Sino-Aortic Denervation Causes Right Atrial Beta Adrenoceptor Down-Regulation 1

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 2 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 280: , 1997 Sino-Aortic Denervation Causes Right Atrial Beta Adrenoceptor Down-Regulation 1 ANGELINA ZANESCO, REGINA C. SPADARI-BRATFISCH and LOUIS A. BARKER Department of Physical Education, State University Paulista, Rio Claro (A.Z.) and Department of Physiology and Biophysics (A.Z., R.C., S-B.) and University of Campinas, Sao Paulo, Brazil and Department of Pharmacology (L.A.B.), LSUMC New Orleans, New Orleans, Louisianna Accepted for publication October 28, 1996 ABSTRACT Rat isolated right atria obtained 1 wk after sinoaortic denervation were less sensitive to the chronotropic actions of -agonists than were tissues obtained from animals that underwent sham surgery or no surgery at all. The potencies, but not the maximal responses for two high efficacy agonists, norepinephrine and isoproterenol, were reduced about 3- to 4-fold. Sinoaortic denervation (SAD) caused about a 3-fold decrease in potency and about a 60% decrease in maximal response for a low efficacy agonist, prenalterol. The changes in the actions of these agonists occurred in the absence of any changes in the subtype of beta receptor mediating the chronotropic response. The results of analyses of the data for prenalterol showed that SAD caused a decrease in the operational efficacy of this SAD disrupts baroreceptor mediated regulation of blood pressure and heart rate (Krieger, 1964; Krieger et al., 1980). Immediately after SAD there is an increase in sympathetic activity that is characterized by a labile hypertension and tachycardia. In the rat, the tachycardia is transient and usually returns to near control rates within 2 wk after SAD. In the conscious rat, the labile hypertension has been reported to persist for months (Vasquez and Krieger, 1980; 1982). However, if the animals are maintained in a quiet, nonstressful, environment, the hypertensive phase converts to a normotensive state within 20 days (Irigoyen, et al., 1995). The mechanisms for the reversal of the tachycardia and hypertension are not fully understood. Cardiac tissues contain both beta-1 and beta-2 adrenoceptors (Lands et al., 1967 and Carlson et al., 1972). In the rat, the chronotropic and inotropic responses to neuronally released and circulating catecholamines are mediated by the beta-1 subtype (Juberg et al., 1985). Generally, in response to Received for publication March 15, Supported by Conselho Nacional de Desenvolvimento Científico Tecnológico-CNPq, Grant /93 4 for participation in the Sandwich Program for superior graduate students. Work completed by A. Z. in partial fulfillment for the requirements for the Ph.D. degree from the State University of Campinas, UNICAMP, Campinas, S.P., Brasil. agonist without any changes in its K D value for beta-1 adrenoceptors. SAD had no effect on the responses of the tissue to blockade of uptake 1 and uptake 2, suggesting no compensatory changes in the removal processes caused the decreased potency. The results of radioligand binding assays showed that SAD caused a decrease in the maximal binding of 125 I-cyanopindolol without altering its K D. Also, the results of competition binding assays confirmed the lack of effect of SAD on the K D for prenalterol. The SAD-induced changes in the actions of agonists acting at right atrial beta-1 receptors were caused by a down-regulation of beta-1 adrenoceptors, which probably occurred in response to SAD-induced increases in sympathetic tone. an increased sympathetic tone, either as a result of a disease state or pharmacological intervention, a desensitization of or down-regulation of beta-1 adrenoceptors, but not beta-2 receptors, occurs in cardiac tissues (Brodde, 1987). However, not all situations that are known to produce an increased sympathetic tone result in a desensitization or down-regulation of cardiac beta-1 receptors. For example, a supersensitivity to ISO mediated by beta-2 adrenoceptors was seen in right atria obtained from rats after certain forms of stress (Callia and DeMoraes, 1984; Bassani and DeMoraes, 1988; Spadari and DeMoraes, 1988) or in an early stage of experimentally induced sepsis (Barker et al., 1990). Only a few studies have addressed the effects of SAD on cardiac beta adrenoceptors. Chloralose anesthetized rats showed a decreased responsiveness to the chronotropic actions of ISO at 5 hr after SAD, a time at which SAD-induced tachycardia was maximal (Vasquez and Krieger, 1982). This reduced sensitivity to ISO was also seen at 14 to 15 days after SAD, a time period at which the SAD-induced tachycardia had abated (Vasquez and Krieger, 1982). The results of further studies using an isolated heart preparation obtained at 5 hr and 14 days after SAD suggested a decrease in the potency and maximal chronotropic response to ISO administered as bolus injections ABBREVIATIONS: AR, adrenoceptor; EDTA, ethylenedaiminetetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid; ICYP, 125 I-cyanopindolol; ISO, isoproterenol; KHB, Krebs-Henseleit buffer; NE, norepinephrine; PREN, prenalterol; SAD, sino-aortic denervation. 677

2 678 Zanesco et al. Vol. 280 into the perfusion line (Cabral and Vasquez, 1984). The results of these studies suggested that SAD, as other models of hypertension, produced an uncoupling between receptor and G-protein and/or a down-regulation of beta adrenoceptors mediating chronotropy. In these studies, possible effects of SAD on the removal processes for catecholamines and/or changes in the subtype of beta adrenoceptor mediating the chronotropic response were not evaluated. Our studies were undertaken to determine the means by which SAD alters the actions of agents acting at right atrial beta receptors. Functional assays using isolated right atrial preparations were conducted to determine the effects of SAD on the actions of two high efficacy agonists, ISO and NE, and a low efficacy agonist, PREN. The advantages of using a low efficacy agonist, such as PREN, is that such agonists are much more affected by changes in receptor number and/or coupling than are high efficacy agonists and comparative functional assays of a low and a high efficacy agonist on the same tissue permit an estimation of the affinity and efficacy of the low efficacy agent (Kenakin, 1993; Leff et al., 1990). Additionally, the effects of SAD were evaluated on the neuronal and nonneuronal uptake processes; and on the active population of beta receptors mediating the chronotropic response. Radioligand binding assays were carried out to determine if agonist affinity and/or the number of right atrial beta receptor binding sites was altered after SAD. Methods Surgical procedures. In conducting this research, the authors adhered to National Institutes of Health guidelines for the use of animals. Male Wistar rats ( g) were used in all experiments. Three groups of animals were used: animals with SAD, animals with sham surgery and naive control animals. All surgical procedures used aseptic techniques and were carried out under anesthesia produced by ketamine, 50 mg/kg, i.m., and xylazine, 5 mg/kg, i.m. Bilateral SAD was performed as described by Vasquez and Krieger (1982). Briefly, after the induction of anesthesia, the external and internal branches of the carotid arteries were exposed. The vagas nerve, the sympathetic trunk and surrounding connective tissue were gently dissected away from the vessels, the superior laryngeal nerve was resected and a section of the sympathetic trunk removed. Sham surgery consisted of the same procedures used to expose and free the arteries, but without denervation. After SAD or sham surgery, cannulae containing sterile saline were placed in the left femoral vein and artery for the subsequent administration of drugs and recording blood pressure and heart rate. The vein and artery were cannulated using sterile PE-50 and PE-10 tubing, respectively. Cannulae were exteriorized at the dorsal neck region. Following surgery, all animals were treated with benzathine penicillin, 100,000 U, i.m., to minimize infections. Twenty-four hr after surgery, the effectiveness of SAD was determined by the administration of sodium nitroprusside, 4 g/kg, i.v., to conscious unrestrained rats. SAD was considered adequate if the animal responded with no increase in heart rate after a decrease in diastolic pressure of 30 to 50 mm Hg. Sham surgery was considered successful if the animals responded with a tachycardia following the depressor challenge. Only animals meeting the criterion for SAD or sham are included in our study. Blood pressure was recorded from the femoral artery using a Statham P23 1D pressure transducer (Grass Instruments, Quincy, MA) and heart rates were measured with a Grass EKG/tachygraph and displayed on a Grass model 7D polygraph. Functional assays using isolated right atria. A modification of the procedures described by Kenakin and Beek (1980) was used. Seven days after surgery, animals were anesthetized with halothane and euthanized by stunning and exsanguination. The hearts were rapidly removed and placed in oxygenated KHB. The right atria were removed and mounted in water jacketed tissue chamber (10 ml volume) containing KHB, ph 7.3 to 7.5, at 34 C and gassed with 95% O 2-5% CO 2. The composition of the KHB was (millimolar): NaCl, 124; KCl, 4.75; MgSO 4, 1.30; CaCl 2, 2.25; NaHCO 3, 25.0; NaH 2 PO 4, 0.6; dextrose, 10.0; sodium ascorbate, 0.3; disodium EDTA, 0.03 and 17 -hydroxyestradiol, Ascorbate and EDTA were added to inhibit the oxidation of catecholamines (Hughes and Smith, 1978). 17 -Hydroxyestradiol was added to block the extraneuronal uptake of catecholamines (Salt, 1972). The neuronal uptake of catecholamines was inhibited by treatment with 10 M phenoxybenzamine for 30 min (O Donnell and Wanstall, 1985). After treatment with phenoxybenzamine, the bathing solution was changed by overflow washes every 15 min for 1 hr to remove unreacted phenoxybenzamine and allow a stable basal rate to develop. Construction and analyses of concentration-response curves. Concentration-response curves for the positive chronotropic actions of ISO, NE and PREN were constructed by the cumulative variation of agonist concentration at one-half log unit increments (Van Rossum, 1963). In those experiments where PREN was used, concentration-response curves for both PREN and ISO were generated on the same tissue. The change in atrial rate was used as the response metameter. Curves were constructed after the treatment with and removal of phenoxybenzamine when stable basal rates were established. In one series of experiments, the effects of SAD on catecholamine uptake mechanisms were evaluated by generating concentrationresponse curves in the absence of uptake blockade and then in the presence of estradiol and after treatment with phenoxybenzamine. The possible effects of SAD on changes in the subtype of -adrenoceptor mediating the chronotropic response were evaluated by constructing concentration-response curves for ISO or NE in the absence and then in the presence of either ICI118,551, a selective beta-2 antagonist (O Donnell and Wanstall, 1980) or CGP A, a selective beta-1 antagonist (Dooley et al., 1986). All concentration-response data were evaluated for a fit to a logistics function in the form: E E max /(1 (10 c /10 x ) n ) (1) where E is the increase in rate above basal; E max is the maximum response that the agonist can produce; c is the logarithm of the EC 50, the concentration of agonist that produces half-maximal response; x is the logarithm of the concentration of agonist; the exponential term, n, is a curve fitting parameter that defines the slope of the concentration-response line, and is the response observed in the absence of added agonist. Nonlinear regression analyses to determine the parameters E max, log EC 50 and n were done using Graph- Pad Prism (GraphPad Software, San Diego, CA) with the constraint that zero. In the text and tables, the potency parameter, log EC 50, is given as the pec 50,-1 (log EC 50 ). The dissociation constant, K D, for the partial agonist, PREN, was estimated by the method of Black et al. (1985). Initially the data sets for PREN and ISO were analyzed as described above. The responses for the individual data sets for PREN were normalized to the maximum response estimated for ISO for each tissue. The transformed data were then fit to the expression: E E max n [10 x ] n /((10 K [10 x ]) n n [10 x ] n ) (2) where E is the fractional response relative to that produced by ISO; E max is the relative maximal tissue response which was constrained to unity; K is the logarithm of the molar equilibrium dissociation constant; is the model definition for efficacy; and n and x are as defined above. The term is defined as the ratio, [R T ]/K E, where [R T ] is the concentration of active receptors and K E is the concentration of agonist-receptor complex producing one-half of the agonist maximal

3 1997 Sino-Aortic Denervation and Beta AR 679 response. The value of n for each data set was constrained to that value obtained in the fitting to equation 1. This method for estimating the K D for an agonist is limited to cases where the agonist maximal response is measurably less than the tissue maximum response (Leff et al., 1990). Two of the data sets for PREN obtained from control tissues were not used in this analysis because they had E max values that were not different from those for ISO on the same tissues. The dissociation constants are reported in the text and tables as pk D values, -1 (log K D ). In the experiments in which competitive antagonists were used to characterize the active population of beta adrenoceptors, concentration-response curves for agonists were constructed in the absence of antagonist and in the presence of antagonist after a 2-hr equilibration period. In all of these studies, only a single concentration of antagonist was used on a single atrium. Concentration-response data were analyzed as described above. Concentrations of agonist producing half-maximal response in the absence, [A], and the presence, [A ], of antagonist were estimated for use in the Schild equation (Arunlakshana and Schild, 1959): log CR 1 n log B log K B (3) where CR is [A ]/[A]; n is slope; [B] is the concentration of antagonist and all other terms are as defined above. The apparent molar equilibrium dissociation constant for the interaction of the antagonist with the receptor, K B, was determined from a linear regression of log (CR -1) on log [B]. The dissociation constants are reported in the text and tables as pk B values, -1 (log K B ). Full Schild analyses were carried out for ISO and NE on tissues obtained from the naive control group. In studies using atria from the SAD and SHAM groups, only single concentrations of ICI118,551 and CGP20712A were used. The concentrations used, 10 nm CGP20712A and 50 nm ICI118,551, were ones that interact with the receptor for which they have the highest affinity and have only minimal interactions at the other site. A prediction of the expected behavior of the concentration of antagonists used was obtained by simulations using the model of Lemoine and Kaumann (1983) for antagonism at two receptors for a common response mediated by a single agonist: log CR 1 log B log 1 K B1 2 K B2 B K B1 K B2 }/ B 2 K B1 1 K B2 where CR is as defined above, sigma-1 and sigma-2 are fractional stimuli of agonist effect mediated through beta-1 and beta-2 adrenoceptors, and K B1 and K B2 are the equilibrium dissociation constants for the interaction of the antagonist with beta-1 and fbeta-2 adrenoceptors. In the simulations the values of sigma for each site were varied between 0 and 1. The pk B values used were those obtained in this laboratory and literature values. Those used for CGP A, were 9.4 (this study) and 5.5 for beta-1 and beta-2 receptors, respectively ((Dooley et al., 1986; Hall et al., 1990). For ICI118,551 the respective values were 7.0 and 9.4 (Bilski et al., 1983; Barker et al, 1990; this study). ICYP binding studies. Right atria were obtained as described above. For each experiment, right atria from three rats were pooled. Membranes were prepared by a modification of the procedure described by Juberg et al. (1985). The right atria were rapidly removed and homogenized with a Tissumizer for 30 sec in 10 ml of 20 mm of NaPO 4 (ph 7.6) containing 154 mm NaCl. The homogenate was centrifuged at 100,000 g for 1 hr at 4 C to isolate all particulate bound receptors (Maisel et al., 1985). The supernatant was discarded and the pellet was resuspended in 1 ml of 0.32 mm sucrose per ten mg wet weight tissue, and stored at 70 C until use. This preparation was used to permit an evaluation of the effects of SAD on the total number of beta adrenoceptor binding sites that would not be influenced by changes produced only by a redistribution between sarcolemal and light vesicular bound receptors. (4) Aliquots of the membrane suspension (100 l containing g protein) were incubated in triplicate at 37 C for 90 min with varying concentrations of ICYP (5 300 pm) or a single concentration of ICYP (100 pm) in the presence of varying concentrations of PREN ( M) in a final volume of 250 l of a buffer containing: HEPES, 50 mm, ph 7.5; MgCl 2, 4 mm; sodium ascorbate, 0.3 mm; and ED- TA(Na) 2, 0.03 mm. Triplicate samples containing propranolol (10 M) were used to define nonspecific binding. Incubations were terminated by adding 10 ml of 50 mm HEPES buffer at 4 C, followed by rapid filtration through glass fiber filters (GF/B Whatman, Clifton, NJ) and a wash with 10 ml of the HEPES incubation buffer. Immediately after filtration, the radioactivity was measured with a Gamma counter (Beckman Instruments, Fullerton, CA, model 9800). Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The apparent number of binding sites and affinity for ICYP were determined by nonlinear regression analyses using the equation (Kenakin, 1993; Klotz, 1982): B B max / 1 10 K /10 x n (5) where B is the amount of ligand bound expressed as fmol/mg protein; B max is the maximal number of sites expressed as fmol/mg protein; K is the logarithm of the molar equilibrium dissociation constant, K D, of the radioligand; x is the logarithm of the concentration of free ligand; and n is the Hill coefficient for the binding of the ligand. The IC 50 value for PREN was determined from a fit to equation 1. The apparent K D for PREN was calculated from its IC 50 value for inhibiting ICYP binding using the expression (Leff and Dougall, 1993): K d(pren) IC 50 / 2 ICYP /K d(icyp) n 1/n 1 (6) where n is the slope factor for the line describing the inhibition of ICYP binding by PREN. Statistical analyses. The program InStat (GraphPad Software, San Diego, CA) was used for statistical analyses. Where appropriate, one-way analyses of variance followed by a Bonferroni multiple comparisons post hoc test were performed to determine if the treatments had an effect. In some cases, a paired Student s t test was used. P.05 was accepted as significant. Drugs. (-)-NE, (-)-ISO and 17 -hydroxyestradiol were obtained from Sigma Chemical Co. (St. Louis, MO). The following compounds were kindly provided as gifts: PREN (Dr. Terry Kenakin), phenoxybenzamine (Dr. Norman Robie), ICI118,551 (ICI, Ltd), and CGP 20712A (Ciba Geigy Pharmaceuticals). 125 I-Cyanopindolol, 2000 Ci/ mmol, was obtained from Amersham Life Science Inc. (Arlington Heights, IL) Results Effect of SAD on uptake mechanisms. The results of the studies on the combined effect of neuronal and nonneuronal uptake blockade are given in table 1. The potentiation of NE after the blockade of neuronal and nonneuronal uptake systems was similar in atria obtained from all of the treatment groups. The left shifts in the concentration-response curves for NE on tissues from the naive control, sham and SAD were not significantly different from each other (oneway ANOVA, F 2.6, P.05). Concentration-response curves for ISO were also significantly (P.05, paired Students t-test) shifted to the left after uptake blockade. The shifts were less than those observed for NE. The leftward shifts for concentration-response curves for ISO on tissues from the naive control, sham and SAD groups were not significantly different from each other (one-way analysis of variance, F 2.0, P.05). All subse-

4 680 Zanesco et al. Vol. 280 TABLE 1 Effect of combined blockade of the neuronal and non-neuronal uptake systems on the potency of ISO and NE at right atrial beta-receptors mediating chronotropy Agonist/Treatment Group Preblockade pec 50 Shift N Postblockade a ISO Naive Sham SAD NE Naive Sham SAD a All postblockade pec 50 values are significantly different from their respective preblockade controls, P.05, Student s paired t test. Data are mean S.E. for N experiments. quent studies reported below were obtained under conditions in which the neuronal and nonneuronal uptake systems were blocked. Effects of SAD on the chronotropic actions of ISO, NE and PREN at right atrial beta adrenoceptors. The concentration-response parameters for ISO, NE and PREN on isolated right atria obtained from control animals and from animals 7 days after sham surgery or SAD are summarized in table 2. The basal rates for atria were (47), (28) and (28) for the naive control, SHAM and SAD groups, respectively. The rates for the SHAM and SAD groups were significantly different from that for the naive control and that for the SAD group was significantly different from that for the SHAM group (analysis of variance, F 65, P.05). Despite the differences in basal rates, there were no differences between the E max values for the chronotropic actions of ISO and NE on atria from any of the three treatment groups. The E max values for the chronotropic actions of PREN were significantly less than those for ISO and NE on atria from all treatment groups (analysis of variance, F 21.0, P.05). When compared to both control groups, the pec 50 values for all three agonists were lower on atria obtained from rats 7 days after SAD (P.05). For ISO, NE and PREN, SAD was associated with a 2.5-, 4.5- and 2.6-fold reduction in the EC 50, respectively. SAD had no effect on the E max values for the full agonists, ISO and NE, but did cause a decrease in the absolute and relative (as compared to ISO) E max values for the partial agonist, PREN. SAD had no effect on the slope parameter for any of the agonists (P.05). The concentrationresponse curves for the actions of ISO and PREN are shown in figure 1 and those for the actions of NE are shown in figure 2. Effects of SAD on the apparent affinity and efficacy of PREN at right atrial beta adrenoceptors. The effects of SAD on the K D value and efficacy of PREN are summarized in table 3. The apparent K D values for PREN determined by the functional and competition binding assays were not significantly different. In the competition binding assays the pic 50 values (calculated pk D values in parenthesis) and slope parameters for PREN were ( ) and , ( ) and and ( ) and , respectively, for the naive, sham and SAD groups. SAD had no significant effects on the pic 50 values, calculated pk D values or the slope parameters. These data show that SAD had no effect on the apparent K D value of PREN for beta adrenoceptors as determined by either functional or radioligand binding assays. The results of the functional assays showed that SAD produced a decrease in the efficacy of PREN. Characterization of the active population of beta adrenoceptors mediating chronotropy. In the initial studies, Schild analyses were done to determine pk B values for the antagonism of ISO and NE by CGP 20712A and ICI 118,551 on atria from naive control animals. The concentration-response curves for the antagonism of ISO by CGP 20712A and ICI 118,551 are shown in figures 3 and 4. Similar results for each antagonist were obtained for the antagonism of NE (data not shown). The slopes of the initial regression analyses were not significantly different from unity. Accordingly, subsequent estimates for the pk B values were obtained with the slopes constrained to unity (Kenakin, 1993). These data are shown in table 4. The pk B values for the antagonism of ISO or NE by CGP 20712A were not significantly different (P.05, unpaired Student s t test). The pk B values obtained from a regression of the combined TABLE 2 Concentration-response parameters for the chronotropic actions of ISO, NE and PREN on isolated right atria Agent/Treatment pec 50 E max R max Slope Parameter N beats min 1 ISO Naive Sham SAD a NE Naive Sham SAD a PREN Naive c,d Sham c,d SAD b a,c,d a Values are mean S.E. for N experiments. R max is the E max of PREN relative to that of ISO. All data were obtained under conditions in which neuronal and non-neuronal uptake were inhibited. a Significantly different from both control groups, P.05. b Significantly different from naive control, P.05, but not sham control. c Significantly different from the corresponding E max values for ISO determined on the same preparation, paired Student s t test, P.05. d Significantly different from population mean E max values for ISO and NE in all treatment groups, P.05.

5 1997 Sino-Aortic Denervation and Beta AR 681 Fig. 1. Concentration-response curves for the chronotropic actions of ISO and PREN on isolated right atria obtained from control (A), sham (B) and SAD (C) treatment groups. Each point is the mean S.E. for six to eight experiments in which curves for ISO and PREN were generated on the same tissue. The assays were conducted under conditions of uptake blockade. The y axis is increase in atrial rate in beats min 1 to provide a direct comparison of the effects of SAD on the actions of ISO and PREN. data for the antagonism of ISO and NE by were and for CGP 20712A and ICI 118,551, respectively. The regressions for the combined data are shown in figures 5, A and B. These results were consistent with antagonism at a single population of active receptors. The next series of experiments were carried out on tissues obtained after sham or SAD to determine if the treatment altered the active population of beta adrenoceptors mediating chronotropy. In these experiments, ISO was used as the agonist and only a single concentration of CGP 20712A or ICI 118,551 was used. ISO was selected as the agonist because it acts equally well at both beta-1 and beta-2 adrenoceptors. The concentrations of the antagonists used were those that would block the beta adrenoceptor subtype for which the antagonist is selective and have little to no measurable effect at the other site, i.e., less than or about equal to 1 times K B for the least sensitive sight. For CGP 20712A a concentration of 10 nm was used to selectively block beta-1 receptors. For ICI 118,551 a concentration of 50 nm was used to block beta-2 Fig. 2. Concentration-response curves for the chronotropic actions of NE on isolated right atria obtained from control, sham and SAD treatment groups. Each point is the mean S.E. for six experiments. The assays were conducted under conditions of uptake blockade. The y axis is fractional response. TABLE 3 Effects of SAD on the affinity and efficacy ( ) of Prenalterol at right atrial beta adrenoceptors Treatment Group Binding Assay a pk d Functional Assay Naive SHAM SAD b 6 Values are mean S.E. for N experiments. In the functional assays, PREN was always compared to ISO on the same tissue under conditions of uptake inhibition. a Calculated from IC 50 values for the inhibition of ICYP binding. b Significantly different from naive and sham, P.05. Fig. 3. Antagonism of the chronotropic actions of ISO by CGP A. A total of 14 experiments were carried out. The control data are the mean S.E. for 14 replications. For curves generated in presence of antagonist, each point is the mean S.E. for three to four repetitions of each concentration of antagonist. The assays were conducted under conditions of uptake blockade. The y axis is fractional response. receptors. In tissues obtained 7 days after sham and SAD surgery, the apparent pk B values for CGP 20712A were (n 6) and (n 6), respectively. These data are shown in figure 6. ICI 118,551 at a concentration of 50 nm did not produce a significant change (P.05, paired Student s t test) in the pec 50 values for the action of ISO on atria from either group of animals. The pec 50 values for ISO obtained in the absence and in the presence of ICI 118,551 were and (n 5) for atria obtained from rats 7 days after sham surgery. The corresponding values for tissues obtained from rats 7 days after SAD were and (n 5). pk d N

6 682 Zanesco et al. Vol. 280 Fig. 4. Antagonism of the chronotropic actions of ISO by ICI 118,551. A total of 11 experiments were carried out. The control data are the mean S.E. for 11 replications. For curves generated in presence of antagonist, each point is the mean S.E. for three or mean range for two (ICI 1E-5) repetitions of each concentration of antagonist. The assays were conducted under conditions of uptake blockade. The y axis is fractional response. Effects of SAD on ICYP binding. The isotherms for ICYP-specific binding to right atrial beta receptors are shown in figures 7, A and B. The specific binding, as a percentage of total binding, ranged from % (15) for 5 pm ICYP to % (15) for 300 pm ICYP. The parameters for ICYP binding are given in table 5. SAD had no effect on the affinity of ICYP for beta adrenoceptors binding sites. Relative to both control groups, SAD caused about a 40% decrease in the number of binding sites as expressed per mg tissue and about 30% decrease in binding sites as expressed per mg protein. The B max for ICYP per gram tissue in atria from SAD group was significantly different from both control groups (P.05). The B max for ICYP per mg protein in tissues from SAD group was significantly different from the sham group, P.05, but not from the naive control group. Discussion Our results confirm and extend the findings made by Cabral and Vasquez (1984) where it was shown that the potency of ISO for producing a positive chronotropic response is reduced after SAD. Our results show that at 1 wk after SAD, independently of uptake blockade, there were decreases in the potencies for the chronotropic actions of ISO acting on isolated right atria. In contrast to the findings made by Cabral and Vasquez (1984), it was observed that the changes in the potency of ISO occurred in the absence of changes in its maximal responses. Because Cabral and Vasquez (1984) had similar observations on hearts taken from animals at 5 hr and 15 days after SAD, it is not likely that the use of tissues Fig. 5. Schild regressions for CGP 20712A (A) and ICI 118,551 (B) antagonism of ISO and NE at beta receptors in right atria obtained from control rats. obtained at different times after SAD is the basis for the differences in the effects seen on the maximum response to ISO. This difference may be due to the preparations used to study the effects of SAD on the chronotropic actions of ISO. Cabral and Vasquez (1984) measured ventricular rates in a perfused isolated heart preparation and administered ISO, over a relatively narrow dose range, as a bolus injection into the perfusate. This procedure would provide only a transient exposure to ISO and a true maximum may not have been observed because of the limited dose range used. In our study, isolated right atria suspended in tissue chambers were used and ISO was added cumulatively to the bath and not removed until the full concentration-response curve had been developed. Also, an effect of SAD on conduction that would cause a dissociation between the rates of atrial and ventricular beating may have contributed to differences in the results. Our study also shows that SAD resulted in the decreased potency, but not maximal response of another high efficacy beta agonist, NE. The potency and the maximal response for the chronotropic actions of the low efficacy beta agonist, PREN, were reduced 1 wk after SAD. The K D value of PREN for right atrial beta receptors was estimated by functional and radioligand binding assays. The effects of SAD on the actions of PREN were associated with a decrease in its efficacy for agonism at atrial beta adrenoceptors. No effects of SAD on the K D value of PREN for beta adrenoceptors were observed. The estimates of the pk D for PREN obtained from the functional and binding assays were not significantly different. Both assays gave similar results, about 6.4 from binding assays and about 6.7 from the functional assays. The pk D values for PREN obtained in the binding assays are in good agreement with those found by Brodde et al. (1984) and Hedberg and Mattsson (1981), 6.2 and 6.4, respectively. The TABLE 4 Estimates of the pk B values for the antagonism of ISO and NE by CGP 20712A and ICI 118,551 at -receptors in right atria from control rats Antagonist Agonist CGP 20712A ICI 118,551 pk B slope N pk B N slope ISO NE Values are mean S.E. N is the number of data points. The pk B estimates were obtained with the slope constrained to unity. The slope values given are those obtained in the initial regression analyses.

7 1997 Sino-Aortic Denervation and Beta AR 683 Fig. 6. Antagonism of the chronotropic actions of ISO by CGP 20712A on tissues obtained from sham (A) and SAD (B) treatment groups. Each point is the mean S.E. for six experiments. The concentration of CGP 20712A used was one that selectively blocks beta-1 adrenoceptors and has no measurable effect on beta-2 adrenoceptors. The assays were conducted under conditions of uptake blockade. The y axis is fractional response. estimates obtained from functional assays were also similar to those, 6.4 and 6.6, obtained by Hedberg and Mattsson (1981). However, our estimates of the pk D value of PREN obtained from the functional assays are lower than those reported by Kenakin and Beek (1980; 1984) and Kenakin and Ferris (1983) whose functional assays yielded pk D values of 7.1 to 7.5. The basis for theses differences in the estimation of the apparent pk D value of PREN for beta adrenoceptors are not known. The results of the studies with selective antagonists showed that the changes in the potency were not related to changes in the beta receptor subtype mediating the response. Based on the results of simulations of Schild-plots for the behavior of an antagonist acting at two receptor subtypes (Lemoine and Kaumann, 1983), it was predicted that if as little as 5 to 10% of the fractional stimuli were mediated by beta-2 receptors in atria, the concentration-response curve for ISO in the presence of 10 nm CGP20712 A would have been shifted to right about 10-fold as opposed to about a 30-fold shift if only beta-1 receptors were involved. For 50 nm ICI118,551, it was predicted that the curve for ISO would have been shifted to the right by about 3- to 4-fold if the fractional stimuli by beta-2 receptors was 0.5 and a shift of less than 2-fold, if it was 0.1 or less. CGP20712 A, at 10 nm which is about thirty times its K B value for beta-1 adrenoceptors and about 10,000-fold less than its K B value for beta-2 receptors (Dooley et al., 1986; Hall et al., 1990), shifted the concentration-response curve for the chronotropic actions of Fig. 7. Saturation binding of ICYP to beta adrenoceptors present the total particulate fraction of right atrial homogenates obtained from control, sham and SAD treatment groups. In both figures the abscissa is the logarithm of the molar concentration of ICYP. In A, the ordinate is expressed as fmol/mg protein and in B, as nmol/g wet weight tissue. Each data point is the mean S.E. for four to five experiments. TABLE 5 Effect of SAD on 125 I-cyanopindolol binding Treatment Group B max pk d n nmol/g tissue fmol/mg protein Naive Sham SAD a b Values are mean S.E. for 4 to 5 determinations. a Significantly different from naive and sham, P.05. b Significantly different from sham, P.05. ISO on atria from both the SAD and SHAM treatment groups to the right by a factor of about 30. The estimates of the pk B for CGP20712 A were 9.47 and 9.55 for tissues obtained from animals after sham surgery or SAD, respectively. These estimates obtained from a single concentration of CGP20112 A were not significantly different from each other or from the pk B value, 9.36, obtained from a Schild analysis for its actions on tissues from naive control rats. The estimated affinity for CGP A agrees with that reported by others (Dooley et al., 1986; Hall et al., 1990) for its interaction with beta-1 receptors. ICI118,551, at 50 nm which is 30 to 100 times its K B value for beta-2 adrenoceptors and slightly less than its K B value for beta-1 receptors (Bilski et al., 1983; Barker et al, 1990), produced dextral shifts of about 1.5-fold in the concentrationresponse curves for the chronotropic actions of ISO on atria

8 684 Zanesco et al. Vol. 280 from both the SAD and SHAM treatment groups. In both cases, the shifts were not significant. Collectively, these results show that the two antagonists behaved the same on tissues obtained from the SAD and SHAM treatment groups and in a manner not different from that seen on atria obtained from naive control animals. These results indicate that agonism at beta-1 adrenoceptors produced 90% or more of the fractional stimuli for the chronotropic response in atria obtained from animals that had undergone SAD. The results of the binding assays showed that SAD had no effect on the affinity of ICYP for beta receptor binding sites. The B max value for ICYP binding expressed either on the basis of wet weight or protein was decreased in atria obtained from animals after SAD as compared to that for atria obtained after sham surgery. A statistical difference between the values for the SAD and naive treatment groups was seen only when the comparison was made on binding sites per gram tissue wet weight. Because the preparation used for the binding studies contained both sarcolemal membrane bound and internalized light vesicular bound receptors (Maisel et al., 1985) possible effects of SAD on the redistribution of sarcolemal bound and light vesicular bound binding sites were not be determined. The changes in the agonist actions of ISO, NE and PREN reported here occurred at a time after SAD in which sympathetic tone is still elevated as evidenced by increased heart rates, mean arterial pressure and plasma levels of catecholamines (Alexander et al., 1980; Vassalo et al., 1991). The effects of chronic exposure to an agonist to cause either an uncoupling between the receptor and effector proteins and/or a down-regulation of its receptor are well documented (Stiles et al., 1984). For example, the decreased sensitivity to ISO seen in tissues from spontaneously hypertensive rats appears to be due to a down-regulation of cardiac beta adrenoceptors in conjunction with an increase in the G protein, Gi, which can mediate an in inhibition of adenylyl-cyclase (Böhm et al., 1994). Classical drug-receptor theory predicts that some decreases in the population of active receptors, by either an uncoupling or a down-regulation, can produce decreases in potency of high efficacy agonists without change in their maximal response. However, the decreases in the population of active receptors that have no measurable effect on the maximal responses for high efficacy agonists can cause a decrease in the potency and in the maximal response of a low efficacy agonist (Kenakin, 1993). The effects of SAD on the concentration-response curves for ISO and PREN reported here are strikingly similar to those reported by Kenakin and Ferris (1983) for the agonist actions of ISO and PREN on rat left atria after a 4-day in vivo treatment with ISO. Kenakin and Ferris (1983) found that the treatment with ISO produced a down-regulation of beta receptors that was associated with a decreased potency of ISO and a decreased potency and maximal response for PREN. Our results show that the effects of SAD on the actions of the high efficacy agonists, ISO and NE, as well as those on the low efficacy agonist, PREN, can be explained largely by a down-regulation of active beta-1 receptors that mediate the chronotropic response. Further study is required to determine if changes in G proteins also contribute to this. In summary, these results suggest that in the rat SAD produces a down-regulation of right atrial beta-1 adrenoceptors. This can explain the decreased sensitivity for chronotropic actions of sympathomimetic amines that have been observed both in vivo and in in vitro and may be, in part, a basis for the transient nature of the tachycardia. Compensatory changes in other reflexes, such as the Bezold-Jarish reflex which is exaggerated after SAD (Chianca and Machado, 1994), may also contribute to the transient nature of the tachycardia that is seen in the whole animal. Also, the effects of surgery, per se, as well as SAD on basal rates, suggest that other mechanisms are involved in the adaptation. In view of the observations which show that chronic beta receptor activation is associated with an up-regulation of cardiac muscarinic receptors (Nomura et al., 1982) and that chronic beta blockade not only increases responsiveness of atria to beta agonists, but also to agents acting at atrial 5-HT 4 and histamine H 1 - and H 2 -receptors (Saunders et al., 1994; 1996), the possible effects of SAD on other receptors involved in regulating cardiac function merit investigation. Acknowledgments The authors thank Drs. Dennis Paul and Renee Bergeron and Ms. Lerna Minor for technical assistance in conducting the radioligand binding assays. The helpful comments and suggestions made by Drs. A. M. Cabral, D. Vassalo and E. C. Vasquez of the Federal University of Espirito Santo, Vitória, BR, and Dr. K. Varner of LSUMC are appreciated. Special thanks are extended to Professor E. M. Krieger of the Heart Institute, São Paulo, BR for initially suggesting this research. 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