Concentration Of Beta-adrenoceptors Research Articles

Iterative optimal design of PET experiments for estimating beta-adrenergic receptor concentration.

To estimate in vivo myocardial beta-adrenergic receptor concentration with sufficient precision and to reduce the experimental complexities in positron emission tomography (PET), an iterative optimal design method is applied. An initial three-injection protocol, utilising [F-18]-labelled (R)- and (S)-fluorocarazolol and unlabelled (S)-fluorocarazolol, is optimised for ligand dosages and administration times to maximise the precision of all model parameters using the D-optimal criterion. Using this experimental protocol, PET data are collected in porcine studies, and model parameters are estimated. All model parameters are identified with satisfactory precision. The in vivo myocardial beta-receptor concentration is 7.5+/-0.6 pmol x ml(-1), which corresponds to the in vitro result of 10.1+/-1.3 pmol x ml(-1). With more accurate parameter values, a simplified two-injection protocol is optimally designed, utilising only radiolabelled and unlabelled (S)-fluorocarazolol, based on a new criterion to maximise the precision of the beta-receptor concentration. This revised optimum design predicts that the in vivo beta-receptor concentration can be estimated with good precision but reduced experiment complexity.

Deficient Activity of Stimulatory Nucleotide-Binding Regulatory Protein in Lymphocytes From Patients With Essential Hypertension

Lymphocytes are widely used as a model for the cardiovascular beta-adrenoceptor-adenylate cyclase system. We evaluated the role of this system in the pathogenesis of hypertension by studying lymphocytes obtained from patients with essential hypertension. Untreated hypertensive patients and normotensive control subjects were studied. The number and affinity of the beta-adrenoceptors were measured by a radioligand binding method with 125I-cyanopindolol. The responses of cyclic adenosine monophosphate (cAMP) to isoproterenol, cholera toxin, and forskolin were also determined. The concentration and affinity of beta-adrenoceptors did not differ significantly in the two groups, nor was a significant difference found in the basal level of cAMP. The effects of isoproterenol on the accumulation of cAMP were reduced in the lymphocytes from the hypertensive compared with the normotensive subjects. There was no significant difference in the effect of forskolin on cAMP accumulation in the two groups. These results indicate that the activity of the stimulatory nucleotide binding regulatory protein (Gs-protein) is reduced in lymphocytes from patients with essential hypertension. This defect of Gs-protein in the lymphocytes may represent a defect of Gs-protein in the cardiovascular system in such patients.

Characterisation of the beta adrenergic response cascade in fetal guinea pig lung.

Suitable models for the study of lung development are needed. The suitability of the guinea pig for studying the role of the beta adrenergic response cascade in fetal lung development has been evaluated. Radioligand binding assays with iodine-125 labelled iodopindolol were performed to identify and characterise the beta adrenergic receptors. To demonstrate that these receptors were functional, isoprenaline and forskolin stimulated generation of cyclic AMP (cAMP) in the lung tissue was quantitated by radioimmunoassay. The concentration of beta receptors increased with gestational age from 23 fmol/mg at 35 days to 140 fmol/mg at 64 days. Competition binding studies were consistent with a predominance of beta 2 receptors. The ability of isoprenaline to stimulate cAMP generation was greater during the saccular phase than during the canalicular phase of lung development. Incorporation of tritium labelled choline into phosphatidylcholine increased significantly between the canalicular and saccular phases. The beta adrenergic response cascade in fetal guinea pig lung exhibits similar characteristics to those previously described in fetal human lung and is therefore a good model in which to study the effects of beta agonists on fetal lung development.

Positron emission tomography with 11C CGP-12177 to assess beta-adrenergic receptor concentration in idiopathic dilated cardiomyopathy.

Positron emission tomography (PET) with 11C-labeled CGP-12177 (CGP) has been shown to have the potential to noninvasively measure beta-adrenergic receptor concentration in dog heart. The present study was undertaken to evaluate the clinical value of this technique. Eight normal subjects and 10 patients with heart failure related to an idiopathic cardiomyopathy were studied. Estimation of beta-receptor concentration was based on a graphic method applied on myocardial PET time-concentration curves obtained after an intravenous injection of 11C-CGP followed 30 minutes later by a coinjection of labeled and unlabeled CGP. The clinical tolerance of these injections was good. Left ventricular concentration of beta-receptors was decreased in patients compared with controls (3.12 +/- 0.51 versus 6.60 +/- 1.18 pmol/mL, respectively; p < 0.001). This 53% decrease agrees with previous in vitro data. In eight of the 10 patients, the beta-receptor concentration obtained from PET was compared with the beta-receptor density determined on left ventricular endomyocardial biopsy samples by in vitro binding technique using 3H-CGP-12177. Results obtained with both techniques were correlated (r = 0.79, p = 0.019). Moreover, decreased beta-receptor concentration correlated with the beta-contractile responsiveness to intracoronary dobutamine infusion (r = 0.83, p = 0.003), indicating a direct link between changes in the receptor number and its biological function. PET appears to be a safe and reliable method of assessing in vivo changes in the number of left ventricular beta-adrenergic receptor sites of patients with idiopathic cardiomyopathy.

Increased 2-Adrenoceptor Number in Peripheral Sympathetic Ganglia of Spontaneously Hypertensive Rats

We determined beta-adrenoceptor concentrations in sympathetic ganglia from adult Spontaneously Hypertensive Rats (SHR) and Wistar-Kyoto (WKY) control rats by quantitative autoradiography. Adjacent tissue sections were incubated with [125I]iodocyanopindolol, with or without excess of unlabeled (-)-propranolol, the beta 1-antagonist CGP 20712A or the beta 2-antagonist ICI 118,551. Most beta-adrenoceptors were of the beta 2-type. Their concentration was higher in the superior cervical and stellate ganglia of SHR when compared to normotensive WKY. Our results indicate that beta 2-adrenoceptor stimulation may be enhanced in sympathetic ganglia of SHR, and could play a role in the maintenance of their increased sympathetic activity.

β-Adrenergic Receptors and cAMP Response Increase during Explant Culture of Human Fetal Lung: Partial Inhibition by Dexamethasone

We studied beta-adrenergic receptors and responses in human fetal lung (15-25 wk gestation) maintained in explant culture with and without added dexamethasone. To determine beta-adrenergic receptor concentration, we performed radioligand binding assays with [125I]-iodocyanopindolol. We also examined the ability of isoproterenol to stimulate cAMP generation as a measure of response to beta-adrenergic receptor occupancy. In control cultures, beta-receptor concentration increased significantly from d 0 to 3 of culture and thereafter remained stable. The kd (approximately 24 pM) of [125I]-iodocyanopindolol did not change with time in culture. The ability of isoproterenol to stimulate cAMP generation over basal levels increased in controls throughout the 5 d in explant culture. Addition of dexamethasone (10 nM) to the culture medium partially blocked the increase in beta-receptor concentration and decreased both cAMP content and generation (basal and stimulated) in a dose-dependent manner (median effective concentration approximately 1 nM). In these same explants, dexamethasone increased the activity of fatty acid synthetase, an enzyme important in surfactant synthesis, more than 2-fold. Our results indicate that beta-adrenergic receptors and isoproterenol stimulation of cAMP generation increase spontaneously in human fetal lung grown in explant culture. Dexamethasone, which accelerates other aspects of human lung development in vitro, decreases beta-adrenergic receptor concentration and inhibits beta-adrenergic responses.

-Adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.

Reserpine but not surgical denervation regulates rat renal beta-adrenergic receptors

The effects of unilateral surgical denervation or reserpine administration on renal beta-adrenergic receptors were examined in rat kidney cortex. The specific binding of [125I]iodocyanopindolol was used to quantitate the beta-adrenoceptors. Denervation had no significant effect on beta-adrenoceptor concentration in denervated compared with contralateral control kidney, 7 days postsurgery. In contrast, reserpine treatment increased beta-adrenoceptor concentration 30% compared with control (P less than 0.05). Tissue norepinephrine levels were depleted to a significant extent with both manipulations. The reserpine effect was investigated further. Reserpine increased both beta 1- and beta 2-adrenergic receptor subtypes to the same extent. The effect of reserpine was primarily on tubular beta-adrenoceptors including those in the proximal tubules; glomerular beta-adrenoceptors were minimally affected by reserpine. Other adrenergic receptor subtypes (alpha 1- and alpha 2-) were also significantly increased by reserpine; however, angiotensin II receptors were not altered, indicating that the reserpine effect was not a general one affecting all membrane receptors. Reserpine treatment increased beta-adrenergic receptor-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by 49% over control in the renal cortex. Denervation had no significant effect on cAMP accumulation. Overall, our results suggest that, in addition to sympathetic nerve terminal norepinephrine, other factors may be involved in the regulation of renal beta-adrenergic receptors.

Adrenoceptors in experimental hypertension.

Cardiac, aortic and renal alpha- and beta-adrenoceptors were examined in three types of experimental hypertension in rats. In spontaneously hypertensive rats (SHR), cardiac alpha 1-adrenoceptor concentration was increased and cardiac and aortic beta-adrenoceptor concentrations were decreased compared with Wistar-Kyoto rats (WKY). A similar decrease in cardiac beta-adrenoceptor concentration was also shown in two-kidney, one clip (2K, 1C) and deoxycorticosterone acetate(DOCA)-salt hypertensive rats. Renal alpha 1- and alpha 2-adrenoceptor concentrations were increased in SHR, but not in 2K, 1C and DOCA-salt hypertension. In contrast, renal beta-adrenoceptor concentration was increased in 2K, 1C and DOCA-salt hypertension, but unchanged in SHR. The observed increase in cardiac alpha 1- and renal alpha 2-adrenoceptor concentrations may partly contribute to the elevation of blood pressure in SHR.

Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp [NH]p, isoproterenol plus GTP or Gpp [NH]p was significantly greater in the SHR. Beta-receptor density measured by [ 3H]DHA binding was unaltered. The dissociation constant, K d, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 ± 0.45 nM) compared with the WKY (8.53 ± 0.82 nM). Activity of G s was assessed by complementing S49 cyc − acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp [NH]p, and isoproterenol plus Gpp [NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized r response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary G S in the hypertensive rats.

Ventricular beta-adrenoceptors in adriamycin-induced cardiomyopathy in the rabbit

beta-Adrenoceptor concentrations have been measured in ventricles of rabbits with adriamycin-induced cardiomyopathy. Despite considerable cardiac hypertrophy (1.6 +/- 0.7 mean +/- S.E.M. g ventricle/kg body wt, n = 6; to 2.2 +/- 0.1) no change in beta-adrenoceptor density was observed (33.8 +/- 2.0 fmol/mg protein in membranes from cardiomyopathic rabbits compared with 36.8 +/- 3.0 for controls). Furthermore, no alteration in the profiles of high affinity agonist binding was observed, and the receptor interaction with the adenylate cyclase stimulatory coupling factor was unimpaired. These results indicate that, despite the marked changes which occur during the development of this model of low-output heart failure, the cardiac beta-adrenoceptor systems are normal.

Characterization of rat liver beta-adrenoceptors during perinatal development as determined by [125I]-iodopindolol radioligand binding assays.

1. The subtype specificity of beta-adrenoceptors in foetal (20 days post coitum) rat liver membrane preparations has been determined by use of [125I]-iodopindolol binding assays and the characteristics of radioligand binding have been resolved. 2. The kinetics of radioligand association and dissociation (in the presence of 5 x 10(-4) M isoprenaline) showed an association rate constant of 1.5 x 10(7) M-1 S-1 and dissociation rate constant of 9.1 x 10(-4) S-1, corresponding to a dissociation constant for [125I]-iodopindolol of 60.7 pM. A similar dissociation constant (75 pM) was determined by saturation binding assays. 3. The rank order of potency for displacement of [125I]-iodopindolol binding was consistent with binding to a predominantly beta 2-adrenoceptor population (i.e. ICI 118551 greater than isoprenaline greater than adrenaline greater than noradrenaline greater than atenolol). Computer analysis of displacement curves in the presence of a beta 1-subtype selective agent (atenolol) or a beta 2-subtype selective agent (ICI 118551) revealed the presence of beta 2- and beta 1-adrenoceptor subtypes in a ratio of about 80:20%. 4. Saturation binding assays by use of [125I]-iodopindolol were carried out at different perinatal ages to determine total beta-adrenoceptor concentrations and beta 2-subtype (in the presence of 5 x 10(-7) M atenolol) adrenoceptor concentrations. Competition binding assays with atenolol confirmed that at all ages apparent beta 2-adrenoceptor binding accounted for 84-95% of the total beta-adrenoceptor binding. The total beta- and beta 2-adrenoceptor binding capacity increased by 2.3 fold from 20 days post coitum to birth, and then decreased postnatally at 1 and 2 days post partum. The dissociation constant for [125I]-iodopindolol binding did not show any change with age. 5. The change in beta 2-adrenoceptor concentration with age is discussed in relation to the changing beta-adrenoceptor-mediated responsiveness of glucose production by rat liver during perinatal development.

Inhibitory effects of atropine and adrenergic antagonists on the changes in autonomic receptors and cyclic nucleotides of rat parotid and submandibular glands caused by sympathetic nerve stimulation.

The changes in cyclic AMP (cAMP) concentration and density of beta-adrenoceptors caused by electrical stimulation of the sympathetic innervation to parotid and submandibular glands of rat did not occur when the alpha- and beta-adrenergic antagonists, phentolamine, and propranolol were administered 20 min prior to initiation of stimulation. They also did not occur when phentolamine, the beta-adrenergic antagonist, was administered alone prior to nerve stimulation, indicating that beta-adrenoceptors mediate these effects. Simultaneous administration of the alpha- and beta-antagonists also prevented the changes in densities of muscarinic receptors and cGMP concentrations usually induced by sympathetic nerve stimulation. Also, the changes in muscarinic receptors and cGMP did not occur when atropine was administered prior to nerve stimulation, nor did they occur with simultaneous administration of atropine, phentolamine + propranolol; with phentolamine alone, or propranolol alone, the effects were blocked to a large extent. Secretion was inhibited completely when both adrenergic antagonists were present during nerve stimulation, but flow rate was unchanged when atropine was present. The changes in both beta-adrenoceptors and muscarinic receptors reflect a desensitization caused by prolonged exposure to neurotransmitters released when the sympathetic nerve is stimulated. The changes are prevented when either atropine or adrenergic antagonists are present during nerve stimulation.

Characterization of beta 1- and beta 2-adrenoceptor subtypes in the rat atrioventricular node by quantitative autoradiography.

We characterized beta-adrenoceptor subtypes in the atrioventricular node of the rat heart by quantitative autoradiography. Consecutive 16-microns-thick sections from single rat hearts containing the atrioventricular node were incubated with increasing concentrations of [125I]iodocyanopindolol. After exposure to [3H]Ultrofilm, optical densities corresponding to the atrioventricular node were determined by computerized densitometry after comparison with [125I]standards. The computer program LIGAND was used for analysis of receptor subtypes. Delineation of beta-adrenoceptor subtypes was achieved by incubating consecutive tissue sections with 50 pM [125I]iodocyanopindolol in the presence of increasing concentrations of the beta 1-selective antagonist atenolol or the beta 2-selective antagonist ICI 118,551. The atrioventricular node contains a higher concentration of beta-adrenoceptors than the adjacent interventricular septum. We estimated that the proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node were about 56% and 44% of the total binding capacity respectively.

Localization and quantification of beta-adrenergic receptors in human brain.

Little information is currently available on the localization of noradrenergic systems in the human CNS. We used quantitative autoradiography with [125I] iodopindolol to examine beta-adrenergic receptors in postmortem human brain. The concentration of beta-receptors was highest in all subfields of the hippocampus, followed by cerebellum, and then thalamic nuclei, basal ganglia, midbrain, and cerebral cortex. Low levels were found in white matter and hypothalamus. This distribution differed from the distribution of beta-receptors reported in membrane homogenates of human brain and also from the distribution of beta-receptors in rat brain determined by autoradiography. The similarities and differences between the distribution of beta-receptors in the human and rat brains may have implications regarding the role of norepinephrine in the CNS of these two species.

Molecular Pharmacology of β-Adrenoceptors

The development of radioligand-binding studies has greatly advanced our knowledge of the molecular pharmacology of beta-adrenoceptors. With this technique it is possible for the first time to directly determine the tissue concentration of beta-adrenoceptors, and by this, the responsiveness of tissues to beta-adrenergic stimulation. One major insight into the molecular pharmacology of beta-adrenoceptors to come from radioligand-binding studies was that the tissue concentration of beta-adrenoceptors is not a fixed number, but is rather dynamically regulated by a variety of drugs, hormones, physiological and pharmacological conditions. Since changes in beta-adrenoceptors assessed in circulating lymphocytes of man mirror changes in density and functional responsiveness of the corresponding myocardial beta-adrenoceptors, alterations in beta-adrenoceptor function can be also studied in human beings. In addition, by means of radioligand-binding studies the coexistence of beta 1- and beta 2-adrenoceptors has been demonstrated in numerous tissues of various species, including human heart and lung. The detailed knowledge of distribution and regulation of beta-adrenoceptors should help to improve the individual effectiveness of drug treatment.

Glucocorticoids preferentially increase fetal alveolar beta-adrenoreceptors: autoradiographic evidence.

To localize fetal rabbit lung beta-adrenoreceptors before and after glucocorticoid treatment, light microscopic autoradiography was performed with the reversible radiolabeled beta-adrenergic antagonist, [3H]dihydroalprenolol, on day 26 of gestation. Autoradiograms of adult lung and fetal myocardium were also prepared. Examination of these autoradiograms showed densely labeled airways, alveoli, and myocardium. Specific labeling, defined as that prevented by incubation with l-propranolol (1 microM), was 90%. Analysis of grain counts in the fetus showed that airways were more densely labeled than alveoli (p less than 0.001), labeling was increased by treatment (p less than 0.001) and treatment increased alveolar (p less than 0.002) but not airway labeling. Adult lungs were much more densely labeled than fetal, and fetal myocardial labeling was not altered by treatment. Adult untreated lung showed the same pattern as fetal untreated lung with airways being more densely labeled than alveoli (p less than 0.001). To validate estimates of relative beta-adrenoreceptor concentration derived from autoradiograms, comparisons to determinations of beta-receptor concentration from scintillation counting of lung section digests and from previously performed radioligand binding studies, using membranes prepared from whole lung homogenates, were made. There is excellent agreement between estimates of relative receptor concentration and specific binding derived from the counting of autographic grains and both scintillation counting of lung section digests and previously performed radioligand binding of lung particulate. In all preparations, specific binding was (90%), increased with glucocorticoid treatment in fetal lung (50-100%), was greater in concentration in adult compared to fetal lung (7-10-fold), and did not increase in fetal myocardium with treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral adrenoceptors after sino-aortic denervation.

The effect of bilateral sino-aortic denervation on cardiac and renal cortical alpha- and beta-adrenoceptor concentrations was studied in rats by radioligand binding experiments using [3H]-prazosin (alpha 1), [3H]-yohimbine (alpha 2) and [125I]-iodocyanopindolol (beta). Cardiac alpha 1- and beta-adrenoceptor concentrations were not altered by sino-aortic denervation. There was a significant increase in beta-adrenoceptor concentration in the renal cortex of sino-aortic denervated rats compared to sham-operated controls.

DEPENDENCE OF β‐ADRENERGIC RESPONSIVENESS ON THYROID STATE OF MALE RATS

The dose of thyroxine (0-150 micrograms/kg body weight) administered subcutaneously daily to surgically thyroidectomized male rats was correlated significantly with their metabolic activity as assessed by rate of oxygen consumption and colonic temperature. There was a significant dose-dependent increase in total serum thyroxine, triiodothyronine and reverse-triiodothyronine concentrations with increasing doses of thyroxine administered. The cardiostimulatory response to administration of isoprenaline (8 micrograms/kg body weight s.c.) was also correlated directly with the dose of thyroxine administered. The concentration of cardiac beta-adrenoceptors in these animals was correlated significantly with total serum thyroxine and triiodothyronine concentrations, basal heart rate, and the chronotropic response to administration of isoprenaline.

Effect of caloric restriction on cardiac reactivity and beta-adrenoceptor concentration.

The effect of a 21-day program of caloric restriction on cardiac reactivity and beta-adrenoceptor number was investigated in male Sprague-Dawley rats. Rats on the restricted diet (Restricted) exhibited significant decreases in body weight, epididymal fat pad, and retroperitoneal fat pad weight as well as the percent of body fat represented by these adipose tissue depots when compared with rats fed ad libitum (Fed). Fed rats exhibited significantly increased total heart weight and total heart protein, but the percent cardiac protein and ratio of heart weight to body weight were similar in Fed and Restricted rats. Isolated atria from Fed and Restricted rats developed similar chronotropic and inotropic responses over a range of isoproterenol concentrations. Although total beta-adrenoceptor number (fmol/heart) was greater in Fed rats, the concentration of beta-adrenoceptors (fmol/mg protein) was remarkably similar regardless of the dietary regimen. Therefore, despite significant decreases in body weight, body fat, and heart weight, the myocardium of Restricted rats maintained the capability of responding to isoproterenol as that of Fed rats, the mechanism of which is at least partially mediated through maintenance of beta-adrenoceptor concentration.