Bromoacetylalprenololmenthane Research Articles

Responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on aortae from normo-, pre- and hypertensive rats.

The objective of this study was to determine the responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on the isolated aorta in the maturation of normotensive and hypertensive rats. The effects of a very slowly reversible antagonist, bromoacetylal-prenololmenthane (BAAM), on the relaxant responses of the aortae of 5- and 14-week-old Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) to isoprenaline were determined. Five-week-old SHRs are pre-hypertensive and the aortic rings are less responsive to isoprenalinethan age-matched WKY (pD2 values: WKY, 8.40; SHRs, 8.03). Similar relaxant responses to forskol in were obtained on the aortae of 5- and 14-week-old WKY and SHRs. The K(A) value for isoprenaline at the aortic beta2-adrenoceptors of the 5-week-old WKY was 2.1 x 10(-7) M, and similar values were obtained on the aortae of 5-week-old SHR and 14-week-old WKY and SHRs. In the maturation of the WKY aortae from 5 to 14 weeks, there was a reduction in the maximum response, a major loss of sensitivity and a loss of beta2-adrenoceptor reserve for isoprenaline. On 5-week-old SHR aorta, the sensitivity to isoprenaline was 2.5-fold lower, and the beta2-adrenoceptor reserve was less than on age-matched WKY. In the development of hypertension on the SHR aorta from 5 to 14 weeks, there was a reduction in the maximum response to isoprenaline. At 14 weeks, the sensitivity and the beta-adrenoceptor reserve to isoprenaline were similar, but the maximum responses were lower on the SHR than WKY. As there are differences in pre-hypertensive SHR and age-matched WKY aortic responses to isoprenaline, it is no longer valid to consider that the loss of responsiveness to isoprenaline in hypertension is solely owing to the hypertension. There are no changes in affinity, but major changes in the sensitivity, maximum responses and aortic beta2-adrenoceptor reserves to isoprenaline in the maturation of normotensive and pre-hypertensive aortae.

β-Adrenoceptor reserve in human lung: a comparison between airway smooth muscle and mast cells

The effects of several β-adrenoceptor agonists on the relaxation of precontracted human bronchial rings and the inhibition of IgE-mediated histamine release from human lung mast cells (HLMC) were studied. For the relaxation of bronchial rings, isoprenaline, fenoterol and terbutaline were full agonists whereas salbutamol was a full agonist in some (two out of six) experiments and a partial agonist in the remainder. For the inhibition of histamine release, relative to isoprenaline, neither fenoterol, terbutaline nor salbutamol was a full agonist. Studies with the irreversible β-adrenoceptor antagonist, bromoacetylalprenolol menthane, indicated that there was a larger β-adrenoceptor reserve for the relaxation of precontracted bronchial rings than for the inhibition of histamine release from HLMC. Further studies indicated that the isoprenaline inhibition of histamine release was considerably more susceptible to desensitizing treatments than the isoprenaline relaxation of bronchial rings. Collectively, these data suggest that a larger β-adrenoceptor reserve exists for the relaxation of smooth muscle than the inhibition of histamine release from HLMC and that differences in receptor reserve may contribute to the relative susceptibilities of the two systems to desensitization.

Affinity constants and beta-adrenoceptor reserves for isoprenaline on cardiac tissue from normotensive and hypertensive rats.

To determine whether there are differences in cardiac beta-adrenoceptor responsiveness, isoprenaline affinity constants and fractional beta-adrenoceptor occupancy-response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-beta-(2-hydroxyl-3-alpha-naphthoxypropy lamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible beta-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD2 values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline KA values were 2-3 x 10(-6) M, which is as expected for isoprenaline at beta1 or beta2-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline KA values were 2-4 x 10(-8) M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3-4% of the beta-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25-35% and 55%, respectively, of the beta-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller beta-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline KA values on the WKY right atrium. The isoprenaline KA value on the SHR right atrium was 1-4 x 10(-8) M. Because the isoprenaline KA values for the left and right atria are markedly different from those previously reported for isoprenaline at beta1 or beta2-adrenoceptors, we suggest that atypical beta-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower beta-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.

The effects of bromoacetylalprenololmenthane on the responses to isoprenaline of the left ventricle from normo-, prehyper- and hypertensive rats.

1. We have studied the effects of bromoacetylalprenololmenthane (BAAM), a very slowly reversible beta-adrenoceptor antagonist, on the responses of the left ventricle of 5 and 22 week old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) to isoprenaline. At 5 weeks the SHRs were prehypertensive and at 22 weeks they had hypertension-induced hypertrophy of the left ventricle. 2. The mean potency of isoprenaline on the left ventricle from 5 week old WKY was similar to that obtained on left ventricle with BAAM at 10(-6) M for 30 min, there was a parallel rightward shift of the isoprenaline concentration-response curves. Treatment with a higher concentration of BAAM (10(-5)M) caused non-parallel rightward shifts of the concentration-response curves, with a depression of the isoprenaline maximum responses. These data were used to derive affinity (KA) values for isoprenaline. The mean isoprenaline KA value on the left ventricle from 5 week old WKY was 2.44 x 10(-6)M, and similar KA values were obtained on the left ventricles from 22 week old WKY and 5 and 22 week old SHRs. On all the left ventricles tested, isoprenaline produced a half maximal response by occupying less than 1%, and a near maximal response by occupying about 5% of the available beta(1)-adrenoceptors. 4. This study has shown that there are no differences in the cardiac responses to isoprenaline at beta(1)-adrenoceptors, isoprenaline KA values or the beta(1)-adrenoceptor reserve for isoprenaline on the SHR left ventricle in prehypertension or in the early stages of hypertension-induced hypertrophy.

Glaucoma, capillaries and pericytes. 4. Beta-adrenergic activation of cultured retinal pericytes.

To characterize the relaxation of pericytes induced by beta-adrenergic stimulation, changes in the contractile tone of pericytes were quantified as a change in the wrinkling of an elastic silicone surface on which they were cultured. Isoproterenol produced relaxation of pericytes in a dose-dependent manner over a range of 5 nM to 1 microM. Low concentrations of the nonselective beta-blockers propanolol and timolol blocked the relaxation produced by isoproterenol. The specific beta 2-adrenergic component of isoproterenol-induced relaxation was shown by blockage with bromoacetyl alprenolol menthane. In contrast, atenolol and betaxolol, as relatively selective beta 1-adrenergic blockers, had no effect on the isoproterenol-induced relaxation.

Loss of maximum attenuation and receptor reserve for isoprenaline at the beta 2-adrenoceptors of the portal veins of hypertensive rats.

To test the hypothesis that vascular beta 2-adrenoceptor hyporesponsiveness in spontaneously hypertensive rats (SHR) is not induced by increased blood pressure or venous hypertrophy. We compared the attenuating effects or isoprenaline, sodium nitroprusside and verapamilon the portal veins from Wistar-Kyoto (WKY) rats and SHR. studied the effects of slowly reversible beta-adrenoceptor antagonists, bromoacetylalprenololmenthane (BAAM) and ICI 147798, on the isoprenaline responses in order to determine the affinity and fractional beta 2-adrenoceptor occupancy-response relationships for isoprenaline. The SHR portal veins did not develop hypertrophy. There was a small reduction in the sensitivity to isoprenaline and a marked reduction in the maximum attenuation of hypertension caused by isoprenaline. The sensitivity and efficacy of sodium nitroprusside and verapamil were not altered by hypertension. BAAM and ICI 147798 inhibited the isoprenaline responses and reduced the maximum attenuation to isoprenaline. In the WKY rat portal vein the dissociation constant (KA) values for isoprenaline were independent of BAAM concentration, and was 1.78 +/- 0.32 x 10(-7) mol/l. Similar isoprenaline KA values were obtained from the ICI 147798 data and in the SHR portal vein. In the WKY rat portal vein, from the BAAM data, it was calculated that isoprenaline produced 50, 95 and 100% maximum responses by occupying 6 +/- 1, 20 +/- 3 and 43 +/- 5% (n = 21) of the available beta 2-adrenoceptors, respectively. Similar occupancy-response relationships were obtained in the WKY rat portal vein from the ICI 147798 data. At each level of isoprenaline response the receptor reserve was significantly smaller in the SHR than it was in the WKY rat portal vein. Thus, from the BAAM data, isoprenaline produced 50, 95 and 100% maximum responses by occupying 14 +/- 3, 33 +/- 6 and 58 +/- 7% (n = 15) of the available SHR portal vein beta 2-adrenoceptors, respectively. The SHR portal vein displays a selective beta 2-adrenoceptor hyporesponsiveness in the absence of a raised blood pressure or hypertrophy. This beta 2-adrenoceptor-associated hyporesponsiveness consisted of a marked loss of maximum attenuation in response to isoprenaline and of beta 2-adrenoceptor reserve for isoprenaline responses.

Receptor availability defines the extent of agonist-mediated G-protein down-regulation in neuroblastoma × glioma hybrid cells transfected to express the β2-adrenoceptor

Sustained exposure of neuroblastoma × glioma hybrid, NG108-15, cells transfected to express the human, β2-adrenoceptor (clone βN22) to isoprenaline or iloprost (an agonist at the endogenously expressed IP prostanoid receptor) resulted in a substantial and selective down-regulation of the α subunit of the G-protein G s. Treatment of these cells with the irreversible β-adrenoceptor antagonist bromoacetyl alprenolol menthane diminished both the potency and the maximal ability of isoprenaline but not of iloprost to cause G sα down-regulation. These results demonstrate that the extent of agonist-mediated G sα down-regulation is dependent upon the availability of receptor to agonist.

Alprenolol and Bromoacetylalprenololmenthane are Competitive Slowly Reversible Antagonists at the β1-Adrenoceptors of Rat Left Atria

We studied the effects of alprenolol and bromoacetylalprenololmenthane (BAAM) on rat left atria. Alprenolol and BAAM at 10(-7), 3 x 10(-7), and 10(-6) M inhibited the cardiac stimulation response slightly, which is indicative of membrane-stabilizing activity independent of beta-adrenoceptor blockade. This membrane-stabilizing activity was readily reversible. Alprenolol and BAAM also caused surmountable antagonism of isoprenaline responses, and this beta 1-adrenoceptor antagonism was slowly reversible. Inhibition of the isoprenaline responses with alprenolol and BAAM at 10(-6) M was at equilibrium after 60 min, which is indicative of reversible antagonism. We conclude that alprenolol and BAAM are competitive slowly reversible beta 1-adrenoceptor antagonists on rat left atria.

Desensitization and functional antagonism by β-adrenoceptor and muscarinic receptor agonists, respectively: a comparison with receptor alkylation for calculation of apparent agonist affinity

1. Apparent affinity constants (KD) for prenalterol, an agonist of low intrinsic efficacy at beta 1-adrenoceptors in rat left atria, have been determined by use of receptor desensitization and functional antagonism induced by isoprenaline and carbachol, respectively. The values obtained have been compared to those values estimated with the irreversible beta-adrenoceptor antagonist, bromoacetylalprenololmenthane (BAAM). 2. The -log KD values for prenalterol estimated by desensitization or irreversible antagonism ranged from 6.8-7.1 and 6.2-7.1, respectively. 3. Carbachol produced functional antagonism of the response to prenalterol even though it was removed before addition of prenalterol. This effect was mediated by M2-muscarinic receptors. Pretreatment of animals with pertussis toxin did not affect the functional antagonism elicited by carbachol. The apparent KD value obtained after pre-exposure to carbachol (6.8) was similar to those estimated by use of either alkylation with BAAM or desensitization with isoprenaline (see above). 4. It is concluded that acute desensitization or functional antagonism of responses to agonists of low intrinsic efficacy provides a means to estimate apparent KD constants. This approach could be useful to characterize receptors for which an irreversible antagonist may not be available.

Effects of bromoacetylalprenolol—menthane (BAAM), an irreversible β—adrenoceptor antagonist, on the rat isolated left atria and portal vein

1. The effects of treatment with bromoacetylalprenololmenthane (BAAM), an irreversible beta-adrenoceptor antagonist, for 30 min on the contractile responses of the rat left atria and portal vein have been determined. 2. On the electrically driven rat left atria, BAAM at 10(-5) M followed by a 60-min wash-out caused parallel rightward shifts of the isoprenaline response curves with no reduction in the maximal response to isoprenaline. 3. Treatment of the atria with BAAM at 3 x 10(-5) M caused non-parallel rightward shifts of isoprenaline response curves with a reduction in the maximum response to isoprenaline. The KA (dissociation constant) for isoprenaline at beta 1-adrenoceptors was 7.3 x 10(-7) M. Calculation of receptor occupancy demonstrated that in order to produce a maximal response of the rat left atria, isoprenaline had to occupy 12% of the beta 1-adrenoceptors. 4. Treatment of the atria with higher concentrations of BAAM (greater than or equal to 6 x 10(-5) M) caused a non-specific action, the reduction of the response to electrical stimulation. 5. On the rat portal vein, BAAM at 3 x 10(-7)-3 x 10(-6) M followed by a 45-min wash-out had no effect on the spontaneous contractile activity, caused non-parallel rightward shifts of isoprenaline response curves with a depression of the isoprenaline maximum responses. Using data derived from experiments with 3 x 10(-7), 10(-6) and 3 x 10(-6) M BAAM, the KA for isoprenaline at beta 2-adrenoceptor was 6.4, 3.7 and 7.7 x 10(-7) M and the calculated receptor occupancy was that in order to produce a maximal response of the rat portal vein, isoprenaline had to occupy 14, 21 and 12%, respectively of the beta 2-adrenoceptors. 6. The present study with the rat left atria and portal vein has shown that it is possible to determine treatments with BAAM that produce no non-specific actions and consequently BAAM may be used as an experimental tool in KA and receptor occupancy determinations in these tissues.

Organ‐bath studies using the irreversible β‐adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM)

1. The effects of the irreversible beta-adrenoreceptor antagonist bromoacetylalprenololmenthane (BAAM) were studied in isolated cardiac and uterine preparations from guinea-pigs and rats and in guinea-pig ileal preparations. 2. In the presence of BAAM (0.1-10 microM) concentration-effect curves to (-)-isoprenaline were shifted to the right in a concentration-dependent manner in all cardiac and uterine tissues. Maximum responses to (-)-isoprenaline were unaffected by BAAM except in guinea-pig left atrial and in some guinea-pig uterine preparations; however, the reductions in the maximum responses were not concentration-dependent. The mean pKB values for BAAM in guinea-pig left atria, right atria, rat whole atria and rat uterus were 7.26, 7.24, 6.84 and 7.90 respectively. 3. In guinea-pig ileal preparations, BAAM (0.1-30 microM) relaxed contractions induced by K+, histamine and acetylcholine in a non-beta-adrenoreceptor-related manner since relaxant responses were unaffected by propranolol (0.5 microM). In other tissues higher concentrations of BAAM (30-100 microM) elicited atrial standstill and depressed K+-induced contractions in uterine smooth muscle. 4. Treatment of tissues with BAAM (10-100 microM) followed by extensive wash-out increased the EC50 values for (-)-isoprenaline 21- to 83-fold. The maximum response to the catecholamine was unaffected by BAAM except in guinea-pig left atrial preparations following treatment with 100 microM BAAM. At these concentrations BAAM markedly increased the effective refractory period. 5. Concentration-effect curves for the partial agonist, oxymethylene-isoprenaline (OM-ISO) were shifted to the right 12- to 355-fold after pretreatment of tissues with BAAM (10-30 microM) followed by wash-out. The maximum response to OM-ISO was unaltered in guinea-pig and rat uteri and was reduced to a similar degree as observed with (-)-isoprenaline in guinea-pig left atria. 6. In general, the non-selective beta-adrenoreceptor antagonist BAAM depressed maximum responses to beta-adrenoreceptor agonists only in cardiac preparations and at concentrations which elicited depressant activity. On the basis of the present study, BAAM does not appear to be a suitable irreversible beta-adrenoreceptor antagonist for use in organ bath experiments.

De novo cardiac beta adrenoceptor synthesis in adult rats under normoxic and hypoxic conditions.

Bromoacetylalprenololmenthane (BAAM), an alkylating irreversible beta adrenoceptor antagonist, was used to study in vivo beta adrenoceptor synthesis in adult rat left ventricle under normoxic and hypoxic conditions. In normoxic conditions cardiac beta adrenoceptor density decreased by 50% 15 h after treatment and recovered to control values after about 250 h. A similar decrease in receptor density and pattern of recovery was found in environmental hypoxia. Neither hypoxia nor treatment with BAAM had a significant effect on the dissociation constant of the radioligand. These results show that cardiac beta adrenoceptor synthesis occurs rather slowly and suggest that alteration of the rate of receptor synthesis in hypoxic states is not a major determinant of reported changes in density.

Interactions of putatively irreversible antagonists with β1 − and β2 − adrenergic receptors

Three compounds that have been suggested to irreversibly inactivate β-adrenergic receptors were studied: NHNPNBE [ N-(2-hydroxy-3-[1-napthoxy]-propyl)- N-bromoacetylethylenediamine], BAAM (bromoacetylalprenololmenthane), and Ro 3-7894 [1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol]. Membranes of rat cerebral cortex were used as a source of predominantly β 1 − adrenergic receptors and membranes of rat cerebellum were used as a source of predominantly β 2 − adrenergic receptors. β− Adrenergic receptor binding sites were studied by Scatchard analysis of saturation isotherms of specific [ 125I]-pindolol ([ 125I]PIN) binding. NHNPNBE added to the incubation medium competitively inhibited specific [125 I]PIN binding in both cerebellum and cerebral cortex with K l values of 1–2 μM. in each tissue. After washout of membranes pretreated with NHNPNBE for 30 min at 37°, no loss of specific [ 125I]PIN binding sites was observed in either cerebellum or cortex except at very high concentrations (30–100 μM). Ro 3-7894 caused a simple competitive inhibition of specific [ 125I]PIN binding in rat cerebellar membranes with a K I , of approximately 14 μM, an effect which was reversed completely by washing. In cerebral cortex, Ro 3-7894 added to the incubation medium apparently decreased the density of [ 125I]PIN binding sites with an IC 50 around 1 μM. This effect was reversed after washing the membranes twice. However, in the presence of Ro 3-7894 some Scatchard plots showed a slight curvature. Further saturation of the [ 125I]PIN binding sites in cerebral cortex showed that the inhibition by Ro 3-7894 was competitive but with a high- and low-affinity component, consistent with Ro 3-7894 being a β 1-selective competitive antagonist. Ro 3-7894 was also β 1ft-selective in other tissues. BAAM added to the incubation medium competitively inhibited specific [ 125I]PIN binding in both cerebellum and cortex with K l values of 0.006 to 0.03 μM, but was about 5-fold more potent in cerebellum. After treatment of membranes with higher concentrations of BAAM for 30 min at 37° and washing twice, there was a dose-dependent decrease in the density of specific [ 125I]PIN binding sites with ic 50 values of approximately 0.3 μM in both tissues. Similar effects were observed in rat heart. These data suggest that NHNPNBE is a simple competitive antagonist at both β 1 − and β 2-adrenergic receptors except at very high concentrations. Ro 3-7894 is a β 1-selective competitive antagonist with no apparent irreversible effects. BAAM is a slightly β 2-selective competitive antagonist which can also irreversibly decrease the density of both β 1- and β 2-adrenergic receptor binding sites at relatively low concentrations.

Pharmacological analysis of beta-adrenoceptor-mediated agonism in the guinea-pig, isolated, right atrium.

The recently developed, operational model of pharmacological agonism defines the efficacy of agonists by tau = [Ro]/KE, where [Ro] is the total functional concentration of receptors and KE is the concentration of agonist-occupied receptors for half-maximal effect. Theoretically, variations in [Ro] and KE affect tau and in turn, E/[A] curve profiles similarly. Using the beta-adrenoceptor mediated chronotropic responses of the guinea-pig isolated right atrial preparation we have investigated the consequences of experimental [Ro] and KE variation. Bromoacetylalprenolol menthane (M-75) produced displacements of isoprenaline and dichloroisoprenaline E/[A] curves consistent with [Ro] reduction. Cholera toxin produced displacements consistent with decreases in KE. The operational model provides a simple conceptual framework for the prediction and interpretation of changes in E/[A] curve profile resulting from experimental interventions at the post-receptor (KE) level as well as at the receptor ([Ro]) level.

Effect of a beta-adrenergic antagonist on blood pressure, heart rate and beta-adrenoceptors in Turkey poults

1. An irreversible beta-adrenergic blocker, bromoacetylalprenololmenthane (BAAM), was administered both peripherally and centrally to turkey poults, Meleagris gallopavo. 2.Peripheral administration of BAAM (60 mg/kg body weight) effected a significant reduction in blood pressure and heart rate. Twenty minutes postinjection, mean blood pressure and heart rate were reduced 34.5 and 24.2%, respectively. Two days later, mean blood pressure values remained significantly depressed at 17.3% below preinjection determinations. 3. Biochemical analysis of heart tissue following peripheral (intraperitoneal) injections of BAAM (60 mg/kg body weight) showed a significant decrease in beta-adrenergic receptors (BAR). Little or no change in the number of BAR in brain tissue was observed. 4. Central (intraventricular) administration of BAAM (0.72 mg/g brain weight) resulted in no change in mean blood pressure or heart rate during a 20 min postinjection period. 5. Biochemical analysis of heart tissue following central injections of BAAM showed little or no change in the number of BAR. There was, however, a significant decrease in the number of BAR in brain tissue. fa ]Scientific Article No. A-3426, Contribution No. 6498, of the Maryland Agricultural Experiment Station (Department of Poultry Science).