x UMK objective and 12.5 x oculars. The light source was a 100 W Quartz Iodine lamp. Internal arteriolar diameters were measured with a Watson imageshearing eyepiececalibrated against a stage micrometer. Arterial blood pressure was recorded from the carotid cannula with a Statham P23Gb transducer and displayed on a Beckman RP dynograph. Vasoactive agents were applied to the surface of the mesenteryin volumes of 0.1 ml. Epinephrine was used both as the bitartrate (Sigma) and as the hydrochloride (Parke Davis). Stock solutions of epinephrine in 5% dextrose solution were kept on ice and diluted 10 times with Ringer’s-gelatin solution immediately before warming and topical application to the mesentery. Doses of epinephrine are expressedin terms of the base.Vasopressin was used either as Pitressin (Parke Davis) or as synthetic lysinevasopressin (Sigma) and also diluted in Ringer’s gelatin. Dexamethasone sodium phosphate (Decadron-Merck, Sharp and Dohme) was diluted in Ringer%-gelatin solution to a concentration of 0.2 mg/ml, and this solution was used for superfusion of the mesentery in the sameway as the normal Ringer’s solution. Observations were made of the effect of epinephrine and vasopressin on arterioles of diameter 21-48 pm. Measurements of diameter were made at a site on the arterioles where the best visualization of the internal wall of the arteriole could be made. Usually four or more topical applications of the vasoconstrictor agent were made before, during and again after superfusion of the mesenterywith the dexamethasone-Ringer’sgelatin solution, After each measurementsufficient time (2-3 min) was allowed for full recovery of the preparation and this interval was not increased by more than about 2 min at the time the superfusion solution was changed. For each application of adrenaline or vasopressinthe maximum decreasein diameter was measured.The doses of adrenaline and vasopressin used were chosen so that they produced constrictor responseswell below maximal. The unpaired t test was used for statistical analysis of the results a P value of less than 0.05 being considered significant. RESULTS Superfusion of the mesoappendix with Ringer’s-gelatin solution containing 0.2 mg/ml dexamethasone did not result in a change in systemic arterial blood pressure, nor produce a significant change in mean arteriolar diameter when all the experiments were considered together. In four individual experiments the mean diameter before and after dexamethasone was significantly greater than the mean diameter during dexamethasone superfusion. The mean decrease in diameter produced by dexamethasone in these four experiments was 2.53% (range 0.95-3.81x) of the control diameter. A statistically significant increasein diameter occurred during dexamethasone superfusion in two of the preparations although the percentage changes in diameter were again very small (2.59 and 2.31 %). The results of the five experiments in which the effect of dexamethasone on the constrictor action of epinephrine was tested are shown in Fig. 1. In all cases,except
320
EPINEPHRINE
(p/ml
1
FIG. 1. The effect of dexamethasone on epinephrine-induced arteriolar constriction in five mesoappendix preparations (experiments l-5). Experiment numbers are reference numbers only. Height of bars indicate mean decrease in arteriolar diameter (‘m k 1 SE). Numbers above each bar indicate the number of observations. Numbers below the bars indicate concentration of epinephrine @g/ml).
6.
’IcI
034
ill 6.
C CON-rRol.
I LIEXAMETHASONE 02 mg/ml
7.
6
6
I
002
5 b
‘1 la .008
68. 9. . 10.
WNTROL
.02
5
1% 5
5
~$Y-fy 5-A A 4L
5
6 .02
CONlROL
5
6
DEXAMETHASONE
11. 12. 6
b 13. 6 9.
14.
15.
4 16.
CONlROL
02 mgp
FIG. 2. The effect of dexamethasone on vasopressin-induced arteriolar constriction in eleven mesoappendix preparations (experiments 6-16). Experiment numbers are reference numbers only. AExperiments in which dexamethasone potentiated the effect of vasopressin. B-Experiments in which dexamethasone did not potentiate the effect of vasopressin. Mean decreases in diameter are shown (pm k 1 SE). Numbers adjacent to SE lines indicate the number of observations. Numbers adjacent to the response lines indicate the concentration of vasopressin (units/ml) used in each experiment.
DEXAMETHASONE
AND ARTERIOLAR
CONSTRICTION
321
one, the mean of the decreasesin arteriolar diameter produced by epinephrine before and after dexamethasonewas significantly less than the constriction obtained during dexamethasonesuperfusion. In one experiment (1 in Fig. 1) the responseto 0.2 pg/ml epinephrine was markedly potentiated by dexamethasone but the response to 0.5 pg/ml epinephrine was not significantly potentiated. This may be explained by the fact that the higher dose of epinephrine produced almost complete closure of the arteriole even in the control period before superfusion with dexamethasone. Mesoappendix preparations from 11 rats were tested for the possible potentiation by dexamethasoneof the arteriolar constriction induced by vasopressin. A statistically significant increase in the response to vasopressin during superfusion with dexamethasone was recorded in five experiments (see Fig. 2A). In one experiment (9 in Fig. 2A) two periods of dexamethasone superfusion, each preceded and followed by control periods of Ringers-gelatin superfusion were used. In the first trial there was an increased response to vasopressin but it was not statistically significant, whereas in the second trial the potentiation was statistically significant. For this reason the results of experiment 9 also appear in Fig. 2B which shows the results obtained in those experiments in which the mean of the decreasesin diameter produced by vasopressin before and after dexamethasone superfusion was not significantly different from the mean decreasein diameter produced by vasopressinduring dexamethasonesuperfuaion. DlSCUSSlON While cortisone, hydrocortisone and methylprednisolone topically applied to the mesoappendix do not produce changes in arteriolar diameter (Altura, 1971; Altura et al., 1974) it has been found by Baez (quoted by Altura, 1971) that topical dexamethasone will increase microvascular tone in 20-25 % of mesoappendix preparations. This proportion is similar to that found in the present experiments where a small but statistically significant decreasein arteriolar diameter occurred in 4 of I6 preparations during superfusion of the mesoappendix with Ringer’s-gelatin solution containing 0.2 mg/ml dexamethasone. However, in two of the other preparations there was a small, although statistically significant, increase in diameter during dexamethasone superfusion. The variability of the effect of dexamethasonealone and the very small changesin diameter would make it unlikely that these are changesof relevance to the overall effect of dexamethasone on the arteriolar response to adrenaline and vasopressin. Moreover since the mean change in diameter for all 16 preparations was not statistically significant and systemic blood pressure was unaffected by superfusion of the mesentery with dexamethasone, decreasesin arteriolar diameter produced by a particular vasoconstrictor agent in the presenceand absenceof dexamethasonemay be compared directly. In the caseof epinephrine, the results presented in this paper confirm the findings of Baez et al. (1970) that topically applied dexamethasone potentiates its constrictor action on arterioles in the rat mesoappendix. Systemicadministration of dexamethasone has also been reported to potentiate arteriolar constriction by epinephrine (Altura, 1966, 1971)although it has not been possible to confirm this in this laboratory (Darke, unpublished observations). The results obtained with vasopressindemonstrate that acute topical administration
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of dexamethasone can potentiate its constrictor action on arterioles in vivo. A significant increase in the vasoconstrictor response to vasopressin was obtained during dexamethasone superfusion in 5 of 1l mesoappendix preparations. In the remaining six experiments a statistically significant increase in the response could not be demonstrated and in one preparation there is a significant decrease in the response. The reason for this variation in the effect of dexamethasone is unclear. It is unrelated to any effect of dexamethasone alone since a statistically significant effect of dexamethasone alone on arteriolar diameter was observed in only three of the vasopressin experiments. Moreover in two of the experiments an increase in diameter during dexamethasone superfusion was associated with nonpotentiation of vasopressin whereas in the other experiment, although the effect of vasopressin was also not potentiated, there was in fact a decrease in diameter during dexamethasone superfusion. The presence or absence of potentiation was also not found to be related to the use of pitressin as opposed to synthetic lysine-vasopressin. Even though potentiation of vasopressin was not found in all preparations the positive results nevertheless mean that any comprehensive explanation of the potentiation of the effect of vasoactive agents by glucocorticoids must involve a mechanism to explain the potentiation of nonadrenergic agents. It is unlikely, as discussed by Altura (197l), that inhibition of COMT by glucocorticoids can completely explain even the potentiation of all adrenergic agents since, as reported by Baez et al. (1970), dexamethasone potentiates the action of methoxamine on arterioles. While it is possible that potentiation of the action of adrenergic agents by glucocorticoids might involve a modification of the affinity of these agents for the a-receptor (Besse and Bass, 1966; Yard and Kadowitz, 1972) it is clear that this cannot be the only site of action of glucocorticoids. Moreover, it appears that it may not be possible to consider all glucocorticoids as having the same effect on the microcirculatory action of vasopressin. Altura et al. (1974) recently found that systemic administration of hydrocortisone and methylprednisole inhibited the actions of vasopressin epinephrine and norepinephrine on mesenteric arterioles, in contrast to the present results with dexamethasone. It is apparent that further experiments are required to elucidate the reason for these differences between individual glucocorticoids with respect to their effect on the actions of vasoactive agents. ACKNOWLEDGMENTS This work was supported by grants from the Manitoba Heart Foundation. I am grateful to Dr. W. D. Dorian, Merck, Sharp and Dohme Ltd., for a generous supply of dexamethasone, and to Dr. P. Gaskell for his helpful criticism of the manuscript. REFERENCES ALTURA, B. M. (1966). Role of glucocorticoids in local regulation of blood flow. Amer. J. Physiol.
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