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The effects of chronic ethanol treatment on endothelium-dependent responses in rat thoracic aorta
Alcohol, Vol. 7, pp. 121-127. ©Pergamon Press pie, 1990. Printed in the U.S.A.
0741-8329/90 $3.00 + .00
The Effects of Chronic Ethanol Treatment on Endothelium-Dependent Responses in Rat Thoracic A o r t a I S H A N N O N P. W I L L I A M S , R O B I N D. A D A M S A N D S. J A M A L M U S T A F A 2
Department o f Pharmacology, School o f Medicine, East Carolina University, Greenville, N C 27858 R e c e i v e d 19 J u n e 1989; A c c e p t e d 9 O c t o b e r 1989
WILLIAMS, S. P., R. D. ADAMS AND S. J. MUSTAFA. The effects of chronic ethanol treatment on endothelium-dependent responses in rat thoracic aorta. ALCOHOL 7(2) 121-127, 1990.--The purpose of this study was to investigate the effects of chronic ethanol consumption on blood pressure and vascular responses, specifically, the possible alterations in endothelium-dependent relaxation which are associated with ethanol-induced hypertension in the rat model. Male rats received ethanol in drinking water for 13 weeks. Systolic pressure was recorded weekly. Following treatment, segments of thoracic aorta with and without intact endothelium were used to generate relaxation-response curves to the endothelium-dependent agents, acetylcholine, ATP and bradykinin, as well as the endothelium-independent agents, adenosine and sodium nitroprusside. Mean systolic pressures at the end of the treatment period were: 127.8 --- 1.2 and 151.1 --- 1.3 mmHg for controls and ethanol-treated rats, respectively. Ethanol treatment did not affect the relaxation produced by either acetylcholine, ATP or sodium nitroprusside in aorta with or without endothelium. In contrast, ring segments with intact endothelium from ethanol-treated rats exhibited augmented relaxation in response to both adenosine and bradykinin compared to controls. Removal of the endothelium abolished the relaxation produced by bradykinin in both groups. Although removal of the endothelium had no effect on the relaxation produced by adenosine in the control group, it attenuated the adenosine-induced relaxation in the ethanol-treated group back to control levels. These data suggest that chronic ingestion of ethanol causes elevated blood pressure and augments the endothelium-dependent relaxation to bradykinin. These findings also suggest that chronic ethanol treatment can cause the appearance of an endothelium-dependent component in the relaxation produced by adenosine. These augmented endothelium-dependent relaxations observed with chronic ethanol consumption would seem to oppose rather than contribute to hypertension that develops with chronic ethanol consumption and may serve in a protective capacity. Ethanol
Chronic treatment
Endothelium-dependent responses
MANY epidemiological studies have shown that chronic ethanol consumption is associated with increased incidence of hypertension (6, 20-22). In addition, there is an increased incidence of sudden cardiac death as well as variant angina and cardiac fibrillation associated with binge drinking (3, 13, 14). Presently, the mechanisms responsible for the precipitation of these events are not understood. Much of the research investigating the chronic effects of ethanol and ethanol-induced hypertension has considered possible alterations in either plasma volume or circulating neurohumoral vasoconstrictor agents as well as peripheral vascular responsiveness to vasoconstrictor agents. Although there has been some consistency between reports, the variability between reports has made ascription of a consistent explanation for the rise of ethanol-induced hypertension most difficult. For instance, an initial study in 1983 showed that with chronic ethanol consumption in the rat model there was an associated elevation in blood pressure (9). This and a subsequent report (10) correlated the rise in blood pressure with an expansion in plasma volume as well as a rise in plasma norepinephrine levels. Other studies, while
Thoracic aorta
showing no evidence for changes in plasma volume (4,35), have reported alterations in vascular responses in the rat associated with ethanol intake from 2-24 weeks duration. But here too, the reports have varied. For instance, while some reports (4) show a developed hypersensitivity to contractile stimulants such as potassium, catecholamines and angiotensin II after 12-24 weeks of ethanol treatment, others have reported no change in the responsiveness to norepinephrine after 12 weeks of ethanol treatment (2). Still others have reported a desensitization to the contractile effects of phenylephrine during 6-18 weeks of ethanol treatment (35). These differences may reflect differences in treatment protocols or experimental design. However, with this much variability to date, a specific alteration in either plasma volume, or the vascular responsiveness to other vasoconstrictor agents which can account for the increased blood pressure associated with chronic ethanol consumption cannot be deduced. Another possibility for exploration into ethanol-induced hypertension could be possible alterations in vascular relaxation and/or the role of the endothelium and endothelium-dependent relaxation
1Supported by National Institutes of Health Grant No. HL 27339. 2Requests for reprints should be addressed to S. Jamal Mustafa, Ph.D.
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WILLIAMS, ADAMS AND MUSTAFA
associated with chronic ethanol consumption. However, due in part to the relatively recent discovery of endothelium-dependent relaxation, most previous reports have failed to consider what, if any, role the endothelium may have played in the reported results. In 1980, it was discovered that the vascular endothelium could produce and release a factor or factors termed endothelium-derived relaxing factor(s) (or EDRF) in response to acetylcholine (16). Since that time many agents including ATP, ADP, bradykinin, substance P and the calcium ionophore, A23187, have been found to produce vasodilation which is endothelium dependent (11, 15, 36). In addition to these reports, other studies have demonstrated that the responses to contracting and relaxing agents may be modified by the presence of the endothelium (8, 29, 34). In relation to the effects of ethanol on endothelium function, previous evidence has shown that acute administration of high concentrations of ethanol produced contraction in the rat thoracic aorta (5). This study further reported that a tolerance to the acute contractile effects of ethanol developed after 2-6 weeks of ethanol ingestion (5). A subsequent report demonstrated a developed tolerance to the contractile effects of ethanol in rat aorta after 2 days of administration of ethanol by oral intubation (24). This subsequent report showed that the tolerance to the contractile effects of ethanol was dependent on the endothelium (24). Additional studies have since shown that this endothelium-dependent tolerance to ethanol-induced contraction was mediated by EDRF (23). Although these studies have shown that ethanol consumption may affect endothelial function, the role of the endothelium and endothelium-dependent relaxation in association with chronic ethanol consumption and ethanol-induced hypertension has not been demonstrated. This study was conducted in order to investigate the effects of chronic ingestion of ethanol on vascular responsiveness with regard to possible alterations in endotheliumdependent relaxation in relationship to the associated hypertension. METHOD
Male Sprague-Dawley (SD) rats (Charles Rivers Laboratories) weighing between 227-304 g were randomly divided into control and ethanol-treated groups. Rats in the ethanol-treated group received ethanol in their drinking water at a concentration of 5% for the first week, 10% weeks 2-3, and 20% by volume weeks 4-13 adapted from the method originally described in 1983 (9). Control rats received tap water ad lib. In order to maintain a similar caloric intake, both the ethanol-treated and control groups received Purina Lab Chow (St. Louis, MO) on a restricted basis of 15 g/day and 18 g/day, respectively. Systolic blood pressures were measured weekly before 9 a.m. using a Model 72 heart rate and blood pressure meter (IITC Inc., Woodland Hills, CA). All blood pressure measurements were taken after 30 min under an elevated room temperature of 30°C. These measurements were always taken in the morning in order to minimize the effects of possible circadian variations on blood pressure. Body weights were also measured weekly. At the end of the treatment period the rats were anesthetized with et-chloralose (100 mg/kg) and pentobarbital (20 mg/kg), given IP. During anesthesia, arterial blood samples were taken via femoral artery cannulation for enzymatic determination of blood ethanol concentrations according to a previously described method (7). Rats were then sacrificed by decapitation followed by immediate removal of thoracic aortae. All rats were sacrificed prior to 10 a.m. to avoid variability due to the well-defined circadian rhythm which ethanol metabolism follows. Aortic vessels from both control and ethanol-treated rats were trimmed free of excess connective tissue and cut into ring segments 3 mm in length. Special care was taken throughout this
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TIME (WEEKS) FIG. 1. Effects of chronic ethanol treatment (EtOH TX) on mean systolic pressure in male rats. n= 8 and 12 rats for control and ethanol-treated groups, respectively. Mean systolic pressure for the ethanol-treated group was significantly (p<0.01) greater than controls beginning at week 3.
procedure to maintain an intact endothelium. In some of the segments the endothelium was mechanically removed by gently rubbing the intimal surface. Aortic ring segments with and without endothelium were mounted on hooks in isolated organ baths containing Krebs buffer 37°C. The Krebs solution was composed of the following (in mM): NaCI 118.1, KC1 4.7, MgSO4 1.2, KH2PO 4 1.2, CaC12 2.5, NaHCO 3 25.0 and glucose 11.1. The baths were maintained at pH 7.4 by continuous aeration with 95% 02 and 5% CO 2. The ring segments were allowed to equilibrate for 1 hr under an initial resting tension of 2 g. After equilibration, vessel segments were precontracted with 30 mM KC1 or 5 x 10 7 M phenylephrine. In vessels precontracted with phenylephrine, concentration-response curves to either acetylcholine, ATP, bradykinin, adenosine or sodium nitroprusside were generated.
Drugs The following pharmacological agents, obtained from Sigma Chemical Co., were used: phenylephrine hydrochloride, potassium chloride, acetylcholine chloride, ATP, bradykinin, adenosine, and sodium nitroprusside. All drugs were initially dissolved in distilled water at concentrations of 10 -2 M from which serial dilutions were made. Ethyl alcohol USP, 95%, was obtained from AAPER Alcohol and Chemical Co., Shelbyville, KY.
Statistical Analysis The steady state contraction produced by either 30 mM KCI or 5 x 10 - 7 M phenylephrine was expressed as increase in gram tension from resting tension. The relaxation responses to acetylcholine, ATP, bradykinin, adenosine and sodium nitroprusside were expressed as % of the steady state contraction obtained with 5 x 10 - 7 M phenylephrine. All data are expressed as means - S.E. Between group differences in the contraction produced by both 30 mM KC1 and 5 x 10 -7 M phenylephrine were assessed using Student's t-test for unpaired data for single comparisons. The within group comparisons of the contractions produced by these same agents in segments with and without intact endothelium were also assessed using Student's t-test for unpaired data for single comparisons. Between group (ethanol-treated vs. control) comparisons at each concentration of both the endothelium-dependent (acetylcholine, ATP and bradykinin) and -independent relaxing agents (adenosine and sodium nitroprusside) in ring segments with
CHRONIC ETHANOL AND THE ENDOTHELIUM
123
TABLE 1 THE EFFECTSOF CHRONICETHANOLCONSUMPTIONON THE CONTRACTIONPRODUCEDBY KCI AND PHENYLEPHRINE IN MALERATS
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0.71 ± 0.09 (g) 0.72 = 0.05 (g)
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1.89 ± 0.22 (g) "1.96 -+ 0.12 (g)
• 1.34 ± 0.07 (g) • 1.31 ± 0.08 (g)
Values represent the mean ± S.E. increase in (g) tension above an initial resting tension of 2 g in response to either 5 x 10-7 M phenylephrine or 30 mM KC1. Endo (+) or Endo ( - ) represent aortic segments with or without endothelium, respectively. *p<0.001 compared to respective within group (ethanol-treated or control) responses in vessels with intact endothelium.
and without intact endothelium were assessed using analysis of variance followed by a Student-Newman-Keuls post hoc test. Within group differences (endothelium present vs. endothelium removed) in the responses at each concentration of either the endothelium-dependent or endothelium-independent relaxing agents were compared using Student's t-test for unpaired data for single comparisons. All differences were considered significant at p<0.05. RESULTS
Ingestion of ethanol in rats for 13 weeks caused elevated systolic blood pressure compared to control rats. The mean systolic pressures at the time of sacrifice, for the ethanol-treated group and control, were 151.1---1.3 and 127.8---1.2, respectively. A difference in pressure was detectable by the third week of treatment and increased steadily throughout the remaining treatment period (Fig. 1). Although dally intake of ethanol was not measured in the present study, others (2) have reported daily ethanol intake of Sprague-Dawley rats subjected to a similar treatment regime to average around 10-11 g/kg/day at the 20% concentration. Both the ethanol-treated and control groups showed steady gains in weight over the course of treatment. At the time of sacrifice body weights averaged 4 6 8 ± 13 g vs. 445 ___17 g for the ethanol-treated and control groups, respectively. Blood ethanol levels for the ethanol-treated rats, also determined at the time of sacrifice, were 40.8 ___6.2 mg/dl. Since the main objective of this study was to examine the effects of chronic ethanol consumption on endothelium-dependent relaxation, only one concentration of the receptor-operated and voltage-dependent contracting agent phenylephrine and KC1, respectively, was tested. And although limited to only one concentration of each agent, these challenges were included to provide a rough assessment of possible changes in the vasoconstrictive responsiveness to these agents with regard to possible alterations in the endothelium-dependent modulation of the contraction produced by these agents. Our findings revealed no differences in the contractions produced by either 30 mM KC1 or 5 x 1 0 - 7 M phenylephrine between ethanol-treated rats and controls in rings of thoracic aorta with or without intact endothelium (Table 1). However, removal of the endothelium augmented the contraction produced by KC1 in both the ethanol-treated rats and controls (Table 1). The contraction produced by phenylephrine was also augmented by removal of the endothelium in the ethanol-treated
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animals. However, in the control rats this difference only approached significance at p =0.052 (Table 1). There was no difference in the concentration-response curves to acetylcholine in rings with intact endothelium from ethanoltreated rats vs. control rats, with the exception of the lowest concentration of acetylcholine (Fig. 2). Removal of the endothelium abolished the response to acetylcholine in both groups (Fig. 2). Also, there was no difference in the relaxation-response curves to a second endothelium-dependent relaxing agent, ATP, in rings with intact endothelium from ethanol-treated vs. control rats. Here again, removal of the endothelium abolished the relaxation produced by ATP, except at the highest concentration, where some relaxation persisted in both groups (Fig. 3). Unlike the responses to acetylcholine and ATP, the relaxation produced by bradykinin, another endothelium-dependent relaxing agent, was augmented in aortic tings with intact endothelium from ethanol-treated animals compared to controls (Fig. 4). Removal of the endothelium
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abolished the relaxation produced by bradykinin in both groups (Fig. 4). Adenosine only produced relaxation at concentrations of 1 × 10 - 7 M or greater. These relaxations were also augmented in vessels with intact endothelium from ethanol-treated rats compared to control rats (Fig. 5). Removal of the endothelium had no effect on the relaxation produced by adenosine in vessels from control rats. However, in vessels from ethanol-treated rats removal of the endothelium reduced the relaxation produced by adenosine back to control levels, such that there was no difference in the relaxation between these tings and those from control rats with or without intact endothelium (Fig. 5). Between group comparisons (ethanol-treated vs. control) of relaxation responses to the endothelium-independent relaxing agent, sodium nitroprusside, revealed no between group differences at any concentration of sodium nitroprusside in rings with or without intact endothelium, respectively (Fig. 6). However, in both the ethanol-treated and control groups, removal of the endothelium augmented the relaxation produced by sodium nitroprusside compared to respective within group responses in vessels
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ADENOSINE (-log M) FIG. 5. Effects of chronic ethanol treatment (EtOH TX) on the relaxation produced by adenosine. Other details are the same as in Fig. 2. **Significantly different from control responses in vessels with intact endothelium, p<0.01.
FIG. 6. Effects of chronic ethanol treatment (EtOH TX) on the relaxation produced by sodium nitroprusside. Other details are the same as in Fig. 2. *Significantly different from the within group responses in vessels without endothelium, p<0.05.
with intact endothelium (Fig. 6). Although removal of the endothelium shifted the relaxation response curve for sodium nitroprusside to the left in both the control and the ethanol-treated groups, the difference between the shifts was significantly less in the ethanol-treated group compared to control (Fig. 6). The difference in shift was assessed by comparing the mean difference in % relaxation at each concentration of sodium nitroprusside in control vessels (% relaxation without endothelium - % relaxation with endothelium) to the respective mean difference in ethanol-treated vessels. DISCUSSION
This study as well as previous studies (9,35) has demonstrated that rats which receive ethanol in their &'inking water at a concentration of 20% by volume over a prolonged period develop an increased systolic pressure compared to control rats which receive water alone. Studies aimed at elucidating the vascular effects of chronic ethanol treatment have considered possible alterations in the vascular responsiveness to vasoconstrictor agents such as catecholamines and angiotensin II (2, 9, 35). However, a considerable amount of variability exists concerning the effects of chronic ethanol on the vascular responsiveness to various vasoconstrictor agents. For instance, one study (4) reported an increased sensitivity to 20 mM KC1 as well as a hypersensitivity to other contractile stimulants such as catecholamines and angiotensin II in aortae from rats maintained on liquid diets containing ethanol (6.8% v/v) or sucrose (controls) from 12-24 weeks. In contrast, others have reported a decreased sensitivity to the contraction produced by phenylephrine at concentrations <3 × 10 -8 M, but reported no change in the maximal contraction produced by phenylephrine in thoracic aorta of rats that received ethanol in their drinking water (20% by vol.) from 6--18 weeks (35). Still others, using in vivo preparations, have reported similar pressor response curves to both phenylephrine and angiotensin II in control and chronically ethanol-fed rat (5% week 1, 10% weeks 2-3 and 20% by vol. in their drinking water weeks 3-12) (2). While these reported differences may represent differences in either treatment protocols or experimental designs, these studies have not considered possible alterations in either endotheliumdependent relaxation or the role of the endothelium in the modulation of the vascular responses of other contracting or
CHRONIC ETHANOL AND THE ENDOTHELIUM
relaxing agents associated with chronic ethanol consumption. In the present study we found no difference in the contraction produced by either 30 mM KCI or 5 x 10 -7 phenylephrine in vessels with or without endothelium from ethanol-treated vs. control rats. These findings are consistent with the previous in vivo f'mdings of others (2). Since the present study only examined the effects of chronic ethanol consumption at a near maximal contracting concentration of phenylephrine, our findings do not preclude possible effects of chronic ethanol consumption on the response to phenylephrine at lower concentrations as was previously reported (35). Differences between the present findings and those which reported a hypersensitivity to various contractile agents associated with chronic ethanol consumption (4) may represent differences in either the treatment protocol or the duration of treatment. Like previous studies (8,29), the present study did demonstrate that removal of the endothelium in aortae from both ethanoltreated and control rats augmented the contraction produced by KC1. An increased sensitivity to potassium after removal of the endothelium may be linked to the elimination of an endothelialassociated hyperpolarization of the smooth muscle (29). Like the response to KC1, removal of the endothelium also augmented the contraction produced by the ot-adrenoceptor agonist, phenylephfine, in the ethanol-treated rats. However, due to slightly greater variability in the control group, the augmented response to phenylephrine with removal of the endothelium only approached significance at p = 0.052. The present findings of no differences in the amount of contraction produced by either KC1 or phenylephfine in aorta with or without endothelium from ethanol-treated animals compared to their respective controls suggest, on a limited basis, that chronic ingestion of ethanol in the rat produced little effect on the vascular responsiveness to these contracting agents. In addition, these findings suggest that although the endothelium may modulate the contraction produced by both KC1 and phenylephrine, this modulation is not altered by chronic ethanol consumption in the rat. In terms of the hypertension which develops following chronic ethanol consumption, our limited data do not support the involvement of changes in either the vascular reactivity to, or the endothelium-dependentmodulation of the contraction produced by either KCI or phenylephrine as contributing factors. However, a more thorough examination of the concentration response relationships of these agents, with regard to endothelial modulation, associated with chronic consumption of ethanol is needed. Our study revealed no differences in relaxation response curves to ATP between the ethanol-treated rats and controls. Removal of the endothelium practically abolished the relaxation produced by ATP, except at the highest concentrations for both groups, where some relaxation persisted. This relaxation produced by ATP in the absence of the endothelium may have resulted from the breakdown of ATP into one of its vasoactive metabolites, possibly adenosine. Like ATP, there were virtually no differences in the endotheliumdependent relaxation produced by the acetylcholine between the ethanol-treated and control groups. Bradykinin, the third endothelium-dependent relaxing agent tested in the current study, was much less potent in producing relaxation than either ATP or acetylcholine. In contrast to other reports showing bradykinin to have an ED5o around 1-10 nM in bovine arteries, the current EDso was found to be around < 1 p,m in rat aorta. These differences were probably due to differences between the two species in response to this agent. More importantly, and in contrast to the lack of an effect of chronic ethanol consumption on the relaxation produced by ATP and acetylcholine, the relaxation produced by bradykinin was found to be significantly augmented in aortae from the ethanol-treated rats compared to controls. In previous studies (18,31), the involvement
125
of prostaglandin metabolites in the relaxation produced by bradykinin has been reported. In one of these previous studies, investigators (18) reported that bradykinin can produce endotheliumdependent relaxation via either the production of a cyclo-oxygenase product or the production and release of EDRF. It is possible that ethanol treatment may have augmented one of these two processes resulting in an augmented relaxation to bradykinin. Since the relaxation produced by both acetylcholine and ATP were not affected by chronic ethanol treatment, it appears that the augmented relaxation to bradykinin does not result from a generalized increase in endothelium-dependent relaxation. In contrast to other reports (32,33), which suggested that the relaxant responses to nitrovasodilators are independent of the presence of endothelial cells, we demonstrated that removal of the endothelium augmented the relaxation produced by sodium nitroprusside. These findings are in agreement with a previous report (34) which also found an augmented response to sodium nitroprusside with removal of the endothelium in aortic rings from spontaneously hypertensive and Wistar-Kyoto normotensive rats. This previous report suggested that this augmented response to sodium nitroprusside in the absence of the endothelium may result from the removal of an endothelium-derived constricting factor (34). In addition, the endothelium may serve as a barrier to the relaxing effects of this agent. In agreement with previous in vivo work (1) the current study did not demonstrate an effect of chronic ethanol consumption on the endothelium-independent relaxation produced by sodium nitroprusside. The previous in vivo findings (1) showed that neither acute nor chronic ethanol treatment (4 weeks) had an effect on the in vivo depressor effects of nitroprusside in rats. The present findings suggest that with chronic ethanol consumption, the ability of the vascular smooth muscle to relax does not seem to be impaired and as such, should not contribute to the hypertension seen with chronic ethanol consumption. However, the present findings did demonstrate that removal of the endothelium shifted the relaxation response curve to the left in both the ethanol-treated and control groups, suggesting that the endothelium can modulate the relaxant effects of agents which were previously thought to act independent of the endothelium. Adenosine, an endogenous metabolite of ATP, is thought to produce vasodilation at the level of the vascular smooth muscle by activating adenosine receptors of the A2 type (26,30). An unexpected finding of the present study was an augmented relaxation to adenosine in aorta from ethanol-treated rats compared to control rats with intact endothelium. This, along with the finding that removal of the endothelium in aortae of ethanol-treated rats reduced the relaxation produced by adenosine back to that of control vessels with or without endothelium, suggests that chronic ingestion of ethanol in the rat causes the appearance of an endothelium-dependent component to the relaxation produced by adenosine. Our findings of no differences in the relaxation produced by adenosine in vessels without endothelium from ethanol-treated animals compared to respective controls suggest that the appearance of the endothelium-dependent component to adenosine was independent of changes in the vascular responsiveness to adenosine at the smooth muscle level. Although the nature of this endothelium-dependent component was not evidenced in the present study, recent evidence has shown that adenosine can cause the release of prostacyclin from perfused rabbit hearts (19) with the coronary vascular endothelium considered to be the main source of this eicosanoid (20). Since chronic ethanol consumption was only associated with an augmented relaxation to bradykinin and adenosine in the absence of changes in the relaxation produced by either the endothelium-dependent relaxing agents, acetylcholine and ATP, or the endothelium-independent relaxing agent, sodium nitroprusside, it is possible that the augmented relaxation
126
WILLIAMS, ADAMS AND MUSTAFA
to bradykinin and adenosine occurs via a common mechanism. Since previous studies have shown that both adenosine and bradykinin can stimulate prostaglandin production, this mechanism may involve an endothelium-dependent production of a prostaglandin metabolite, possibly prostacyclin (18,19). This possibility is further supported by evidence which has shown that acutely, moderate concentrations of ethanol (34.8 mM) can raise prostacyclin levels in vivo and in vitro (25) while they may selectively reduce platelet (14.3 and 28.6 mM ethanol) thromboxane production (28). While chronic ethanol consumption (4-5 weeks 36% of energy as ethanol) in rats has been associated with an actual decrease in basal release of prostacyclin, it has been shown to cause the acute appearance of ethanol-induced prostacyclin production in rat aorta (17). These two effects were observed in the absence of an effect of chronic ethanol consumption on acetaldehyde-stimulated prostacyclin formation (17). Although the nature of the ethanol-induced prostacyclin formation was not evidenced by this previous study (17), the findings allow the possibility of other alterations in the stimulated release of prostacyclin produced by chronic ethanol consumption. Other possibilities for the augmented relaxation produced by adenosine could include either an increase in the number or sensitivity of the adenosine receptors or an uncovering of additional adenosine
receptors. This latter possibility stems from the established effect of ethanol to cause perturbations of membranes (12). However, this latter possibility would have been limited to alterations in adenosine receptors at the level of the endothelium since, in the absence of the endothelium, there was no difference between adenosine responses in the ethanol-treated group compared to respective responses in the control group. In summary, the present study has supported previous studies which have shown that chronic consumption of ethanol in the rat can elevate systolic pressure. We have also shown that in addition to the elevation of systolic pressure, there is a concurrent augmentation of endothelium-dependent relaxation produced by bradykinin and adenosine, the nature of which remains to be elucidated. But regardless of the nature of these augmented endotheliumdependent relaxation responses, these alterations associated with chronic ethanol consumption would seem to oppose rather than contribute to the elevation of systolic pressure which is associated with chronic ethanol consumption. In conclusion, although chronic ethanol consumption was shown to have an effect on endothelium-dependent relaxation, this effect seemed to be in opposition to the hypertension which accompanies chronic ethanol consumption.
REFERENCES 1. Abdel-Rahman, A-R. A.; Dar, M. S.; Wooles, W. R. Effect of chronic alcohol administration on arterial baroreceptor function and pressor and depressor responsiveness in rats. J. Pharmacol. Exp. Ther. 232:194-201; 1985. 2. Abdel-Rahman, A-R. A.; Wooles, W. R. Ethanol-induced hypertension involves impairment of baroreceptors. Hypertension 10:67-73; 1987. 3. Alderman, E. L.; Coltart, D. J. Alcohol and the heart. Br. Med. Bull. 38:77-80; 1982. 4. Altura, B. M.; Altura, B. T. Symposium on cardiovascular effects of ethanol-part II: peripheral and cerebrovascular actions of ethanol, acetaldehyde and acetate: relationship to divalent cations. Alcohol.: Clin. Exp. Res. 11:99-111; 1987. 5. Altura, B. T.; Pohorecky, L. A.; Altura, B. M. Demonstration of tolerance to ethanol in non-nervous tissue: effects on vascular smooth muscle. Alcohol.: Clin. Exp. Res. 4:62-69; 1980. 6. Arkwright, P. D.; Belin, L. J.; Rouse, I.; Armstrong, B. K.; Vandongen, R. Effects of alcohol use and other aspects of lifestyle on blood pressure levels and prevalence of hypertension in a working population. Circulation 66:60-66; 1982. 7. Bonnichsen, R. K.; Lundgren, G. Comparison of the ADH and the Windmark procedures in forensic chemistry for determining alcohol. Acta Pharmacol. Toxicol. 13:256-266; 1957. 8. Bullock, G. R.; Taylor, S. G.; Weston, A. H. Influence of the vascular endothelium on agonist induced contractions and relaxations in rat aorta. Br. J. Pharmacol. 89:819-830; 1986. 9. Chan, T. C. K.; Sutter, M. C. Ethanol consumption and blood pressure. Life Sci. 33:1965-1973; 1983. 10. Chan, T. K. C.; Wall, R. A.; Sutter, M. C. Chronic alcohol consumption, stress and hypertension. Hypertension 7:519-524; 1985. 11. Cherry, P. D.; Furchgott, R. F.; Zawadzki, J. V.; Jothianandan, D. The role of endothelial cells in the relaxation of isolated arteries by bradykinin. Proc. Natl. Acad. Sci. USA 79:2106-2110; 1982. 12. Chin, J. H.; Goldstein, D. B. Membrane-disordering action of ethanol. Variation with membrane cholesterol content and depth of the spin label probe. Mol. Pharmacol. 19:425--431, 1981. 13. Ettinger, P. W.; Wu, C. F.; DeLaCruz, C.; Weisse, A. B.; Ahmed, S. S.; Regan, T. J. Arrhythmias and the "Holiday Heart" alcohol associated cardiac rhythm disorders. Am. Heart J. 95:555-562; 1978. 14. Friedman, H. S.; Geller, S. A. The effect of alcohol on the heart, skeletal, and smooth muscles. In: Leiber, C. S., ed. Medical disorders of alcoholism, Philadelphia: Sannders; 1982: 436--479. 15, Furchgott, R. F. The requirement for endothelial cells in the relaxation of arteries by acetylcholine and some other vasodilators. Trends
Pharmacol. Sci. 2:173-176: 1981. 16. Furchgott, R. F.; Zawadzki, J. V. The obligatory role of endothelial cells in the relaxation produced by acetylcholine. Nature 288:373376; 1980. 17. Guivernau, M.; Baraona, E.; Lieber, C. S. Acute and chronic effects of ethanol and its metabolites on vascular production of prostacyclin in rats. J. Pharma~ol. Exp. Ther. 240:59-64; 1987. 18. Ignarro, L. J.; Byms, R. E.; Buga, G. M.; Wood, K. S. Mechanisms of endothelium-dependent vascular smooth muscle relaxation by bradykinin and VIP. Am. J. Physiol. 253(Heart Circ. Physiol. 22):H1074-H1082; 1987. 19. Karwatowska-Prokopczuk, E.; Ciabattoni, G.; Wennmalm, A. Effects of adenosine on the formation of prostacyclin in the rabbit isolated heart. Br. J. Pharrnacol. 94:721-728; 1988. 20. Khetarpal, V. K.; Volicer, L. Alcohol and cardiovascular disorders. Drug Alcohol Depend. 7:1-30; 1981. 21. Klatsky, A. L.; Friedman, G. D.; Armstrong, M. A. The relationship between alcoholic beverage use and other traits to blood pressure: a new Kaiser Permanente study. Circulation 73:628-636; 1986. 22. Klatsky, A. L.; Friedman, G. D.; Siegelaub, A. B.; Gerard, M. J. Alcohol consumption and blood pressure: Kaiser-Permanente multiphasic health examination data. N. Engl. J. Med. 296:1194-2000: 1977. 23. Knych, E. T. Endothelium-dependent transfer of ethanol tolerance in the aorta. Life Sci. 40:1903-2908; 1987. 24. Knych, E. T.; Guimaraes, C. M. S.; Boivin, S. Tolerance to ethanol-induced contractions of vascular smooth muscle: role of endothelium. Life Sci. 35:611-617; 1984. 25. Landolfi, R.; Steiner, M. Ethanol raises prostacyclin in vivo and in vitro. Blood 64:679-682; 1984. 26. Leung, E.; Johnson, C. I.; Woodcock, E. A. An investigation of the receptors involved in the coronary vasodilatory effects of adenosine analogs. Clin. Exp. Pharmacol. Physiol. 12:515-519; 1985. 27. Macintyre, D. E.; Pearson, J. D.; Gordeon, J. L. Localization and stimulation of prostacyclin production in vascular cells. Nature 271:549-551; 1978. 28. Mikhailids, D. P.; Jeremy, J. Y.; Barradas, M. A.; Green, N.; Dandona, P. Effect of ethanol on vascular prostacyctin (prostaglandin Iz) synthesis, platelet aggregation, and platelet thromboxane release. Clin. Res. 287:1495-1498; 1983. 29. Mikkelsen, E. O.; Costa, G.; Nyborg, N. C. B. Influence of the endothelium on the response to calcium agonists, calcium, potassium and noradrenaline in rat aorta. Pharmacol. Toxicol. 62:22-28; 1988. 30. Mustafa, S. J.; Askar, A. O. Evidence suggesting an R-a type receptor
C H R O N I C E T H A N O L A N D THE E N D O T H E L I U M
in bovine coronary arteries. J. Pharmacol. Exp. Ther. 232:49-56; 1985. 31. Needleman, P.; Marshall, G. R.; Sobel, B. E. Hormone interactions in the isolated rabbit heart. Circ. Res. 37:802-808; 1975. 32. Rapoport, R. M.; Murad, F. Agonist induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP. Circ. Res. 52:352-357; 1983. 33. Rubanyi, G.; Vanhoutte, P. M. Endothelium-removal decreases relaxations of canine coronary arteries caused by beta-adrenergic agonist and adenosine. J. Cardiovasc. Pharmacol. 7:139-144; 1985.
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34. Shirasaki, Y.; Su, C.; Lee, T. J.-F.; Kolm, P.; Cline, W. H., Jr.; Nickols, A. Endothelial modulation of vascular relaxation to nitrovasodilators in aging and hypertension. J. Pharmacol. Exp. Ther. 239(3):861-866; 1986. 35. Strickland, J. A.; Wooles, W. A. Effect of acute and chronic ethanol on the agonist responses of vascular smooth muscle. Eur. J. Pharmacol. 152:83-91; 1988. 36. Zawadzki, J. V.; Furchgott, R. F.; Cherry, P. D. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by substance. P. Fed. Proc. 40:689; 1981.
0741-8329/90 $3.00 + .00
The Effects of Chronic Ethanol Treatment on Endothelium-Dependent Responses in Rat Thoracic A o r t a I S H A N N O N P. W I L L I A M S , R O B I N D. A D A M S A N D S. J A M A L M U S T A F A 2
Department o f Pharmacology, School o f Medicine, East Carolina University, Greenville, N C 27858 R e c e i v e d 19 J u n e 1989; A c c e p t e d 9 O c t o b e r 1989
WILLIAMS, S. P., R. D. ADAMS AND S. J. MUSTAFA. The effects of chronic ethanol treatment on endothelium-dependent responses in rat thoracic aorta. ALCOHOL 7(2) 121-127, 1990.--The purpose of this study was to investigate the effects of chronic ethanol consumption on blood pressure and vascular responses, specifically, the possible alterations in endothelium-dependent relaxation which are associated with ethanol-induced hypertension in the rat model. Male rats received ethanol in drinking water for 13 weeks. Systolic pressure was recorded weekly. Following treatment, segments of thoracic aorta with and without intact endothelium were used to generate relaxation-response curves to the endothelium-dependent agents, acetylcholine, ATP and bradykinin, as well as the endothelium-independent agents, adenosine and sodium nitroprusside. Mean systolic pressures at the end of the treatment period were: 127.8 --- 1.2 and 151.1 --- 1.3 mmHg for controls and ethanol-treated rats, respectively. Ethanol treatment did not affect the relaxation produced by either acetylcholine, ATP or sodium nitroprusside in aorta with or without endothelium. In contrast, ring segments with intact endothelium from ethanol-treated rats exhibited augmented relaxation in response to both adenosine and bradykinin compared to controls. Removal of the endothelium abolished the relaxation produced by bradykinin in both groups. Although removal of the endothelium had no effect on the relaxation produced by adenosine in the control group, it attenuated the adenosine-induced relaxation in the ethanol-treated group back to control levels. These data suggest that chronic ingestion of ethanol causes elevated blood pressure and augments the endothelium-dependent relaxation to bradykinin. These findings also suggest that chronic ethanol treatment can cause the appearance of an endothelium-dependent component in the relaxation produced by adenosine. These augmented endothelium-dependent relaxations observed with chronic ethanol consumption would seem to oppose rather than contribute to hypertension that develops with chronic ethanol consumption and may serve in a protective capacity. Ethanol
Chronic treatment
Endothelium-dependent responses
MANY epidemiological studies have shown that chronic ethanol consumption is associated with increased incidence of hypertension (6, 20-22). In addition, there is an increased incidence of sudden cardiac death as well as variant angina and cardiac fibrillation associated with binge drinking (3, 13, 14). Presently, the mechanisms responsible for the precipitation of these events are not understood. Much of the research investigating the chronic effects of ethanol and ethanol-induced hypertension has considered possible alterations in either plasma volume or circulating neurohumoral vasoconstrictor agents as well as peripheral vascular responsiveness to vasoconstrictor agents. Although there has been some consistency between reports, the variability between reports has made ascription of a consistent explanation for the rise of ethanol-induced hypertension most difficult. For instance, an initial study in 1983 showed that with chronic ethanol consumption in the rat model there was an associated elevation in blood pressure (9). This and a subsequent report (10) correlated the rise in blood pressure with an expansion in plasma volume as well as a rise in plasma norepinephrine levels. Other studies, while
Thoracic aorta
showing no evidence for changes in plasma volume (4,35), have reported alterations in vascular responses in the rat associated with ethanol intake from 2-24 weeks duration. But here too, the reports have varied. For instance, while some reports (4) show a developed hypersensitivity to contractile stimulants such as potassium, catecholamines and angiotensin II after 12-24 weeks of ethanol treatment, others have reported no change in the responsiveness to norepinephrine after 12 weeks of ethanol treatment (2). Still others have reported a desensitization to the contractile effects of phenylephrine during 6-18 weeks of ethanol treatment (35). These differences may reflect differences in treatment protocols or experimental design. However, with this much variability to date, a specific alteration in either plasma volume, or the vascular responsiveness to other vasoconstrictor agents which can account for the increased blood pressure associated with chronic ethanol consumption cannot be deduced. Another possibility for exploration into ethanol-induced hypertension could be possible alterations in vascular relaxation and/or the role of the endothelium and endothelium-dependent relaxation
1Supported by National Institutes of Health Grant No. HL 27339. 2Requests for reprints should be addressed to S. Jamal Mustafa, Ph.D.
121
122
WILLIAMS, ADAMS AND MUSTAFA
associated with chronic ethanol consumption. However, due in part to the relatively recent discovery of endothelium-dependent relaxation, most previous reports have failed to consider what, if any, role the endothelium may have played in the reported results. In 1980, it was discovered that the vascular endothelium could produce and release a factor or factors termed endothelium-derived relaxing factor(s) (or EDRF) in response to acetylcholine (16). Since that time many agents including ATP, ADP, bradykinin, substance P and the calcium ionophore, A23187, have been found to produce vasodilation which is endothelium dependent (11, 15, 36). In addition to these reports, other studies have demonstrated that the responses to contracting and relaxing agents may be modified by the presence of the endothelium (8, 29, 34). In relation to the effects of ethanol on endothelium function, previous evidence has shown that acute administration of high concentrations of ethanol produced contraction in the rat thoracic aorta (5). This study further reported that a tolerance to the acute contractile effects of ethanol developed after 2-6 weeks of ethanol ingestion (5). A subsequent report demonstrated a developed tolerance to the contractile effects of ethanol in rat aorta after 2 days of administration of ethanol by oral intubation (24). This subsequent report showed that the tolerance to the contractile effects of ethanol was dependent on the endothelium (24). Additional studies have since shown that this endothelium-dependent tolerance to ethanol-induced contraction was mediated by EDRF (23). Although these studies have shown that ethanol consumption may affect endothelial function, the role of the endothelium and endothelium-dependent relaxation in association with chronic ethanol consumption and ethanol-induced hypertension has not been demonstrated. This study was conducted in order to investigate the effects of chronic ingestion of ethanol on vascular responsiveness with regard to possible alterations in endotheliumdependent relaxation in relationship to the associated hypertension. METHOD
Male Sprague-Dawley (SD) rats (Charles Rivers Laboratories) weighing between 227-304 g were randomly divided into control and ethanol-treated groups. Rats in the ethanol-treated group received ethanol in their drinking water at a concentration of 5% for the first week, 10% weeks 2-3, and 20% by volume weeks 4-13 adapted from the method originally described in 1983 (9). Control rats received tap water ad lib. In order to maintain a similar caloric intake, both the ethanol-treated and control groups received Purina Lab Chow (St. Louis, MO) on a restricted basis of 15 g/day and 18 g/day, respectively. Systolic blood pressures were measured weekly before 9 a.m. using a Model 72 heart rate and blood pressure meter (IITC Inc., Woodland Hills, CA). All blood pressure measurements were taken after 30 min under an elevated room temperature of 30°C. These measurements were always taken in the morning in order to minimize the effects of possible circadian variations on blood pressure. Body weights were also measured weekly. At the end of the treatment period the rats were anesthetized with et-chloralose (100 mg/kg) and pentobarbital (20 mg/kg), given IP. During anesthesia, arterial blood samples were taken via femoral artery cannulation for enzymatic determination of blood ethanol concentrations according to a previously described method (7). Rats were then sacrificed by decapitation followed by immediate removal of thoracic aortae. All rats were sacrificed prior to 10 a.m. to avoid variability due to the well-defined circadian rhythm which ethanol metabolism follows. Aortic vessels from both control and ethanol-treated rats were trimmed free of excess connective tissue and cut into ring segments 3 mm in length. Special care was taken throughout this
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TIME (WEEKS) FIG. 1. Effects of chronic ethanol treatment (EtOH TX) on mean systolic pressure in male rats. n= 8 and 12 rats for control and ethanol-treated groups, respectively. Mean systolic pressure for the ethanol-treated group was significantly (p<0.01) greater than controls beginning at week 3.
procedure to maintain an intact endothelium. In some of the segments the endothelium was mechanically removed by gently rubbing the intimal surface. Aortic ring segments with and without endothelium were mounted on hooks in isolated organ baths containing Krebs buffer 37°C. The Krebs solution was composed of the following (in mM): NaCI 118.1, KC1 4.7, MgSO4 1.2, KH2PO 4 1.2, CaC12 2.5, NaHCO 3 25.0 and glucose 11.1. The baths were maintained at pH 7.4 by continuous aeration with 95% 02 and 5% CO 2. The ring segments were allowed to equilibrate for 1 hr under an initial resting tension of 2 g. After equilibration, vessel segments were precontracted with 30 mM KC1 or 5 x 10 7 M phenylephrine. In vessels precontracted with phenylephrine, concentration-response curves to either acetylcholine, ATP, bradykinin, adenosine or sodium nitroprusside were generated.
Drugs The following pharmacological agents, obtained from Sigma Chemical Co., were used: phenylephrine hydrochloride, potassium chloride, acetylcholine chloride, ATP, bradykinin, adenosine, and sodium nitroprusside. All drugs were initially dissolved in distilled water at concentrations of 10 -2 M from which serial dilutions were made. Ethyl alcohol USP, 95%, was obtained from AAPER Alcohol and Chemical Co., Shelbyville, KY.
Statistical Analysis The steady state contraction produced by either 30 mM KCI or 5 x 10 - 7 M phenylephrine was expressed as increase in gram tension from resting tension. The relaxation responses to acetylcholine, ATP, bradykinin, adenosine and sodium nitroprusside were expressed as % of the steady state contraction obtained with 5 x 10 - 7 M phenylephrine. All data are expressed as means - S.E. Between group differences in the contraction produced by both 30 mM KC1 and 5 x 10 -7 M phenylephrine were assessed using Student's t-test for unpaired data for single comparisons. The within group comparisons of the contractions produced by these same agents in segments with and without intact endothelium were also assessed using Student's t-test for unpaired data for single comparisons. Between group (ethanol-treated vs. control) comparisons at each concentration of both the endothelium-dependent (acetylcholine, ATP and bradykinin) and -independent relaxing agents (adenosine and sodium nitroprusside) in ring segments with
CHRONIC ETHANOL AND THE ENDOTHELIUM
123
TABLE 1 THE EFFECTSOF CHRONICETHANOLCONSUMPTIONON THE CONTRACTIONPRODUCEDBY KCI AND PHENYLEPHRINE IN MALERATS
0
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0.71 ± 0.09 (g) 0.72 = 0.05 (g)
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1.89 ± 0.22 (g) "1.96 -+ 0.12 (g)
• 1.34 ± 0.07 (g) • 1.31 ± 0.08 (g)
Values represent the mean ± S.E. increase in (g) tension above an initial resting tension of 2 g in response to either 5 x 10-7 M phenylephrine or 30 mM KC1. Endo (+) or Endo ( - ) represent aortic segments with or without endothelium, respectively. *p<0.001 compared to respective within group (ethanol-treated or control) responses in vessels with intact endothelium.
and without intact endothelium were assessed using analysis of variance followed by a Student-Newman-Keuls post hoc test. Within group differences (endothelium present vs. endothelium removed) in the responses at each concentration of either the endothelium-dependent or endothelium-independent relaxing agents were compared using Student's t-test for unpaired data for single comparisons. All differences were considered significant at p<0.05. RESULTS
Ingestion of ethanol in rats for 13 weeks caused elevated systolic blood pressure compared to control rats. The mean systolic pressures at the time of sacrifice, for the ethanol-treated group and control, were 151.1---1.3 and 127.8---1.2, respectively. A difference in pressure was detectable by the third week of treatment and increased steadily throughout the remaining treatment period (Fig. 1). Although dally intake of ethanol was not measured in the present study, others (2) have reported daily ethanol intake of Sprague-Dawley rats subjected to a similar treatment regime to average around 10-11 g/kg/day at the 20% concentration. Both the ethanol-treated and control groups showed steady gains in weight over the course of treatment. At the time of sacrifice body weights averaged 4 6 8 ± 13 g vs. 445 ___17 g for the ethanol-treated and control groups, respectively. Blood ethanol levels for the ethanol-treated rats, also determined at the time of sacrifice, were 40.8 ___6.2 mg/dl. Since the main objective of this study was to examine the effects of chronic ethanol consumption on endothelium-dependent relaxation, only one concentration of the receptor-operated and voltage-dependent contracting agent phenylephrine and KC1, respectively, was tested. And although limited to only one concentration of each agent, these challenges were included to provide a rough assessment of possible changes in the vasoconstrictive responsiveness to these agents with regard to possible alterations in the endothelium-dependent modulation of the contraction produced by these agents. Our findings revealed no differences in the contractions produced by either 30 mM KC1 or 5 x 1 0 - 7 M phenylephrine between ethanol-treated rats and controls in rings of thoracic aorta with or without intact endothelium (Table 1). However, removal of the endothelium augmented the contraction produced by KC1 in both the ethanol-treated rats and controls (Table 1). The contraction produced by phenylephrine was also augmented by removal of the endothelium in the ethanol-treated
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animals. However, in the control rats this difference only approached significance at p =0.052 (Table 1). There was no difference in the concentration-response curves to acetylcholine in rings with intact endothelium from ethanoltreated rats vs. control rats, with the exception of the lowest concentration of acetylcholine (Fig. 2). Removal of the endothelium abolished the response to acetylcholine in both groups (Fig. 2). Also, there was no difference in the relaxation-response curves to a second endothelium-dependent relaxing agent, ATP, in rings with intact endothelium from ethanol-treated vs. control rats. Here again, removal of the endothelium abolished the relaxation produced by ATP, except at the highest concentration, where some relaxation persisted in both groups (Fig. 3). Unlike the responses to acetylcholine and ATP, the relaxation produced by bradykinin, another endothelium-dependent relaxing agent, was augmented in aortic tings with intact endothelium from ethanol-treated animals compared to controls (Fig. 4). Removal of the endothelium
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124
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abolished the relaxation produced by bradykinin in both groups (Fig. 4). Adenosine only produced relaxation at concentrations of 1 × 10 - 7 M or greater. These relaxations were also augmented in vessels with intact endothelium from ethanol-treated rats compared to control rats (Fig. 5). Removal of the endothelium had no effect on the relaxation produced by adenosine in vessels from control rats. However, in vessels from ethanol-treated rats removal of the endothelium reduced the relaxation produced by adenosine back to control levels, such that there was no difference in the relaxation between these tings and those from control rats with or without intact endothelium (Fig. 5). Between group comparisons (ethanol-treated vs. control) of relaxation responses to the endothelium-independent relaxing agent, sodium nitroprusside, revealed no between group differences at any concentration of sodium nitroprusside in rings with or without intact endothelium, respectively (Fig. 6). However, in both the ethanol-treated and control groups, removal of the endothelium augmented the relaxation produced by sodium nitroprusside compared to respective within group responses in vessels
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FIG. 6. Effects of chronic ethanol treatment (EtOH TX) on the relaxation produced by sodium nitroprusside. Other details are the same as in Fig. 2. *Significantly different from the within group responses in vessels without endothelium, p<0.05.
with intact endothelium (Fig. 6). Although removal of the endothelium shifted the relaxation response curve for sodium nitroprusside to the left in both the control and the ethanol-treated groups, the difference between the shifts was significantly less in the ethanol-treated group compared to control (Fig. 6). The difference in shift was assessed by comparing the mean difference in % relaxation at each concentration of sodium nitroprusside in control vessels (% relaxation without endothelium - % relaxation with endothelium) to the respective mean difference in ethanol-treated vessels. DISCUSSION
This study as well as previous studies (9,35) has demonstrated that rats which receive ethanol in their &'inking water at a concentration of 20% by volume over a prolonged period develop an increased systolic pressure compared to control rats which receive water alone. Studies aimed at elucidating the vascular effects of chronic ethanol treatment have considered possible alterations in the vascular responsiveness to vasoconstrictor agents such as catecholamines and angiotensin II (2, 9, 35). However, a considerable amount of variability exists concerning the effects of chronic ethanol on the vascular responsiveness to various vasoconstrictor agents. For instance, one study (4) reported an increased sensitivity to 20 mM KC1 as well as a hypersensitivity to other contractile stimulants such as catecholamines and angiotensin II in aortae from rats maintained on liquid diets containing ethanol (6.8% v/v) or sucrose (controls) from 12-24 weeks. In contrast, others have reported a decreased sensitivity to the contraction produced by phenylephrine at concentrations <3 × 10 -8 M, but reported no change in the maximal contraction produced by phenylephrine in thoracic aorta of rats that received ethanol in their drinking water (20% by vol.) from 6--18 weeks (35). Still others, using in vivo preparations, have reported similar pressor response curves to both phenylephrine and angiotensin II in control and chronically ethanol-fed rat (5% week 1, 10% weeks 2-3 and 20% by vol. in their drinking water weeks 3-12) (2). While these reported differences may represent differences in either treatment protocols or experimental designs, these studies have not considered possible alterations in either endotheliumdependent relaxation or the role of the endothelium in the modulation of the vascular responses of other contracting or
CHRONIC ETHANOL AND THE ENDOTHELIUM
relaxing agents associated with chronic ethanol consumption. In the present study we found no difference in the contraction produced by either 30 mM KCI or 5 x 10 -7 phenylephrine in vessels with or without endothelium from ethanol-treated vs. control rats. These findings are consistent with the previous in vivo f'mdings of others (2). Since the present study only examined the effects of chronic ethanol consumption at a near maximal contracting concentration of phenylephrine, our findings do not preclude possible effects of chronic ethanol consumption on the response to phenylephrine at lower concentrations as was previously reported (35). Differences between the present findings and those which reported a hypersensitivity to various contractile agents associated with chronic ethanol consumption (4) may represent differences in either the treatment protocol or the duration of treatment. Like previous studies (8,29), the present study did demonstrate that removal of the endothelium in aortae from both ethanoltreated and control rats augmented the contraction produced by KC1. An increased sensitivity to potassium after removal of the endothelium may be linked to the elimination of an endothelialassociated hyperpolarization of the smooth muscle (29). Like the response to KC1, removal of the endothelium also augmented the contraction produced by the ot-adrenoceptor agonist, phenylephfine, in the ethanol-treated rats. However, due to slightly greater variability in the control group, the augmented response to phenylephrine with removal of the endothelium only approached significance at p = 0.052. The present findings of no differences in the amount of contraction produced by either KC1 or phenylephfine in aorta with or without endothelium from ethanol-treated animals compared to their respective controls suggest, on a limited basis, that chronic ingestion of ethanol in the rat produced little effect on the vascular responsiveness to these contracting agents. In addition, these findings suggest that although the endothelium may modulate the contraction produced by both KC1 and phenylephrine, this modulation is not altered by chronic ethanol consumption in the rat. In terms of the hypertension which develops following chronic ethanol consumption, our limited data do not support the involvement of changes in either the vascular reactivity to, or the endothelium-dependentmodulation of the contraction produced by either KCI or phenylephrine as contributing factors. However, a more thorough examination of the concentration response relationships of these agents, with regard to endothelial modulation, associated with chronic consumption of ethanol is needed. Our study revealed no differences in relaxation response curves to ATP between the ethanol-treated rats and controls. Removal of the endothelium practically abolished the relaxation produced by ATP, except at the highest concentrations for both groups, where some relaxation persisted. This relaxation produced by ATP in the absence of the endothelium may have resulted from the breakdown of ATP into one of its vasoactive metabolites, possibly adenosine. Like ATP, there were virtually no differences in the endotheliumdependent relaxation produced by the acetylcholine between the ethanol-treated and control groups. Bradykinin, the third endothelium-dependent relaxing agent tested in the current study, was much less potent in producing relaxation than either ATP or acetylcholine. In contrast to other reports showing bradykinin to have an ED5o around 1-10 nM in bovine arteries, the current EDso was found to be around < 1 p,m in rat aorta. These differences were probably due to differences between the two species in response to this agent. More importantly, and in contrast to the lack of an effect of chronic ethanol consumption on the relaxation produced by ATP and acetylcholine, the relaxation produced by bradykinin was found to be significantly augmented in aortae from the ethanol-treated rats compared to controls. In previous studies (18,31), the involvement
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of prostaglandin metabolites in the relaxation produced by bradykinin has been reported. In one of these previous studies, investigators (18) reported that bradykinin can produce endotheliumdependent relaxation via either the production of a cyclo-oxygenase product or the production and release of EDRF. It is possible that ethanol treatment may have augmented one of these two processes resulting in an augmented relaxation to bradykinin. Since the relaxation produced by both acetylcholine and ATP were not affected by chronic ethanol treatment, it appears that the augmented relaxation to bradykinin does not result from a generalized increase in endothelium-dependent relaxation. In contrast to other reports (32,33), which suggested that the relaxant responses to nitrovasodilators are independent of the presence of endothelial cells, we demonstrated that removal of the endothelium augmented the relaxation produced by sodium nitroprusside. These findings are in agreement with a previous report (34) which also found an augmented response to sodium nitroprusside with removal of the endothelium in aortic rings from spontaneously hypertensive and Wistar-Kyoto normotensive rats. This previous report suggested that this augmented response to sodium nitroprusside in the absence of the endothelium may result from the removal of an endothelium-derived constricting factor (34). In addition, the endothelium may serve as a barrier to the relaxing effects of this agent. In agreement with previous in vivo work (1) the current study did not demonstrate an effect of chronic ethanol consumption on the endothelium-independent relaxation produced by sodium nitroprusside. The previous in vivo findings (1) showed that neither acute nor chronic ethanol treatment (4 weeks) had an effect on the in vivo depressor effects of nitroprusside in rats. The present findings suggest that with chronic ethanol consumption, the ability of the vascular smooth muscle to relax does not seem to be impaired and as such, should not contribute to the hypertension seen with chronic ethanol consumption. However, the present findings did demonstrate that removal of the endothelium shifted the relaxation response curve to the left in both the ethanol-treated and control groups, suggesting that the endothelium can modulate the relaxant effects of agents which were previously thought to act independent of the endothelium. Adenosine, an endogenous metabolite of ATP, is thought to produce vasodilation at the level of the vascular smooth muscle by activating adenosine receptors of the A2 type (26,30). An unexpected finding of the present study was an augmented relaxation to adenosine in aorta from ethanol-treated rats compared to control rats with intact endothelium. This, along with the finding that removal of the endothelium in aortae of ethanol-treated rats reduced the relaxation produced by adenosine back to that of control vessels with or without endothelium, suggests that chronic ingestion of ethanol in the rat causes the appearance of an endothelium-dependent component to the relaxation produced by adenosine. Our findings of no differences in the relaxation produced by adenosine in vessels without endothelium from ethanol-treated animals compared to respective controls suggest that the appearance of the endothelium-dependent component to adenosine was independent of changes in the vascular responsiveness to adenosine at the smooth muscle level. Although the nature of this endothelium-dependent component was not evidenced in the present study, recent evidence has shown that adenosine can cause the release of prostacyclin from perfused rabbit hearts (19) with the coronary vascular endothelium considered to be the main source of this eicosanoid (20). Since chronic ethanol consumption was only associated with an augmented relaxation to bradykinin and adenosine in the absence of changes in the relaxation produced by either the endothelium-dependent relaxing agents, acetylcholine and ATP, or the endothelium-independent relaxing agent, sodium nitroprusside, it is possible that the augmented relaxation
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WILLIAMS, ADAMS AND MUSTAFA
to bradykinin and adenosine occurs via a common mechanism. Since previous studies have shown that both adenosine and bradykinin can stimulate prostaglandin production, this mechanism may involve an endothelium-dependent production of a prostaglandin metabolite, possibly prostacyclin (18,19). This possibility is further supported by evidence which has shown that acutely, moderate concentrations of ethanol (34.8 mM) can raise prostacyclin levels in vivo and in vitro (25) while they may selectively reduce platelet (14.3 and 28.6 mM ethanol) thromboxane production (28). While chronic ethanol consumption (4-5 weeks 36% of energy as ethanol) in rats has been associated with an actual decrease in basal release of prostacyclin, it has been shown to cause the acute appearance of ethanol-induced prostacyclin production in rat aorta (17). These two effects were observed in the absence of an effect of chronic ethanol consumption on acetaldehyde-stimulated prostacyclin formation (17). Although the nature of the ethanol-induced prostacyclin formation was not evidenced by this previous study (17), the findings allow the possibility of other alterations in the stimulated release of prostacyclin produced by chronic ethanol consumption. Other possibilities for the augmented relaxation produced by adenosine could include either an increase in the number or sensitivity of the adenosine receptors or an uncovering of additional adenosine
receptors. This latter possibility stems from the established effect of ethanol to cause perturbations of membranes (12). However, this latter possibility would have been limited to alterations in adenosine receptors at the level of the endothelium since, in the absence of the endothelium, there was no difference between adenosine responses in the ethanol-treated group compared to respective responses in the control group. In summary, the present study has supported previous studies which have shown that chronic consumption of ethanol in the rat can elevate systolic pressure. We have also shown that in addition to the elevation of systolic pressure, there is a concurrent augmentation of endothelium-dependent relaxation produced by bradykinin and adenosine, the nature of which remains to be elucidated. But regardless of the nature of these augmented endotheliumdependent relaxation responses, these alterations associated with chronic ethanol consumption would seem to oppose rather than contribute to the elevation of systolic pressure which is associated with chronic ethanol consumption. In conclusion, although chronic ethanol consumption was shown to have an effect on endothelium-dependent relaxation, this effect seemed to be in opposition to the hypertension which accompanies chronic ethanol consumption.
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