Antidepressant Treatments, Including Sibutramine Hydrochloride and Electroconvulsive Shock, Decrease β1 but Not β2-Adrenoceptors in Rat Cortex

The beta 1- and beta 2-adrenoceptor populations in rat cortex were individually quantified by labelling all of the receptors with [3H]dihydroalprenolol and displacing with isoprenaline (200 microM) or CGP 20712A (1-(2-[(3-carbamoyl-4-hydroxy)phenoxy]ethylamino)-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol methanesulphonate; 100 nM) to define total beta-adrenoceptors and beta 1-adrenoceptors, respectively. Binding parameters for beta 2-adrenoceptors were calculated by the difference. Oral administration of the monoamine reuptake inhibitors sibutramine HCl (3 mg/kg), amitriptyline (10 mg/kg), desipramine (10 mg/kg), or zimeldine (10 mg/kg) for 10 days decreased the total number of beta-adrenoceptors present in rat cortex. This effect was entirely due to a reduction in the number of beta 1-adrenoceptors. Similarly, 10 days of treatment with the monoamine oxidase inhibitor tranylcypromine (10 mg/kg p.o.) or five electroconvulsive shocks (ECSs; 200 V, 2 s) spread over this period also down-regulated beta-adrenoceptors by reducing the content of the beta 1-subtype. By contrast, treatment with clenbuterol (5 mg/kg p.o.) for 10 days reduced the number of cortical beta-adrenoceptors by an effect on the beta 2-adrenoceptor population. The effects of short-term treatment with these drugs were also investigated, and, using the doses shown above, the results of 3 days of administration or a single ECS were determined. Sibutramine HCl and desipramine were alone in producing a reduction in number of beta-adrenoceptors after 3 days. Once again, this was exclusively due to a loss of beta 1-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise training normalizes enhanced glutamate-mediated sympathetic activation from the PVN in heart failure

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the mechanisms are not known. We hypothesized that ExT would normalize the augmented glutamatergic mechanisms mediated by N-methyl-d-aspartic acid (NMDA) receptors within the paraventricular nucleus (PVN) that occur with HF. Four groups of rats were used: 1) sham-operated (Sham) sedentary (Sed), 2) Sham ExT, 3) HF Sed, and 4) HF ExT. HF was induced by left coronary artery ligation, and ExT consisted of 3 wk of treadmill running. In alpha-chloralose-urethane-anesthetized rats, the increase in renal sympathetic nerve activity in response to the highest dose of NMDA (200 pmol) injected into the PVN in the HF Sed group was approximately twice that of the Sham Sed group. In the HF ExT group the response was not different from the Sham Sed and Sham ExT groups. Relative NMDA NR1 receptor subunit mRNA expression was 63% higher in the HF Sed group compared with the Sham Sed group but in the HF ExT group was not different from the Sham Sed and Sham ExT groups. NR1 receptor subunit protein expression was increased 87% in the HF Sed group compared with the Sham Sed group but in the HF ExT group was not significantly different from the Sham Sed and Sham ExT groups. Thus one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of glutamatergic mechanisms within the PVN.     

Alpha- and beta-adrenergic receptors of the rat salivary gland. Elevation after chemical sympathectomy.

Binding of [3H]dihydroergokryptine and [3H]dihydroalprenolol to membrane preparations from rat submaxillary gland was measured to characterize the alpha- and beta-adrenergic receptors, respectively. Kinetic analysis of the data revealed a high affinity binding site for each radioligand. Inhibition of binding at each site was stereospecific for the active isomer of the catecholamine used. The greater ability of a beta1 than beta2 specific beta-adrenergic antagonist to displace [3H]dihydroalprenolol binding indicated that this binding site was of the beta1 type. Chemical sympathectomy with reserpine or 6-hydroxydopamine resulted in a significant increase in both [3H]dihydroalprenolol and [3H]dihydroergokryptine binding in the rat submaxillary gland. 3scatchard analysis of the data indicated that these increases in binding were due to a change in total number of binding sites for [3H]dihydroergokryptine and [3H]dihydroalprenolol with little change in apparent affinities. This suggests that changes in alpha- and beta-adrenergic receptor density may be important in the development of supersensitivity in salivary glands after reserpine and 6-hydroxydopamine treatment.     

β2-肾上腺素受体在新生大鼠心肌成纤维细胞中的分布及其促增殖作用

为了明确β-肾上腺素受体(AR)亚型在新生大鼠心肌成纤维细胞中的分布及其在成纤维细胞增殖反应中的作用,采用放射配体结合实验和[3H]-thymidine掺人法检测了新生大鼠心肌成纤维细胞的β-AR密度和DNA合成速率。结果显示,在培养心肌细胞和心肌成纤维细胞中β-AR密度(Bmtax)和解离常数(Kp)无显著性差异;竞争抑制曲线分析结果提示,心肌成纤维细胞对CGP 20712A和ICI ll8551单位点拟合均显著优于两位点拟合(P＜0．01),表现为对选择性β1-AR拮抗剂CGP 20712A的低亲和性(IC50值：10．1μmol/L)和对选择性β2-AR拮抗剂ICI 118551的高亲和性(IC50值：0.147μmol/L)。异丙肾上腺素(ISO)促心肌成纤维细胞增殖作用可被ICI 118551和心得安(非选择性β-AR拮抗剂)完全抑制,而CGP20812A则无此作用。上述结果提示,在培养心肌成纤维细胞中β-AR亚型占绝对优势,并且ISO引起的心肌成纤维细胞增殖反应是由β2-AR介导的。     

Dependence of changes in beta-adrenoceptor signal transduction on type and stage of cardiac hypertrophy.

To examine whether cardiac hypertrophy is associated with changes in beta-adrenoceptor signal transduction mechanisms, pressure overload (PO) was induced by occlusion of the abdominal aorta and volume overload (VO) by creation of an aortocaval shunt for 4 and 24 wk in rats. After hemodynamic assessment of the animals, the left ventricular (LV) particulate fraction was isolated for measurement of beta(1)-adrenoceptors and adenylyl cyclase activity, and cardiomyocytes were isolated for monitoring of the intracellular Ca(2+) concentration. Although PO and VO produced cardiac hypertrophy and increased LV end-diastolic pressure at 4 wk, cardiac function was increased in animals subjected to PO but remained unaltered in animals subjected to VO. Cardiac hypertrophy and increased LV end-diastolic pressure were associated with depressed cardiac function at 24 wk of PO or VO, but clinical signs of congestive heart failure were evident only in animals subjected to VO. Isoproterenol-induced increases in cardiac function, activation of adenylyl cyclase activity, and increase in intracellular Ca(2+) concentration, as well as beta(1)-adrenoceptor density, were unaltered by PO at 4 wk, augmented by VO at 4 wk, and attenuated by PO and VO at 24 wk. These results suggest that alterations in beta(1)-adrenoceptor signal transduction are dependent on the type and stage of cardiac hypertrophy.     

Characteristics of beta-adrenergic receptors in isolated cells and in crude membranes of brown adipose tissue

In relation to decreased metabolic sensitivity to catecholamines observed, in vitro, in brown fat of cold-acclimated rats, beta-adrenergic receptors were studied in isolated cells and in a crude membrane preparation from rat interscapular brown adipose tissue. [3H] dihydroalprenolol binding had the same characteristics in both types of preparation; competition studies of [3H] dihydroalprenolol binding led to the characterization of beta 1 subtype adrenergic receptors with a lower affinity of beta-adrenergic agonists for [3H] dihydroalprenolol binding sites in membranes than that found in isolated cells. Cold acclimation produced, in isolated cells only, a decrease of 41% in the [3H] dihydroalprenolol binding sites and a beta-adrenergic agonist affinity increase. It is concluded that beta-adrenergic receptor decrease could be a factor, at the hormone receptor interaction level, in the regulation of the transmission of biological action responsible for the cold-induced decrease in catecholamine responsiveness in brown adipose tissue. For a study of the desensitization process in brown fat, isolated cells seem to offer certain advantages over a crude membrane preparation.     

Heterogeneous cardiac sympathetic innervation in heart failure after myocardial infarction of rats

We examined cardiac neuronal function and beta-receptor with a dual-tracer method of [(131)I]meta-iodobenzylguanidine (MIBG) and [(125)I]iodocyanopindolol (ICYP) in rat heart failure after myocardial infarction (MI). In rats with MI, left ventricular (LV) systolic function decreased, and LV dimension and right ventricular (RV) mass increased gradually. MIBG accumulations of the noninfarcted LV (remote region) and RV decreased by 15% at 1 wk compared with sham-operated rats, and these accumulations were restored by 71% and 56%, respectively, at 24 wk compared with age-matched sham rats despite sustained depletion of myocardial norepinephrine contents in these regions. ICYP accumulation of the remote region and of the RV did not decrease at any stages. Myocardial MIBG distribution was heterogeneous at 1 wk when it was lower in the peri-infarcted region than in the remote region, associated with reduced ICYP accumulation in the peri-infarcted region. The heterogeneous distribution of both isotopes disappeared at 12 wk. Thus cardiac sympathetic neuronal alteration was coupled with downregulation of beta-receptors in rat heart failure after MI. The abnormal adrenergic signaling occurred heterogeneously in terms of ventricular distribution and time course after MI.     

Exercise training normalizes enhanced sympathetic activation from the paraventricular nucleus in chronic heart failure: role of angiotensin II

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the underlying mechanisms are not known. We hypothesized ExT would normalize the augmented activation of the paraventricular nucleus (PVN) via an angiotensinergic mechanism during HF. Four groups of rats used were the following: 1) sham-sedentary (Sed); 2) sham-ExT; 3) HF-Sed, and 4) HF-ExT. HF was induced by left coronary artery ligation. Four weeks after surgery, 3 wk of treadmill running was performed in ExT groups. The number of FosB-positive cells in the PVN was significantly increased in HF-Sed group compared with the sham-Sed group. ExT normalized (negated) this increase in the rats with HF. In anesthetized condition, the increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to microinjection of angiotensin (ANG) II (50～200 pmol) in the PVN of HF-Sed group were significantly greater than of the sham-Sed group. In the HF-ExT group the responses to microinjection of ANG II were not different from sham-Sed or sham-ExT groups. Blockade of ANG II type 1 (AT(1)) receptors with losartan in the PVN produced a significantly greater decrease in RSNA, MAP, and HR in HF-Sed group compared with sham-Sed group. ExT prevented the difference between HF and sham groups. AT(1) receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT(1) receptor protein expression was not significantly different from sham-Sed or sham-ExT groups. In conclusion, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of angiotensinergic mechanisms within the PVN.     

Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and beta-adrenergic receptors in spontaneously hypertensive rats

[3H]Dihydroalprenolol binding and adenylate cyclase activity in the myocardial membranes of Kyoto Wistar normotensive rats and spontaneously hypertensive rats were compared at various stages of postnatal development ranging from 2 to 36 weeks. Basal as well as agonist-stimulated myocardial adenylate cyclase activity was consistently decreased in spontaneously hypertensive rats as compared to normotensive rats as early as 2 weeks of age with significant differences (P < 0.05) observed after 6 weeks of age. When results were expressed as percent stimulation over the basal activity, only isoproterenol plus GTP-stimulated enzyme activity was reduced by 25--30% in spontaneously hypertensive rats, suggesting a specific loss of stimulation by isoproterenol in hypertensive animals. The number of [3H]dihydroalprenolol binding sites of KD for dihydroalprenolol binding were comparable between spontaneously hypertensive and normotensive rats at 3, 6 and 12 weeks of age. The competition of isoproterenol with [3H]dihydroalprenolol for the specific binding sites showed that the affinity of isoproterenol binding was decreased 3--4-fold in spontaneously hypertensive compared with normotensive rats. With postnatal development in age, basal as well as agonist-stimulated activities decreased progressively in both spontaneously hypertensive and normotensive rats. Similarly, the number of [3H]dihydroalprenolol binding sites decreased with the development in age, whereas affinity of dihydroalprenolol binding increased up to 12 weeks of age. These results therefore suggest that adenylate cyclase activity and the number of beta-adrenergic receptors in rat heart, decrease with age and that in hypertension, specific decrease in isoproterenol stimulation of cyclase appears at all stages of development.     

High glucose and diabetes increase the release of [3H]-D-aspartate in retinal cell cultures and in rat retinas

Several evidences suggest that glutamate may be involved in retinal neurodegeneration in diabetic retinopathy (DR). For that reason, we investigated whether high glucose or diabetes affect the accumulation and the release of [(3)H]-D-aspartate, which was used as a marker of the glutamate transmitter pool. The accumulation of [(3)H]-D-aspartate did not change in cultured retinal neural cells treated with high glucose (30 mM) for 7 days. However, the release of [(3)H]-D-aspartate, evoked by 50 mM KCl, significantly increased in retinal cells exposed to high glucose. Mannitol, which was used as an osmotic control, did not cause any significant changes in both accumulation and release of [(3)H]-D-aspartate. In the retinas, 1 week after the onset of diabetes, both the accumulation and release of [(3)H]-D-aspartate were unchanged comparing to the retinas of age-matched controls. However, after 4 weeks of diabetes, the accumulation of [(3)H]-D-aspartate in diabetic retinas decreased and the release of [(3)H]-D-aspartate increased, compared to age-matched control retinas. These results suggest that high glucose and diabetes increase the evoked release of D-aspartate in the retina, which may be correlated with the hypothesis of glutamate-induced retinal neurodegeneration in DR.     

Differential expression of alpha2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats

The aim of the present study was to compare vascular dysfunction between the early (12 wk old) and later (36 wk old) stages of spontaneous diabetes in Goto-Kakizaki (GK) rats. We also evaluated the aortic expression of the alpha(2D)-adrenoceptor and endothelial nitric oxide synthase (eNOS). Vascular reactivity was assessed in thoracic aortas from age-matched control rats and 12- and 36-wk GK rats. Using RT-PCR and immunoblots, we also examined the changes in expression of the alpha(2D-)adrenoceptor and eNOS. In aortas from GK rats (vs. those from age-matched control rats): 1) the relaxation response to ACh was enhanced at 12 wk but decreased at 36 wk; 2) the relaxation response to sodium nitroprusside was decreased at both 12 and 36 wk, 3) norepinephrine (NE)-induced contractility was decreased at 12 wk but not at 36 wk, 4) the expressions of alpha(1B)- and alpha(1D)-adrenoceptors were unaffected, whereas those of alpha(2D)-adrenoceptor and eNOS mRNAs were increased at both 12 and 36 wk; and 5) NE- and ACh-stimulated NO(x) (nitrite and nitrate) levels were increased at 12 wk, although at 36 wk ACh-stimulated NO(x) was lower, whereas NE-stimulated NO(x) showed no change. These results clearly demonstrate that enhanced ACh-induced relaxation and impaired NE-induced contraction, due to NO overproduction via eNOS and increased alpha(2D)-adrenoceptor expression, occur in early-stage GK rats and that the impaired ACh-induced relaxation in later-stage GK rats is due to reductions in both NO production and NO responsiveness (but not in eNOS expression).     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

Beta-adrenoceptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10 days treatment with growth hormone (GH) (l mg/kg body wt/day) and somatostatin (SRIF) (0.25 mg/kg body wt/day) subcutaneously on the activity of β-adrenoceptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using [³H]dihydroalprenolol ([³H]DHA) as radioligand.2. The administration of GH significantly increased the β-adrenoceptor binding affinity and the administration of SRIF decreased the β-adrenoceptor binding capacity in the hypothalamus.3. In the pituitary the β-adrenoceptor binding affinity was significantly decreased after both hormonal applications.4. In the cerebral cortex the β-adrenoceptor binding affinity was significantly decreased after the GH treatment and increased after the SRIF treatment.5. The present study provides direct evidence for GH and SRIF effects on the activity of rat β-adrenoceptors and supports the view about the involvement of β-adrenergic mechanisms in the neurotransmitter regulation of GH secretion in the rat.     

Hormone action at the membrane level. VIII. Adrenergic receptors in rat heart and adipocytes and their modulation by thyroxine.

The regulation of adrenergic receptors in rat heart was measured in rats made hyperthyroid by injection with thyroxine and made hypothyroid by addition of propylthiouracil to the drinking water. Hyperthyroid rats display cardiac hypertrophy and a decrease in epididymal fat pad weight. The maximal beta-receptor level of ventricular membranes, as determined by (-)-[3H]dihydroalprenolol binding, was increased 60% by thyroxine treatment and decreased about 30% by propylthiouracil treatment. The affinity of the beta receptor was unchanged after thyroxine or propylthiouracil treatment. The maximal activity of the isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) varied with thyroid state in a manner parallel to the increase in beta-adrenergic binding sites. Thyroxine treatment also increases by 2-fold the beta receptors in isolated rat fat cells. Propylthiouracil treatment lowered the level of alpha receptors in heart by 30% as measured by [3H]dihydroergocryptine binding, but increased the affinity about 2.5-fold. The highest level of alpha receptors was seen in control hearts. These studies indicate that thyroxine may control the turnover of beta-adrenergic receptors in heart and fat cells and regulate physiological responses in these tissues via a hormone-hormone interplay system. Thyroxine treatment reduced the activity of the membrane-bound Mg2+-ATPase (EC 3.6.1.3) and 5'-mononucleotidase (EC 3.1.3.5) but appears to increase the activity of the (Na+ + K+)ATPase (EC 3.6.1.4).     

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.     

High number of high-affinity binding sites for (-)-[3H]dihydroalprenolol on isolated hamster brown-fat cells. A study of the beta-adrenergic receptors.

The beta-adrenergic receptors of hamster brown adipocytes have been characterised by binding of the radioactive ligand (-)-[3H]dihydroalprenolol, directly to isolated intact cells in suspension. The brown fat cell contains 57,000 specific and saturable binding sites which have a dissociation constant (Kd) for [3H]dihydroalprenolol of 1.4 nM as determined by Scatchard analysis. The kinetically derived Kd, determined from forward and reverse rate constants, is 5 nM. Both of these values are in agreement with the dissociation constant (Kd = 2.2 nM) for alprenolol, determined from competition studies with [3H]dihydroalprenolol in these cells. Beta-adrenergic agonists competed for the specific binding sites with a typical beta 1-adrenergic specificity. The order of potency of agonists agrees well with the ability of these agents to stimulate respiration in isolated brown adipocytes: 50% stimulation of respiration occurs with apparently less than 10% occupancy of binding sites. Both the high affinity and high number of specific binding sites of [3H]dihydroalprenolol in brown fat cells presumably reflect the generally accepted dominating role of catecholamines in the regulation of brown fat metabolism and non-shivering thermogenesis.     

(+/-)-[3H]Epinephrine and (-)[3H]dihydroalprenolol binding to beta1- and beta2-noradrenergic receptors in brain, heart, and lung membranes.

(+/-)-[3H]Epinephrine binds to beta-receptors in calf cerebellar and rat lung membranes in the presence of 1.0 mM pyrocatechol and 1.0 microM phentolamine, with dissociation constants at 4 degrees C of 11 nM and 24 nM, respectively. (+/-)-[3H]Epinephrine associates to equilibrium within 20 min in both tissues, and over 50% of the binding is rapidly dissociable. Inhibition of binding by agonists and antagonists is highly stereoselective, and the structure-activity relationships of adrenergic agents in inhibiting (+/-)-[3H]epinephrine binding suggest an interaction with beta2 type noradrenergic receptors. (-)-Isoproterenol has an apparent Ki of 2 nM, (-)-epinephrine is 1.5 to 3 times weaker, and (-)-norepinephrine is 30 to 60 times weaker. Salbutamol and terbutaline, selective beta2-agonists, are potent inhibitors of binding, as are several nonspecific antagonists. Properties of the sites labeled by (+/-)-[3H]epinephrine in calf cerebellum and rat lung are closely similar. (-)-[3H]Dihydroalprenolol binding in calf cerebellum and rat lung also shows beta2 characteristics. Antagonists have similar potencies in inhibiting (-)-[3H]dihydroalprenolol and (+/-)-[3H]epinephrine binding in both tissues, but agonists are in general more potent inhibitors of (+/-)-[3H]epinephrine. Sodium and lithium selectively lower the affinity of (+/-)-[3H]epinephrine at its binding sites and the affinities of agonists, but not antagonists, at the (-)-[3H]dihydroalprenolol site. Specific (+/-)-[3H]epinephrine binding was not detectable in calf cortex and rat heart, where (-)-[3H]dihydroalprenolol binding suggests a beta1-receptor. A physiological significance of (+/-)-[3H]epinephrine binding is suggested by the strong correlation for agonists and antagonists between affinities in inhibiting binding, and in stimulating or inhibiting a beta-receptor-coupled adenylate cyclase in frog erythrocytes.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.