1987) or a desensitization to the contractile effect of ..... EtOH-treated rats were significantly potentiated rather than depressed by ... lation of the enzyme. Chronic ...
Gen Pharmac Vol 23, No 2, pp 245-248, 1992 Pnnted m Great Bntmn All nghts reserved
0306-3623/92$5 00 + 0 00 Copyrtght © 1992PergamonPresspie
EFFECTS OF CHRONIC ETHANOL CONSUMPTION ON ~-ADRENERGIC-INDUCED CONTRACTIONS IN RAT THORACIC AORTA G. PINARDI,! C BRIEVA,! R VINET2 and M. PENNA1. 'Department of Pharmacology, Faculty of Medicine, Umverslty of Chile, P O Box 70 000, Santmgo 7, and 2Department of Pharmaceutical Soences, Umverstty of Valparaiso, Castlla 4059, Valparaiso, Chtle (Recewed 9 July 1991) Abstract--1 The effects of chromc oral admtmstrauon of ethanol (EtOH, 80 mM dally dunng 15-18 days) on the modulatmn exerted by the endothehum on the contracuons reduced by phenylephnne (Ph) and clomdme (C) were studied m rat aorUc nngs wtth and wRhout endothehum 2 The maximal contraction reduced by a 70 mM KCI depolarizing solution was slmdar in control and EtOH treated-nngs 3 EtOH pretreatment slgmficantly enhanced the contractde response to Ph m vessels wRh intact endothehum, but did not ssgmficantlyaffect the response of endothehum-denuded nngs 4 Tlus effect reflects a reduction of the modulatmn exerted by the endothehal cells on al-adrenerglc vasoconstrictton 5 C &d not contract control aorUc nngs wRh endothehum, but after EtOH treatment, the response reached about 20% of the maximal KCl-mduced contraction 6 EtOH pretreatment slgmficantlyenhanced the contractile response to C both m endothehum-denuded and intact aorUc nngs 7 The results indicate that chrome EtOH consumption slgmficantlypotentmtes a-andrenergqc-mduced contractmns m rat aortic nngs, probably through interference wtth the productmn and/or the release of EDRF
INTRODUCTION It has been well estabhshed that ethanol (EtOH) has many cardmvascular effects, affecting heart rate, blood pressure and myocardml contracUhty (Nakano and Kessmger, 1972) Many epldermologlcal studies have shown that chromc alcohol consumption is assocmted with elevated artenal blood pressure (Khetarpal and Volicer, 1981; Klatsky et al., 1986), but the mechanisms responsible for these effects are not completely understood. Much of the research investigating the chromc effects of EtOH have dealt wRh vascular responsweness to vasoconstrictor agents. In 1985 a report correlated the rise m arterial pressure with an expansion m plasma volume and an increase m plasma norepmephnne levels (Chan et al, 1985) Since then, many conflicting reports showing alteratmns m vascular responses in the rat after chromc EtOH mgestmn (2-24 weeks duratmn) have appeared For instance, Altura and Altura (1987) reported hypersensltwlty to contracting agents like potassmm, catecholammes and angmtensm II, while others have reported no change m the responsiveness to norepinephrine (Abdel-Rahman and Wooles, 1987) or a desensitization to the contractile effect of phenylephnne (Stnckland and Wooles, 1988). These differences may reflect different experimental designs or different protocols of chronic EtOH administration. The recognition of the role of endothelium in the modulation of vascular responsiveness to con*To whom all correspondence should be addressed oP 23/~-0
tractang and relaxing agents (Bullock et al., 1986, Shlrasakl et al, 1986, Vmet et al., 1991a, b) has thrown a new hght on the vascular effects of EtOH Knych et al (1984) reported that the developing of tolerance to the contractile effect of EtOH m the isolated rat aorta was endothehum-depcndent, being probably medmted by the endothelium-denved relaxmg factor (EDRF) (Knych, 1987) These studies show that EtOH may affect endothehal functmn, but the role of the endothehum in ethanol-related hypertension has not been demonstrated (Wdhams et al, 1990). Furthermore, there are few reports regarding the effect of EtOH on the endothehum-dependent vascular responses evoked by vasoconstnctor substances. The present study was camed out to investigate the effects of chromc EtOH consumptmn on the endothelial modulation of ~-adrenerglc-induced contracUons m the rat aorta M A T E R I A I ~ AND METHODS
Rats of UCHA strata (Umversity of Clule, SanUago) wmghmg200 to 300 g were kdled by a blow to the head, and two nngs were prepared from a segment of the thoraac aorta as prevmusly described (Vmet et al, 1991a, b) Bnefly, one nng was depnved of its endothehal layer by inserting the points of a small forceps and gently rolling the ring over filter paper during 15 sec. Paired nngs, one wlth intact endothehum and one without endothehum, were equihbrated for 60 rain m a u s s u e bath under an optimal resting tension of 1.5 g in a modified Krebs-Henseleit solution of the following compomton (raM): NaCI 122.0, KCI 4.7, CaCI, 2.0, MgCh 1 2, KH2PO4 1.2, NaHCO3 15.0, glucose
245
G PINARDIet al
246
11 5 and EDTA 0 026 at pH 7 4 The solution was maintamed at 37°C and was continuously bubbled with a 95% 02-5% CO 2 gas mixture The nngs were contracted at least three times with a depolarizing KC1 solution (70 0 mM) When the K+-mduced contractions reached a steady level, the nngs were repeatedly washed and re-eqmhbrated for an addmonal 30 min before testing the agonlsts Cumulative dose-response curves were obtained by a stepwlse increase in the concentration of phenylephrme (Ph, selective ~radrenerglc agonlst) and clonldlne (C, selectwe ~t2-adrenerglc agonist) Additions were made as soon as a steady response was obtained from the preceding dose Each pmr of nngs was exposed to only one agonlst The functional Integrity of the endothehum was assessed at the end of each experiment by recording the relaxing response to 10-5-I0 -a M of acetylchohne in the rings previously contracted with l0 7M noreplnephnne (Vlnet et al, 1991a) Dose-response curves were recorded in aortas obtained from rats submitted to the administration of 3 68 g/kg body weight of EtOH (80 mM) as a 20% soluUon m d~stflled water by gabage m one dally dose during 15 to 18 days The experiments were performed 24hr after the last dose of EtOH Control rats were submitted to the same schedule, receiving the same volume of sahne solution instead of EtOH The contractile tension developed by the aortas was expressed as the percentage of the maximal contraction (Em~) obtained by K+-depolarIzatlon in each nng The dose producing 50% of the maximal effect was calculated according to Fleming et al (1972) and was expressed a s p D : ( - l o g ED~0) Statlstmal analysis of the results was done using mean values + SEM and the s~gntficance was assessed by Student's t-test for paired and unpaired data Statistical slgmficance was accepted at the P < 0 05 level RESULTS The removal o f the endothelial layer did n o t affect the maximal c o n t r a c t i o n o b t a m e d m aortic n n g s with intact e n d o t h e h u m by depolartzatlon with K r e b s - H e n s e l e l t solution c o n t a m m g 7 0 m M KCI Similarly, the K + - i n d u c e d c o n t r a c t i o n was not slgmficantly different m aortic rings with and without e n d o t h e h u m obtained f r o m ethanol-treated rats and was also not slgmficantly different from the contraction o b t a i n e d m control n n g s (Table 1) Ftgure 1 shows the d o s e - r e s p o n s e curves to Ph in aorUc rings with and without e n d o t h e h u m o b t a i n e d f r o m rats submitted to 15-18 days o f 80 m M E t O H ingestion, c o m p a r e d with the corresponding d o s e - r e s p o n s e curves from control antmals. As has been prevtously described, m control ammals the contractile response to P h was slgntficantly e n h a n c e d by endothellum removal, while the maximal contractile response to KCl was not affected E . ~ and p D : values were increased slgmficantly (Table 2) The a d m m l s t r a U o n o f 8 0 m M E t O H for 15-18 days shifted the Ph d o s e - r e s p o n s e curve to the left m the n n g s with intact e n d o t h e h u m , slgmficantly increasing the pD2 value, whde E,~x was only slightly increased (Table 2) By contrast, the rings without e n d o t h e h u m Table 1 MaximalcontracUonreducedby 70mM KCI soluuon m rat aortic tangs with and without endothehum after chromc ethanol administration (EtOH, 80 raM) With endotbehum W~thoutendothehum Control (n = 18) 15540-+692 15262+672 EtOH (n ffi 14) 1426 8 ± 90 2 1420 4 + 99 8 Values are mean _+SEM of mg of developedtension/ragof wet Ussue weight and are not Slgmficantly¢hfferent(P > 0 05)
too
L
80
"O
g
40
2o
0 9
8
7
6
5
PhenyLephr,ne(-Log M ) Fig 1 The effect of chronic ethanol admlmstrauon (EtOH, 80mM datly for 15-18 days) on the phcnylephrme dose-response curves in rat aortic nngs wtth and without endothehum The developed tension of each cumulattve dose is expressed as percentage of the maximal contracttle response achieved by a 70 mM KCI depolanzmg solutton m each nng Doses are expressed as the log of the fnal molar concentraUon m the bath Values are mean +_SEM of 18 control and 7 EtOH pmred experiments Curves of rings with intact endothehum are slgntficantly different Curves of rings without endothelium are not significantly different Control with intact endothehum (O), control without endothehum (O), EtOH with intact endothehum ( I ) , EtOH without endothehum (1"7) showed only an increase m the p D 2 value due to augmented responsweness to low doses o f the agomst and the curves reached approximately the same maximal response These c o m b i n e d effects seem to reflect a reducUon m the modulating influence exerted by the endothehal cells o n ctt-adrenergm-mduced contractions The d o s e - r e s p o n s e curves to C are dlustrated m Fig 2 C was not able to reduce a contracUon m control aortic rings with intact e n d o t h e h u m , but Table 2 Effectofchromc ethanol administration(EtOH, 80 mM) on E,,~ and pD 2 values for phenylephnne(Ph) and clomdme(C) m rat aorUc nngs with (E+) and without ( E - ) endothehum E,,~ E+
p D2 E-
E+
EPh control (n=18) 502_+39* 1023_+15 696_+004* 763_+005 Ph + EtOH (n=7) 673_+92 901_+94 744+01"~ 795_+012t C control (n=9) 00 300_+ 72t -682_+009 C + EtOH (n=7) 178+881" 592-+105 646±018" 714-+015 E~x = maxlmalcontracUonwlth agomst × 100/maximalcontraction wlth 70 mM KCI soluuon PD2 = -log ED~ *P < 0 01 between vesselswlth and wlthout endothehum tP < 0 05 betweencontrol and EtOH-treated vessels ~P < 0 01 between control and EtOH-treated vessels
Ethanol, EDRF and ~-adrenerg~c contractions endothehum removal resulted in a dose-response curve reaching 30% of the maximal contraction reduced by KCI (Table 2). After the chromc administration of 80 ram EtOH, the contractile response to C m nngs with intact endothehum reached approximately 20% of the maximal KCl-mduced contraction. The nngs w~thout endothehum from EtOH treated rats showed a s~gmficant shifting of the dose-response curve to the left, with a s~gnificant increase m E,,ox (59.2% of the maximal KCi contraction, Table 2) The maximal contraction induced by C m these aortic nngs was s~gmficantly lower than the contraction reduced by Ph
247
aratlons obtained from rats intoxicated with EtOH display a slgmfi~nt reducUon m the magnitude of ethanol-induced contracttons, demonstrating the development of tolerance (Altura et a l , 1980). Tolerance to the contractile effects of EtOH was not assessed in these expenments, but Knych et al 0984) demonstrated that EtOH at high concentrations (0 4 to l 0 M) Induced a dose-dependent contracUon of rat aortic nngs, which was slgrnficantly reduced m EtOH-trcated rats The presence of a funcUonal endothehal hmng was required for the development of tolerance, since the removal of endothchal cells from aortas obtained from rats intoxicated with EtOH for 2 days significantly shifted the EtOH doseresponse curve to the left and slgmficantly Increased DISCUSSION the maximal contraction, whereas In nngs with intact Tamp~er et al 0988) reported that a strata of cndothehum the dose-response curve was shifted to non-dnnkmg rats (UCHA, Umvers~ty of Chflc, San- the nght and the maximal contraction was not tinge) submitted to the oral admm~stratlon of affected (Knych et a l , 1987) Altura et al (1976, 1978, 2 76g/kg/day (60mM) of EtOH during 15 days 1987) postulated that the vascular effects of EtOH developed tolerance to ethanol-reduced sleeping Umc may be related to alterations in calcmm content of the and hypothermm However, this tolerance seems to blood vessel, arguing that chromc EtOH admlmsbe more cons~stcnt when the dose of EtOH is m- trat~on reduce the aortic calcmm content, a fact creased to 3.68g/kg/day (80mM) during 15 days which could account for the reduced EtOH-mduced (personal commumcatlon) Therefore, m the present contractile response observed m tolerant ammals work the latter schedule of EtOH admm~stratlon was In the present expenments, the responsiveness to used m order to obtain the developmcnt of a reason- Ph, an ~l-selectlve adrenerglc agomst, was slgmfiable degree of tolerance to somc effects of EtOH cantly enhanced m the aortic nngs w~th intact endoEthanol produces dose-dependent contractions of tbehum from EtOH-trcated rats as compared to ~solated aortas of naive rats However, aortic prep- controls This result differs from the finding of Wflhams et al (1990), who reported that ethanol 100 treatment did not modify the response to Ph in nngs with or without endothchum However, they used only one concentration of Ph (5 x l0 -7 M) to conclude that the endothehum-dependent modulation of 80" Ph-mduced contractions is not modified by EtOH. From our results, ~t ~s clear that the influence of endothehum m the Ph responsiveness is significantly reduced after EtOH treatment. After the destruction of the endothehal hnmg, both dose-response curves in control and EtOH-treated ammals are s~mflar and E~x is not slgmficantly affected. On the other hand, Stnckland and Wooles 0988) m agreement with our study, reported that chromc EtOH consumpUon mcreased the contractile response to low conceng trations of Ph I-The enhancement of ~l-adrenerglc-mduced contracuons by EtOH administration appears to be related to a reduced release of E D R F from endotbehal cells Since the receptor-evoked and the basal release of E D R F are highly dependent on extraccllu9 8 7 6 5 lar Ca 2+ concentrauon (Vmet et al., 1991b), the reduced modulating action on al-adrencrglc-induced CLonldine (-log M ) contractions m aortic nngs w~th intact endothehum Fig. 2 The effect of chronic ethanol administration (EtOH, supports a probable influence of EtOH on Ca 2+ 80 mM daily for 15-18 days) on the clomdlne dose-response avaflabihty Ethanol leads to a reduction in the curves m rat aortic nngs with and without endothehum The mtracellular Ca ~+ concentration, e~ther by increased developed tension of each cumulative dose IS expressed as binding of Ca 2+ to the membrane (Mlchaehs and percentage of the maximal contractile response acl'aeved by a 70 mM KC1 depolanzIng solution m each nng. Doses are Myers, 1979) or by decreasing the reflux of Ca 2+ rote expressed as the log of the final molar concentration In the the nerve terminal ( H a r m and Hood, 1980). Therebath Values are mean 4- SEM of the number of 9 control fore, EtOH may exert an inhibitory acUon on the and 7 EtOH pmred expenments Control rings w~th intact release of E D R F by affecting Ca 2+ entry rote the endothehum did not contract Curves of nngs with intact endothehal cell The reduced production and/or reendothehum and curves of nngs without endothehum are lease of E D R F after EtOH treatment also mh~b~ts the significantly different Control voth intact endothehum ( 0 ) , relaxation evoked by acetylchohnc and ATP, without control without endothehum (©), EtOH with intact endoaffecting the relaxation produced by sodmm mtrothelium ( , ) , EtOH w~thout endothehum (Q)
G PINARDIet al
248
prusslde (Hatake et a l , 1989) The removal of the endothehal layer ehmmates the effect of E t O H on • 1-adrenergtc-lnduced contractions, a fact that points to the fundamental role of E D R F in these responses The effect of E t O H administration on E D R F is more evident when ~2-adrenerglc contractions are analyzed As shown previously (Vmet et a l , 1990a, b), C was not able to contract the aortic nngs with intact endothehum from control rats, but the nngs from EtOH-treated rats displayed a contractile response with an Emox approaching 20% of the maximal contraction Induced by KC1 Furthermore, the removal of the endothelium increased very significantly the responsiveness to C in EtOH-treated animals as compared to control rats The Em~x value was significantly different in both groups, indicating that E t O H consumption greatly affects the endothehumdependent modulation of cq-adrenoceptor-mduced contractions Hatake et al (1989) reported that E t O H added to the tissue bath significantly enhanced contractions Induced by C In endothehum-mtact aortic strips, but depressed them m endothehum-denuded strips In the present work, C-mduced contracuons of endothellum-denuded aortic rings obtained from EtOH-treated rats were significantly potentiated rather than depressed by ethanol Contractions due to activation of cq-adrenoceptors are almost completely dependent on extracellular Ca 2÷ concentration, which Is probably not affected by E t O H This view is reinforced by the fact that K+-induced contracUons, which depend exclusively on extracellular calcium concentration, were not modified by E t O H treatment (Table 1) However, the uptake of Ca 2÷ into the endothelial cells may be reduced by ethanol, resulting in a reduction in the production and/or the spontaneous release of E D R F An alternate explanation could reside in the inhibitory action exerted by E t O H on the guanylate cyclase system (Stenstrom et a l , 1986), whmh could prevent the E D R F stimulation of the enzyme Chronic ethanol consumption can potentiate ctadrenerglc-lnduced contractions m rat aorta by reducing the modulation prowded by the endothehum, probably lnterfenng with the synthesis, the release or the action of E D R F Endothehum-dependent relaxatlons of vascular smooth muscle seem to be also affected by E t O H (Hatake et a l , 1989, Wllhams et a l , 1990), even ffreports are rather contradictory Nevertheless, it seems clear that endothehum-dependent vascular responses are altered by ethanol, a fact which can be of some importance m the pathophyslology of hypertension associated with chronic E t O H consumption Acknowledgements--This work was supported by Project 1174-89, Fondecyt, and ProJect B 2680-8935, DTI, University of Chile REFERENCES
Altura B M and Altura B T (1987) Symposium on car&ovascular effects of ethanol-part II penpheral and cerebrovascular actions of EtOH, acetaldehyde and acetate relauonshlp to divalent cauons Alc Chn exp Res 11, 99-I 11 Altura B. M , Carella A. and Altura B T. (1978) Acetaldehyde on vascular smooth muscle possible role in vasodilator action of ethanol Eur J Pharmac $2, 73-83
Altura B M , Edgarlan H and Altura B T (1976) Differenttal effects of ethanol and manmtol on contraction of artenal smooth muscle J Pharmac exp Thdr 197, 352-361 Altura B M , Pohorecky L A and Altura B M (1980) Demonstration of tolerance to ethanol in non-nervous tissue effect on vascular smooth muscle Alc Chn exp Res 4, 62-69 Bullock G R , Taylor F S G and Weston A H (1986) Influence of the vascular endothehum on agonlst induced contractions and relaxations m rat aorta Br J Pharmac 89, 819-830 Chan T K C , Wall R A and Sutter M C (1985) Chronic alcohol consumption, stress and hypertension Hypertension 7, 519-524 Fleming W W, Westfall D P, de la Lande I S and Jellet L B (1972) Log-normal &strlbut2on of eqmeffectlve doses of noreplnephrme and acetylchohne m several Ussues J Pharmac exp Thdr 181, 339-345 Harris R A and Hood W F (1980) Inhibition ofsynaptosomal calcium uptake by ethanol J Pharmac exp Thdr 213, 562-568 Hatake K , Wakabayashl I, Kaklshlta E, TanlgucM T , Ouch1 H , Sakakl N and Hlshlda S (1989) InMbltory effect of ethanol on endothehum-dependent vascular responsiveness Eur J Pharmac 168, 277-283 Khetarpal V K and Vohcer L (1981) Alcohol and car&ovascular disorders Drug Alc Depend 7, 1-30 KlatskyA L, FrledmanG D and ArmstrongM A (1986) The relationship between alcohohc beverage use and other traits to blood pressure a new Kaiser Permanent study Circulation 73, 628-636 Knych E T (1987) Endothehum-dependent transfer of ethanol tolerance m the aorta Life Sct 40, 19031908 Knych E T , Gulmaraes C M S and Bolwn S (1984) Tolerance to ethanol-reduced contractions of vascular smooth muscle role of endothehum Life Sct 35, 611-617 Mlchaells E K and Myers S L (1979) Calcium bmdmg to brain synaptosomes Effects of chronic ethanol intake Btochem Pharmac 13, 2081-2087 Nakano J and Kessmger J M (1972) Car&ovascular effects of ethanol, its congeners and synthetic bourbon in dogs Eur J Pharmac 17, 195-201 Shlrasakl Y , Su C , Lee T J - F , Kolm P, Chne W H Jr and Nlckols A (1986) Endothehal modulation of vascular relaxation to mtrovasodllators m aging and hypertension J Pharrnac exp ThOr 239, 861-866 Stenstrom S, Enloe L, Pfenntng M and Rlchelson E (1986) Acute effects of ethanol and other short-chain alcohols on the guanylate cyclase system of munne neuroblastoma cells (Clone N1E-II5) J Pharmac exp ThOr 236, 458-463 Strlckland J A and Wooles W A (1988) Effect of acute and chromc ethanol on the agomst responses of vascular smooth muscle Eur J Pharmac 152, 83-91 Tampter L, Urrutm C and Qulntandla M E (1988) Effect of different treatment regimens with ethanol and pchlorophenylalanlne on tolerance development in UChA and UChB rats Alcohol 5, 343-347 Vlnet R , Bneva C , Pmardl G and Penna M (1991a) Modulation of ~-adrenergm-lnduced contractions by endothehum-derlved relaxing factor In rat aorta Gen. Pharmac 22, 137-142 Vlnet R , Bneva C , Pmardl G and Penna M (1991b) Influence of extracellular C a 2+ o n the modulation of • -adrenergic-mduced contracUons In rat aorta Gen Pharmac 22, 365-369 Wllhams S. P , Adams R D and Mustafa S. J (1990) The effects of chronic ethanol treatment on endothchumdependent responses in rat thoracic aorta Alcohol 7, 121-127
