The ligand binding assay and its role in understanding adrenergic receptor function.
Explore the source record for details and available documents.
Humans↗
Biomedical subjects
C J Homcy
Publications and source records attributed to C J Homcy.
Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit.
The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.
Adrenergic alpha-Agonists↗
Characterization of the mammalian beta-2 receptor in 8 M urea.
Explore the source record for details and available documents.
Animals↗
Ischemic heart disease in systemic lupus erythematosus in the young patient: report of six cases.
To clarify the clinical spectrum of coronary arterial abnormalities in systemic lupus erythematosus, the data were reviewed on six patients who had a diagnosis of lupus at ages 15 to 29 years and who had ischemic heart disease before age 35. Two patients had coronary arteritis diagnosed on postmortem examination. In a third patient alterations in coronary arterial anatomy occurred with angiographic improvement temporally related to the initiation of steroid therapy. The other three patients had severe diffuse atherosclerotic coronary disease that was identified in two at postmortem examination. In the third patient the course of the disease strongly suggested coronary atherosclerosis, and eventually coronary bypass grafting was performed for relief of angina. In summary, clinically important extramural coronary arteritis and atherosclerosis both occur, although rarely, in young patients with lupus. Coronary artery disease may occur with or without coexisting active extracardiac lupus manifestations. Short-term steroid therapy and follow-up angiography for those with angina and in whom coronary arteritis is suspected warrant consideration. When stable coronary arterial anatomy is demonstrated on follow-up angiography, management is determined by the patient's symptoms irrespective of the prior history of lupus and, if indicated, cardiac surgery for symptomatic relief can be safely performed.
Adolescent↗
Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.
The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization.
Cell Fractionation↗
An antiidiotypic antibody that recognizes the beta-adrenergic receptor.
Antialprenolol rabbit antibodies were fractionated on an acebutolol affinity resin, followed by L-propranolol elution so as to separate a class of binding sites that mimic the beta-adrenergic receptor. Allotype-identicaL rabbits were immunized with this fraction. After 6 mo, antisera exhibited antiidiotypic activity inhibiting [3H]alprenolol binding to the original antibody and to rabbit antiacebutolol antibodies, which had a spectrum of ligand-binding properties identical to the original idiotype. Those antisera demonstrating the original idiotype. Those antisera demonstrating the most potent antiidiotypic activity also blocked [3H]alprenolol binding to the beta-adrenergic receptor of turkey membrane, canine pulmonary membrane, and rat reticulocyte. An idiotype affinity-purified fraction showed similar activity, inhibiting beta-receptor binding with a calculated dissociation constant (KD) of 53 nM. Isoproterenol-mediated adenylate cyclase activity was also inhibited in a competitive manner. The universality of recognition of these antiidiotypic antisera indicate that the three-dimensional structure of a receptor's binding site can be modeled by a subset of an elicited antibody population.
Acebutolol↗
Cellular mechanisms of impaired adrenergic responsiveness in neonatal dogs.
The myocardial responsiveness of conscious, instrumental dogs to exogenously administered isoproterenol and norepinephrine was investigated in neonatal, 6-wk-old, and adult animals. Comparable base-line values for peak left ventricular derivative of pressure with respect to time were observed in all age categories. However, when compared with adult responses, the sympathomimetic amine-induced increases in neonatal left ventricular dP/dt were significantly blunted at each concentration of adrenergic agonist examined, whereas the 6-wk-old puppies displayed an intermediate inotropic response. To investigate the cellular mechanisms of this blunted neonatal response, we correlated physiologic and biochemical measurements of the myocardial responses to catecholamines in each age category. When compared with adult myocardial membrane preparations, neonatal cardiac membranes were characterized in vitro by an increased density of beta-adrenergic binding sites, comparable affinity for adrenergic agonists and antagonists, and an enhanced coupling of adenylate cyclase activation to receptor occupancy. Simultaneous changes in either the serum catecholamine concentration or the membrane content of other intrinsic proteins failed to account for the observed neonatal increase in beta-adrenergic receptor density. These findings are most consistent with a compensatory mechanism of the cardiac cell membrane, whereby an inherent depression in the adrenergic responsiveness of the immature myocardium appears to induce the increase in receptor density and activation of adenylate cyclase.
Adenylyl Cyclases↗
Photoaffinity label for the beta-adrenergic receptor: synthesis and effects on isoproterenol-stimulated adenylate cyclase.
An azide derivative of the beta-adrenergic antagonist acebutolol has been synthesized and its effect examined on the isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity of rat reticulocytes. It behaved as an effective competitive antagonist (Kd = 2 X 10(-7) M) prior to photolysis. However, when the reticulocyte preparation pretreated with acebutolol azide was photolyzed, a noncompetitive inhibition of isoproterenol-stimulated adenylate cyclase was obtained. Photolysis of the azide derivative in buffer alone did not convert it to a product of higher affnity. Labeling of the beta-adrenergic receptor appeared to be irreversible; multiple washings could not reverse the inhibition produced during photolysis with the label whereas washing would completely reverse the antagonism produced by the same concentration of label prior to photolysis. The effect appears to be specific for the beta-adrenergic receptor because the inhibition could be blocked stereoselectively by propranolol and there was no inhibition of fluoride- or GMP-P(NH)P-stimulated adenylate cyclase. furthermore, no effect was observed on the glucagon-mediated stimulation of adenylate cyclase of liver membranes, whereas the catecholamine response in the same membranes was inhibited.
Acebutolol↗
Anti-alprenolol antibodies in the rabbit. A new probe for the study of beta-adrenergic receptor interaction.
We immunized rabbits with an antigen prepared by covalent linkage of alprenolol, a beta-adrenergic receptor antagonist, to bovine serum albumin. Competitive inhibition of [3H]dihydroalprenolol binding to antisera with a variety of unlabeled ligands revealed broad antibody specificity for beta-adrenergic antagonists and agonists. The antiserum was subjected to affintiy fractionation on hydroxybenzylpindolol-Sepharose 4B. Successive elution with 100 mM Tris HCl, 1M NaCl, 4 M LiBr, and 5 M guanidine yielded fractions with increasing affintiy for hydroxybenzylpindolol. The ligand-binding properties of these affinity-fractionated antibodies suggest that certain of these fractions recognize structural aspects of individual beta-adrenergic ligands which are irrelevant to their biological activity, whereas others can be used to distinguish shared functional properties, such as the ethanolamine side chain, within the structural heterogeneity of beta-adrenergic drugs. In particular, elution of hydroxybenzylpindolol-adsorbed antibody with (-)-propranolol allowed identification of an antibody fraction specific for the (-)-stereoisomer. Thus, affinity fractionation of antibodies raised against beta-adrenergic ligands can provide useful analogues for the further study of the recognition properties of the beta-adrenergic receptor.
Alprenolol↗
Beta receptor occupancy. Assessment in the intact animal.
Organ uptake of 125I-hydroxybenzylpindolol, a potent beta adrenergic antagonist, was determined after intravenous administration. Pretreatment with the beta agonist, epinephrine, inhibited an almost identical fraction of 125I-hydroxybenzylpindolol binding as did the antagonist, propranolol. Specific beta receptor binding accounted for 50% of total uptake in the lung and demonstrated the following characteristics. The dose-response curve for propranolol inhibition of 125I-hydroxybenzylpindolol binding duplicated that reported for its physiologic action. Simultaneous serum propranolol levels as determined by a sensitive radioimmunoassay allowed an apparent dissociation rate constant approximately 7 nM to be obtained that correlated closely with the results reported from membrane binding studies. Alpha blockade had no effect and inhibition of 125I-hydroxybenzylpindolol binding by propranolol demonstrated stereospecificity. After chemical sympathectomy with reserpine or 6-OH dopamine, there was a 100% increase in receptor specific binding. Finally, a scintillation camera was employed to visually and quantitatively detect 125I-hydroxybenzylpindolol displacement from the lung during intravenous propranolol administration in the living animal. Reversal of binding was rapid and an in vivo inhibition curve was generated. Such a method provides the potential for longitudinally assessing beta receptor occupancy and apparent affinity directly in man.
Adrenergic beta-Antagonists↗
Photoaffinity labeling of the beta-adrenergic receptor: lack of evidence for spare receptors in the reticulocyte.
Explore the source record for details and available documents.
Acebutolol↗
Atrial flutter with exit block.
The mechanism of atrial flutter is controversial. A 76-year-old woman with rheumatic heart disease was referred to our clinic with an unusual rhythm disturbance which initially appeared to be classic atrial flutter at a rate of 300 beats/min. Later tracings, however, demonstrated a rate exactly one-half that of the earlier ECGs, with an identical p-wave morphology and vector. This latter rhythm also behaved in a manner expected for a flutter mechanism in that both spontaneously and with carotid pressure high-degree atrioventricular block occurred without alteration of the underlying atrial mechanism. Finally, the two rates interchanged spontaneously over several days without any significant interval changes in medical therapy. These findings were initially explained as probable digoxin toxicity. The underlying mechanism, however, was more likely atrial flutter with exit block and in this patient may have represented another facet of her sick sinus syndrome. This unusual phenomenon is discussed in terms of previous reports and possible implications for the mechanism of atrial flutter.
Aged↗
Demonstration of the hydrophilic character of adenylate cyclase following hydrophobic resolution on immobilized alkyl residues. Critical role of alkyl chain length.
Explore the source record for details and available documents.
Adenylyl Cyclases↗
Fatty acid oxidation and esterification in isolated rat hepatocytes: regulation by dibutyryl adenosine 3',5'-cyclic monophosphate.
Isolated rat hepatocytes rapidly utilized [(14)C]palmitate and, in particular, synthesized large amounts of neutral lipids from palmitate. Incorporation into cellular lipids occurred at a linear rate proportional to the medium concentration of fatty acids. Oxidation of [(14)C]palmitate to CO(2) increased with time and was much slower than palmitate esterification. Since [(14)C]acetate and [(14)C]glucose were oxidized to CO(2) at a linear rate, the lag in fatty acid oxidation to CO(2) did not involve enzymatic steps subsequent to acetate formation. The relative contribution of palmitate to esterification and to CO(2) formation depended upon the molar ratio of palmitate to albumin (v) and the length of incubation. Dibutyryl cyclic AMP (1 mM) reduced the oxidation of palmitate and acetate to CO(2) by about 50 and 90%, respectively, but did not alter palmitate esterification. However, equivalent concentrations of sodium butyrate produced similar decreases in CO(2) formation. Dibutyryl cyclic AMP (1 mM) also stimulated palmitate oxidation to water-soluble products, principally ketone bodies, by 50-100%. Sodium butyrate exerted no effect, while monobutyryl cyclic AMP and cyclic AMP both stimulated this pathway significantly. These results indicate that both v and dibutyryl cyclic AMP regulate the metabolism of fatty acids by isolated hepatocytes and suggest that hormonal stimulation of adenyl cyclase controls hepatic lipid metabolism.
Acetates↗
Specificity of serine proteases for cleavage sites on proatrial natriuretic factor.
An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of 35S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF103-126, which cross reacts with proANF, ANF99-126 and ANF103-126, and a newly prepared antisera to synthetic ANF99-105, which uniquely recognizes ANF99-126, but not proANF or ANF103-126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF99-126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF103-126. This cleavage activity of serum is blocked by leupeptin (40 micrograms/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF99-126 while kallikrein cleaves only at the dibasic site to produce ANF103-126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.
Amino Acid Sequence↗
Comparison of the effects of clofibrate and halofenate (MK-185) in isolated rat hepatocytes.
Explore the source record for details and available documents.
Acetates↗
Synthesis and characterization of a radioiodinated photoaffinity probe for the alpha 1-adrenergic receptor.
A radioiodinated aryl azide analog, 2-[4-(4-azido-3- iodobenzoyl ) piperazin -1-yl]-4-amino-6, 7- dimethoxyquinazoline [(125I] CP65 ,526), of the highly selective alpha 1-adrenergic antagonist prazosin was synthesized and characterized using rat hepatic plasma membranes. Prior to photolysis, this ligand bound with high affinity (Kd 0.3 nM), stereoselectively and in a saturable manner to sites with an alpha 1-adrenergic specificity. When membranes pretreated with [125I] CP65 ,526 were irradiated with ultraviolet light, the ligand incorporated irreversibly into the receptor-binding sites, also with typical alpha 1-adrenergic specificity. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of such labeled membranes followed by radioautography revealed major bands at Mr = 77,000, 68,000, and 59,000 daltons. Labeling of each of these bands was inhibitable by a variety of adrenergic ligands, stereoselectively and with a specificity typical of the alpha 1-adrenergic receptor. Smaller peptides with molecular weights of 42,000 and 31,000 daltons also displayed prazosin-inhibitable [125] CP65 ,526-binding. However, as the labeling of these protein species was not inhibitable by other adrenergic agonists or antagonists, they are unlikely to represent subunits of the receptor. Further evidence that [125I] CP65 ,526 incorporates covalently upon photolysis was the ability to specifically label immunoglobulin heavy and light chains of an antiserum that recognized both this ligand and the parent compound, prazosin. This new, radioiodinated, high-affinity probe should thus be uniquely valuable for the molecular characterization of the alpha 1-adrenergic receptor.
Affinity Labels↗
beta-Adrenergic and cholinergic receptors in hypertension-induced hypertrophy.
Perinephritic hypertension was produced in dogs by wrapping one kidney with silk and removing the contralateral kidney 1 week later. Mean arterial pressure rose from 104 +/- 3 to 156 +/- 11 mm Hg, while left ventricular free wall weight, normalized for body weight, was increased by 49%. Muscarinic, cholinergic receptor density measured with [3H]-quinuclidinyl benzilate, fell in hypertensive left ventricles (181 +/- 19 fmol/mg, n = 6; p less than 0.01) as compared with that found in normal left ventricles (272 +/- 16 fmol/mg, n = 8), while receptor affinity was not changed. The beta-adrenergic receptor density, measured by binding studies with [3H]-dihydroalprenolol, rose in the hypertensive left ventricles (108 +/- 10 fmol/mg, n = 7; p less than 0.01) as compared with that found in normal left ventricles (68.6 +/- 5.2 fmol/mg, n = 15), while beta-adrenergic receptor affinity decreased in the hypertensive left ventricles (10.4 +/- 1.2 nM) compared with that found in the normal left ventricles (5.0 +/- 0.7 nM). Plasma norepinephrine levels were similar in the two groups, but myocardial norepinephrine levels were depressed (p less than 0.05) in dogs with hypertension. Moderate left ventricular hypertrophy induced by long-term aortic banding in dogs resulted in elevations in beta-adrenergic receptor density (115 +/- 14 fmol/mg) and decreases in affinity (10.4 +/- 2.2 nM) similar to those observed in the dogs with left ventricular hypertrophy induced by hypertension. Thus, our results suggest that perinephritic hypertension in the dog induces divergent effects on cholinergic and beta-adrenergic receptor density. The increased beta-adrenergic receptor density and decreased affinity may be a characteristic of left ventricular hypertrophy rather than hypertension.
Animals↗