Dissociation Constant of the Norepinephrine-Receptor Complex in Normotensive and Hypertensive Rats

Transcription

1 Dissociation Constant of the Norepinephrine-Receptor Complex in Normotensive and Hypertensive Rats By Robert B. Strecker, Walter C. Hubbard, and Andrew M. Michelakis ABSTRACT Previous reports have suggested that smooth muscle obtained from the thoracic aorta of spontaneously hypertensive rats is less responsive to vasoconstrictive agents than that obtained from normotensive rats. The present study was undertaken to determine whether the responsiveness of aortic muscles from normotensive and spontaneously hypertensive rats correlates with a difference in the affinity of the adrenergic receptors for norepinephrine and whether antihypertensive therapy alters the affinity of the adrenergic receptors for norepinephrine. The affinity of the adrenergic receptors for norepinephrine was determined by computing the dissociation constant of the norepinephrine-receptor complex (K nr ). The values computed for K DR in aortic muscles from normotensive and spontaneously hypertensive rats that had received no antihypertensive therapy were 1.07 x 10~ 7 M and 1.17 x 10~ 7 M, respectively. The values computed for K,, R in aortic muscles from normotensive and spontaneously hypertensive rats that had received antihypertensive therapy were 1.38 x 10~ 7 M and 1.29 x 10~ 7 M, respectively. The differences in these values for K nr are not significant. These results indicate that the difference in the contractility of aortic muscles from normotensive and spontaneously hypertensive rats is not related to an alteration in the affinity of the adrenergic receptors for norepinephrine and that the affinity of the adrenergic receptors for norepinephrine is not altered by antihypertensive therapy. Thus, it appears that the etiology of hypertension cannot be directly correlated with a difference in the affinity of the adrenergic receptors for norepinephrine. Although essential hypertension has been the subject of numerous studies, the etiology of this disease is not completely understood. It has been postulated that essential hypertension develops secondary to an increase in resistance at the arteriolar level (1-3). This increase in resistance may be due to one or a combination of the following three mechanisms. First, there may be an enhanced vasoexcitatory influence (neurogenic, blood-borne, or local) to which the arterioles respond normally (4-7). Second, a change in the inherent responsiveness of the vascular smooth muscle itself may cause an increase in resistance in response to normal vasoexcitatory influences (8-11). Finally, a structural change in the vascular smooth muscle may result in an increase in resistance at normal levels of smooth muscle activity (12-14). An abnormality in the interaction between receptors located in the membrane of vascular smooth muscle and the adrenergic neurotransmit- From the Clinical Pharmacology Program, Departments of Pharmacology and Medicine, Michigan State University, East Lansing, Michigan 48824, and Vanderbilt University, Nashville, Tennessee This study was supported in part by U. S. Public Health Service Grants HL and HL from the National Heart and Lung Institute and by a grant from the Michigan Heart Association. Dr. Strecker was supported in part by Graduate Training Grant GZ-1728 from the National Science Foundation. Received November 22, Accepted for publication August 28, ter norepinephrine could contribute to the etiology and the maintenance of essential hypertension. This possibility was investigated in the present paper by computing the dissociation constant of the norepinephrine-receptor complex (K nh ) in normotensive and spontaneously hypertensive rats. A significant difference in K nh would indicate an alteration in the affinity of the adrenergic receptors for norepinephrine. We also computed K DR in normotensive and spontaneously hypertensive rats that had been maintained on antihypertensive therapy which effectively controlled high blood pressure to determine whether such treatment altered the affinity of the adrenergic receptors for norepinephrine. K DH was calculated for isolated strips of smooth muscle from the aorta of normotensive and spontaneously hypertensive rats. The Wistar strain of spontaneously hypertensive rats developed through selective breeding techniques by Okamoto and Aoki (15) was chosen for this study, because it appears to be a good model for the study of mechanisms involved in essential hypertension. Wistar normotensive rats were used, because they constitute the strain of rats that is generally most homogeneous with respect to the spontaneously hypertensive rats that were employed. Methods Male Wistar rats obtained from Carworth Farms were used in this study. The normotensive and spontaneously 658

2 K D R IN NORMOTENSIVE AND HYPERTENSIVE RATS 659 hypertensive rats were further subdivided into four groups: (1) spontaneously hypertensive rats that received no antihypertensive therapy (SHRU), (2) spontaneously hypertensive rats that received antihypertensive therapy (SHRT), (3) normotensive rats that received no antihypertensive therapy (NRU), and (4) normotensive rats that received antihypertensive therapy (NRT). At the beginning of the 3-month treatment period, each group comprised 12 rats. The mean weight of the normotensive rats was 187 ± 6 (SE) g; that of the spontaneously hypertensive rats was 150 ± 4 g. The age of both groups of rats at the beginning of the study was 12 weeks. During the period of study, blood pressure determinations were made periodically with an electrosphygmograph (model ESG-300, Narco Biosystems) attached to an E & M physiograph (DMP-4A, Narco Biosystems). The rats were housed two to a cage. Untreated rats were allowed to drink distilled water ad libitum, but treated rats were only allowed to drink an antihypertensive drug solution ad libitum. This solution was prepared by dissolving the following agents in 1 liter of distilled water: (1) hydralazine HC1 (Appresoline, Ciba) (75 mg), (2) chlorothiazide (Diuril, Merck, Sharpe and Dohme) (1 g), and (3) reserpine (McKesson Laboratories) (1.4 mg). These regimens were continued for 3 months. The aorta was isolated from each of the rats in the following manner. The rat was killed by a blow to the back of the head and exsanguinated by severing the jugular veins and the carotid arteries. The abdomen and the thorax were opened, the thoracic organs were dissected away, and the aorta was removed. The section of the aorta used was the portion from 1 cm above the diaphragm to 1 cm below the aortic arch. The excised aorta was immediately placed in cold (0-4 C) modified Tyrode's solution saturated with 100% oxygen. The fat and other perivascular tissues were then removed, and the aorta was cut on a spiral as described by Furchgott (16). Two strips cm long and 2-3 mm wide were obtained from each aorta. The muscles were then suspended in muscle baths with a capacity of 15 ml. The isometric contractions of the aortic strips were recorded using an assembly consisting of a microdisplacement myograph transducer (F-50, Narco Biosystems) attached to an E & M physiograph (DMP-4A). The temperature of each bath was kept constant at C. The bathing medium was constantly aerated with 100% oxygen. An initial tension of 0.5 g was applied to each strip. If necessary, periodic readjustments of the tension to 0.5 g were made during a 45-minute equilibration period. At the end of the equilibration period, a norepinephrine dose-response curve was obtained by adding norepinephrine to the bathing medium. The initial concentration of norepinephrine placed in the bathing medium was 6.0 x 10""M. The concentration of norepinephrine was subsequently increased in discrete steps until a final concentration of 3.2 x 10" 6 M was reached. This concentration of norepinephrine induced the maximum response that the muscle was capable of producing under these conditions. The strips were then washed with the modified Tyrode's solution and allowed to relax and equilibrate for 30 minutes. At this time, the alpha-antagonist phenoxybenzamine (10~ 8 M) was placed in the muscle bath for a period of 2 minutes, the strips were washed four times with the modified Tyrode's solution, and a second norepinephrine dose-response curve was obtained. The millimolar composition of the modified Tyrode's solution was: NaCl 125, KC1 5.4, CaCl 2 1.8, MgCl 2 1.2, glucose 11, and Tris (Tris-[hydroxy-methyl]-aminomethane) (Trizma Base, Sigma) 23. The ph of the solution was adjusted to 7.5 with 6N HC1, and the solution was saturated with 100% oxygen prior to use in distilled, deionized water. A norepinephrine stock solution was prepared daily by dissolving 10 mg of /-norepinephrine (free base) in 10 ml of O.lN HC1. A stock solution of phenoxybenzamine was prepared daily by dissolving 1 mg of the powder in 10 ml of 2N HC1. Both of these stock solutions were diluted with the modified Tyrode's solution to give the final concentration desired. The addition of these agents to the muscle bath after dilution did not cause any change in the ph of the bathing medium. Data were analyzed on the Sigma 7 computer with a standard program for analysis of variance (17). Linear regression analysis was also employed (18). To determine whether there was a difference in the affinity of the adrenergic receptors for norepinephrine, we first computed K DR in the manner described by Furchgott (19, 20) and Furchgott and Bursztyn (21). They formulated the following expression: err[d] [Response] = f(s) = f(e[dr]) = / ( \ (1) \K nr + [D]J where [Response] refers to the magnitude of the contractile response of the muscle, / refers to a real, single-valued function, S is the magnitude of the stimulus, e is the intrinsic efficacy, [DR] is the concentration of the norepinephrine-receptor complex, R T is the total concentration of receptor sites in the tissue, [D] is the concentration of norepinephrine, and K DR is the dissociation constant for the notepinephrine-receptor complex. If e or R T is reduced by some fraction, (1 - q), then the magnitude of the response will be reduced. If the original response can be reinstated by increasing the concentration of norepinephrine, then /qer T [D'] \ =. / er T [D] \ \K DR+ [D']) \K nr +[D]J where [D 1 ] is the concentration of norepinephrine that, in the experimental situation, elicits a response equal to that elicited by [D] under control conditions. Solving Eq. 2 for 1/[D] gives the following expression: (2) 1 1 q[d'] qk D (3) A plot of 1/[D] vs. 1/[Z)'] defines a straight line with a -slope equal to (1/q) and an intercept equal to (1 - q)/(k nr ). Therefore, Ano = Slope - 1 Intercept Results The blood pressure of the spontaneously hypertensive and the normotensive rats at the beginning of the study was 150 ± 7 (SE) mm Hg and 110 ± 5 mm Hg, respectively. During the course of the study, the blood pressure of the SHRU group increased to 180 ± 9 mm Hg. The blood pressure of (4)

3 660 STRECKER, HUBBARD. MICHELAKIS Q. j w I40O i = NRT 5 = NRU i=nrt-p0b 5 =NRU-P0B o LOG NOREPINEPHRINE CONCENTRATION FIGURE 1 Norepinephrine dose-response curves obtained employing aortic strips from Wistar normotensive rats. NRT = aortic strips obtained from normotensiue rats that received antihypertensive therapy, NRU = aortic strips obtained from normotensive rats that did not receive antihypertensive therapy, NRT-P0B = aortic strips obtained from normotensive rats that received antihypertensive therapy and were exposed to phenoxybenzamine, and NRU-POB = aortic strips obtained from rats that did not receive antihypertensive therapy and were exposed to phenoxybenzamine. The points represent means ± SE (where possible without overlap) for 11 experiments. the NRU group increased to 135 dt 7 mm Hg. In contrast, the blood pressure of the SHRT group decreased to 113 ± 4 mm Hg and that of the NRT decreased to 109 ± 5 mm Hg during the first month of the 3-month treatment period; the blood pressures then remained at these levels throughout the rest of the treatment period. These results indicate that treatment of the rats was effective in controlling the hypertension. Thus, it was possible to determine K DR in spontaneously hypertensive rats to which effective antihypertensive therapy had been administered. The dose-response relationships between the norepinephrine concentration and the mechanical response of the aortic strips from the NRU, NRT, SHRU, and SHRT groups are shown in Figures 1 and 2. The data in these figures are plotted as the negative logarithm of the molar concentration of norepinephrine in the bathing medium vs. the contractile response of the muscle (expressed as milligrams of tension development). Comparison of the norepinephrine dose-response curves shown in Figure 1 and 2 reveals that the tension produced by the aortic muscles from spontaneously hypertensive rats was significantly less than that produced by the aortic muscles from normotensive rats (P < 0.001). This difference was evident for both treated and untreated spontaneously hypertensive and normotensive rats. In both cases, the magnitude of the response of aortic muscles from spontaneously hypertensive rats was approximately 50% of the response of aortic muscles from normotensive rats. However, the norepinephrine concentration required to induce a contraction equal to half of the maximum contractile response was not changed. These results indicate that an impairment of tension development in response to norepinephrine exists in muscle strips of spontaneously hypertensive rats relative to that in muscle strips of normotensive rats. This impairment persisted even when the high blood pressure was controlled. p 800 n 3 UJ 2; 600- o 5 400H Id (/) 8 j2 w i = SHRT 5 = SHRU i = SHRT-POB <j> = SHRU-POB -LOG NOREPINEPHRINE CONCENTRATION FIGURE 2 Norepinephrine dose-response curves obtained employing aortic strips from Wistar spontaneously hypertensive rats. SHRT = aortic strips obtained from spontaneously hypertensive rats that received antihypertensive therapy, SHRU = aortic strips obtained from spontaneously hypertensive rats that did not receive antihypertensive therapy, SHRT-POB = aortic strips obtained from spontaneously hypertensive rats that received antihypertensive therapy and were exposed to phenoxybenzamine, and SHRU-POB = aortic strips obtained from spontaneously hypertensive rats that did not receive antihypertensive therapy and were exposed to phenoxybenzamine. The points represent means ± SE (where possible without overlap) of eight experiments.

4 K DR IN NORMOTENSIVE AND HYPERTENSIVE RATS % 45' x 40 " B K D R= I.38XIO" 7 M A K DR = l.07xi0 ~7 M of K DR obtained for the aortic muscles from the NRT and SHRT groups were 1.38 x 10~ 7 M and 1.29 x 10" 7 M, respectively (Figs. 3 and 4). These values of K DH for the aortic muscles from normotensive and spontaneously hypertensive rats that had received antihypertensive therapy are not significantly different. Moreover, the values of K DR for aortic muscles from rats that had received antihypertensive therapy are not significantly different from those for aortic muscles from rats that had received no antihypertensive therapy. These results indicate that antihypertensive therapy does not alter the affinity of the adrenergic receptor for norepinephrine. Discussion A basic question regarding the functional properties of blood vessels from normotensive and hypertensive animals is whether they exhibit differences in responsiveness to excitatory stimuli. Furthermore, if they do exhibit such differences, what is I/D 1 (M)XIO 6 FIGURE 3 Double reciprocal plots of 1/\D\ vs. 1/[D' } from the norepinephrine dose-response curves in Figure 1. Line A was obtained by determining three norepinephrine concentrations that elicited equal responses of the aortic strips from untreated normotensive rats (//[ )]) and untreated normotensive rats exposed to phenoxybenzamine (I/[D']). Line B was obtained by determining three norepinephrine concentrations required to elicit equal responses of the aortic strips from treated normotensive rats (l/[d\) and treated normotensive rats exposed to phenoxybenzamine (1/[D'\). Each line was derived by linear regression analysis. By interpolation, determinations were made in Figures 1 and 2 of the norepinephrine concentrations required to induce contractile responses of the muscle after exposure to phenoxybenzamine ([D 1 ]) that were equal to the contractile responses of the muscle induced by norepinephrine prior to exposure to phenoxybenzamine ([D]). The values of K DR obtained for the aortic muscles from the NRU and SHRU groups were 1.07 x 10" 7 M and 1.17 x 10~ 7 M, respectively (Figs. 3 and 4). These values of K DR for the aortic muscles from normotensive and spontaneously hypertensive rats that received no antihypertensive therapy are not significantly different. These results indicate that the responsiveness of aortic muscles from normotensive and spontaneously hypertensive rats to norepinephrine is not due to a difference in the affinity of the adrenergic receptor for norepinephrine. The values to O X I/D(l *tv // I/ I/ t Q A K 0R = KQR = l.29xi0" 7 M I.I7XIO ~7 WI n l/d'(m)xi0 6 FIGURE 4 Double-reciprocal plots of l/[d] vs. l/[d']from the norepinephrine dose-response curves in Figure 2. Line A was obtained by determining three norepinephrine concentrations required to elicit equal responses of the aortic strips from untreated spontaneously hypertensive rats (//[ ))) and untreated spontaneously hypertensive rats exposed to phenoxybenzamine (// [D']). Line B was obtained by determining three norepinephrine concentrations required to elicit equal responses of the aortic strips from treated spontaneously hypertensive rats (1/[D]) and treated spontaneously hypertensive rats exposed to phenoxybenzamine (//[ )']). Each line was derived by linear regression analysis.

5 K DR IN NORMOTENSIVE AND HYPERTENSIVE RATS PATERSON LH: Physical factors which influence vascular caliber and blood flow. Circ Res 18(suppl I):I-3-13, OKAMOTO K, AOKI K: Development of a strain of spontaneously hypertensive rats. Jap Circ J 27: , FURCHGOTT RF: Spiral-cut strip of rabbit aorta in in vitro studies of response of arterial smooth muscle. Methods Med Res 8: , LINDQUIST EF: Design and Analysis of Experiments in Psychology and Education. Boston, Houghton Mifflin, POPHAM WJ: Educational Statistics. New York, Harper & Row, FURCHGOTT RF: Use of/3-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes. Adv Drug Res 3:21-55, FURCHGOTT RF: Pharmacological differentiation of adrenergic receptors. Ann NY Acad Sci 131: , FURCHGOTT RF, BURSZTYN P: Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors. Ann NY Acad Sci 144: , CLINESCHMIDT BV, GELLER RG, GOVIER WC, SJOERDSMA A: Reactivity to norepinephrine and nature of the alpha adrenergic receptor in vascular smooth muscle of a genetically hypertensive rat. Eur J Pharmacol 10:45-50, SPECTOR S, FLEISCH JH, MALING HM, BRODIE BB: Vascular smooth muscle reactivity in normotensive and hypertensive rats. Science 166: , SHIBATA S, KURAHASHI K, KUCHII M: Possible etiology of contractility impairment of vascular smooth muscle from spontaneously hypertensive rats. J Pharmacol Exp Ther 185: , PAGE TH, DUSTAN HP: Persistence of normal blood pressure after discontinuing treatment in hypertensive patients. Circulation 25: , THURM RH, SMITH WM: On resetting of "barostats" in hypertensive patients. JAMA 201: , FREIS ED, RAGAN D, PILLSBURY H: Alteration of the course of hypertension in the spontaneously hypertensive rat. Circ Res 31:1-7, 1972

6 K DR IN NORMOTENSIVE AND HYPERTENSIVE RATS PATERSON LH: Physical factors which influence vascular caliber and blood flow. Circ Res 18(suppl I):I-3-13, OKAMOTO K, AOKI K: Development of a strain of spontaneously hypertensive rats. Jap Circ J 27: , FURCHGOTT RF: Spiral-cut strip of rabbit aorta in in vitro studies of response of arterial smooth muscle. Methods Med Res 8: , LINDQUIST EF: Design and Analysis of Experiments in Psychology and Education. Boston, Houghton Mifflin, POPHAM WJ: Educational Statistics. New York, Harper & Row, FURCHGOTT RF: Use of/3-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes. Adv Drug Res 3:21-55, FURCHGOTT RF: Pharmacological differentiation of adrenergic receptors. Ann NY Acad Sci 131: , FURCHGOTT RF, BURSZTYN P: Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors. Ann NY Acad Sci 144: , CLINESCHMIDT BV, GELLER RG, GOVIER WC, SJOERDSMA A: Reactivity to norepinephrine and nature of the alpha adrenergic receptor in vascular smooth muscle of a genetically hypertensive rat. Eur J Pharmacol 10:45-50, SPECTOR S, FLEISCH JH, MALING HM, BRODIE BB: Vascular smooth muscle reactivity in normotensive and hypertensive rats. Science 166: , SHIBATA S, KURAHASHI K, KUCHII M: Possible etiology of contractility impairment of vascular smooth muscle from spontaneously hypertensive rats. J Pharmacol Exp Ther 185: , PAGE TH, DUSTAN HP: Persistence of normal blood pressure after discontinuing treatment in hypertensive patients. Circulation 25: , THURM RH, SMITH WM: On resetting of "barostats" in hypertensive patients. JAMA 201: , FREIS ED, RAGAN D, PILLSBURY H: Alteration of the course of hypertension in the spontaneously hypertensive rat. Circ Res 31:1-7, 1972