Decreased number of beta-adrenergic receptors in hypertensive vessels.

Responsiveness to inotropic agents is altered in hypertension and may contribute to its initiation and maintenance. A biochemical basis for this change was provided by the observation that the number of beta-adrenergic receptors, as reflected in specific [3H]dihydroalprenolol binding, was diminished in both arteries and veins of spontaneously hypertensive rats. There was no change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in beta-adrenergic receptor numbers is not secondary to blood pressure elevation but may, instead, contribute to the pathogenesis of hypertension.     

Evidence for heterogeneous distribution of α1, α2- and β-adrenergic binding sites on rat-liver cell surface

Fractionation of preparations of rat-liver membranes on linear sucrose gradients revealed different profiles for the binding of α₁-, α₂- and β-adrenergic radioligands. The peaks of binding activities of [³H]prazosin and [³H]epinephrine were clearly separated from those of [³H]yohimbine and [¹²⁵I]iodocyanopindolol which appeared at lower sucrose densities. Enzyme marker activities in the sucrose subfractions indicated the presence of plasma membranes in all of the subfractions. Furthermore, the binding peaks of the various adrenergic radioligands cannot be correlated with the presence of membranes derived from microsomes, lysosomes or Golgi apparatus. Pretreatment of rat livers with concanavalin A, in order to prevent the fragmentation of the plasma membranes during isolation, resulted in the shift of the binding of [³H]yohimbine and [¹²⁵I]iodocyanopindolol to sucrose-gradient subfractions of higher densities, clearly separate from fractions containing microsomes and Golgi apparatus. There was no distinct separation of the binding peaks of prazosin, yohimbine, and cyanopindolol in sucrose-gradient subfractions from concanavalin A-pretreated livers. These results are consistent with the hypothesis that α₁-, α₂-, and β-adrenergic binding sites are associated with plasma membranes, and are heterogeneously distributed on the rat-liver cell surface.     

The effect of age and cholesterol on the rat lung beta-adrenergic system

To assess the influence of membrane lipid composition on beta-adrenergic receptor number and adenylate cyclase activity in aging, we investigated the effect of cholesteryl hemisuccinate on these parameters in lung membranes of 3-, 12-, and 24-month-old CDF (F-344) rats. When cholesteryl hemisuccinate (0.5 mg/ml) was incubated with lung membranes, beta-adrenergic receptor density was increased by 70%. This effect was the same for each age group studied and indicated that the density of both basal and CHS-sensitive receptors is unaltered in rat lung with age. Forskolin, NaF, p[NH]ppG, and isoproteronol-stimulated adenylate cyclase activity is 30% lower in lung membranes from aged rats. Since enzyme activity is affected by the lipid environment and membrane composition often changes with age, we assessed adenylate cyclase activity following cholesteryl hemisuccinate incorporation. There was up to a 75% decrease in adenylate cyclase activity following cholesteryl hemisuccinate incorporation in lung membranes in each of the three age groups. In untreated membranes, there was no significant difference in cholesterol or lipid phosphate content with age. These data suggest that cholesterol content does not account for alterations in senescent rat lung adenylate cyclase activity.     

Reduced number of beta-adrenergic receptors in the myocardium of spontaneously hypertensive rats.

Inotropic response to β-adrenergic stimulation of the myocardium is decreased in hypertension. A biochemical basis for this decrease was provided by the observation that the number of β-adrenergic receptors — as reflected in specific [³H]dihydroalprenolol binding — was diminished in the myocardium of spontaneously hypertensive rats without a change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in β-adrenergic receptor numbers is not secondary to blood pressure elevation and may be related to increased sympathetic drive in spontaneously hypertensive rats.     

Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization

In order to purify milligram quantities of turkey β-adrenergic receptor (βAR) for structural analysis, we have expressed mutant βARs using the baculovirus system. The initial βAR construct was truncated at both N- and C-termini thus removing an N-glycosylation site. Cys 116 was mutated to leucine and a histidine tag was added at the C-terminus resulting in the βAR construct 20-424/His6. Expression of this construct in Sf9 cells produced 0.5 mg of unpurified receptor per liter of culture which necessitated the use of a fermenter for large-scale production. The yield was improved more than 2-fold to 1.2 mg/l culture by using Tni cells which facilitated the production of receptor on a 4 litre scale in shake cultures. The receptor was purified to homogeneity with 35% recovery giving a yield of 2 mg receptor. A further deletion at the N-terminus (βAR 34-424/His6) eliminated proteolysis which had been observed with the original construct and also increased expression more than 5-fold to 360 pmol/mg solubilized membrane protein. This expression level is one of the highest reported for a G protein-coupled receptor (GPCR) and has enabled us to purify 10 mg βAR for large-scale crystallization experiments.     

Role of cyclic AMP-dependent protein kinase activation in regulating rat submandibular mucin secretion

The extent of activation of rat submandibular gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was determined in vitro using dispersed cells to assess the involvement of this enzyme in submandibular mucin secretion. cAMP-dependent protein kinase activation, as determined by activity ratio method, was markedly increased following β-adrenergic receptor activation. 0.5 M NaCl was required in the homogenization buffer for stabilization of the hormonally activated cAMP-dependent protein kinase. A role for cAMP-dependent protein kinase activation in regulating mucin secretion was strongly suggested by the following: (1) the kinase activity ratio increased rapidly after β-adrenergic receptor stimulation; (2) dose-response relationship of the kinase activation following β-adrenergic receptor activation correlated with isoproterenol induced mucin release; (3) termination of β-adrenergic mediated mucin secretion caused a rapid decrease in the kinase activity ratio; (4) dibutyryl cyclic AMP stimulation caused an increase in the kinase ratio; whereas (5) pure cholinergic and pure α-adrenergic receptor stimulation had no effect on endogenous kinase activity. Although cAMP-dependent protein kinase activation may not be the only regulator of mucin secretion, these data suggest an important regulatory role for this kinase activation during rat submandibular mucin release.     

Acetylcholine receptors of rat lymphocytes

The conditions of the binding of acetylcholine have been studied in lymphocytes isolated from rat peripheral lymph nodes. Acetylcholine appeared to penetrate the lymphocyte membrane. We have confirmed the presence of muscarinic receptors, which, however, are not involved in transport of acetylcholine through the membrane. The receptors of the nicotine type on lymphocytes are demonstrated by the decrease of acetylcholine binding in the presence of a specific antagonist, tubocurarine. These nicotinic receptors may be involved in acetylcholine transport into the cells.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte: Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K⁺ concentration on both [³H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between β-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [³H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K⁺ concentrations, and at each K⁺ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K⁺ concentrations. In contrast to basal potassium influx, which is 50–70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K⁺, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to β-adrenergic receptor occupancy over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy β-adrenergic receptor sites function independently of each other.Analysis of the relation of catecholamine-dependent potassium transport to the number of β-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and beta-adrenergic receptors in cultured hepatocytes. Dependence on protein and RNA synthesis and suppression by isoproterenol

Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.     

Characterization of β-adrenergic receptor subtypes in androgen-induced mouse kidney hypertrophy using a new high-affinity ligand, [125I]iodocyanopindolol

In fully developed androgen-induced hypertrophy of female mouse kidney, β-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [¹²⁵I]iodocyanopindolol, with no change in apparent dissociation constants (K_d range 20–25 pM). Membrane protein relative to total kidney protein, Na⁺/K⁺-dependent ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5) activities and cholesterol content per unit membrane protein did not differ significantly in preparations from control and treated animals. The binding of iodocyanopindolol to kidney membranes was characterized with respect to association and dissociation kinetics, and also in regard to the less-specific contributions of other major catecholamine or indolamine receptors, using mixtures of the corresponding specific competitors. β1-selective drugs, practolol and metoprolol, and β2-selective agents, IPS-339 and zinterol, were competed with iodocyanopindolol to assess the receptor type specificity, and the ensuing binding profiles were dissected by a nonlinear regression analysis as described by Munson, P.J. and Rodbard, D. (Anal. Biochem. (1982) 107, 220–239). Most of the androgen-induced β-adrenergic receptors had the binding properties corresponding to β2-subtype. No consistent increase in the density of β1-adrenergic recepotors could be shown.     

Increased number of β-adrenergic receptors in the hypertrophied myocardium

Development of cardiac hypertrophy is associated with depletion of endogenous catecholamine stores and increased inotropic response to exogenous catecholamines. A biochemical basis for these changes is provided by the observation that the number of cardiac β-adrenergic receptors—as reflected in specific [³H]dihydroalprenolol binding—is increased in hypertrophy without a change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. This change in β-receptor numbers may be an important adaptive mechanism for preserving the contractile performance of the hypertrophied myocardium.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

Inactivation of hepatic glucocorticoid receptors by heparin

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4°C. However in the presence of heparin (40 μg per ml cytosol) receptor binding decreased about 75%. At 25°C liver receptor specific binding was found to have a half0life of about 60 min in control cytosol. However, in the presence of heparin (40 μg per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25°C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4°C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4°C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4°C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucococorticoid-receptor complexes in low salt gradients from 7–8 S to about 3–4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6—7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of beta-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma

An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several beta-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the beta-adrenergic blockers stereoselective pharmacokinetics.     

Rapid reconstitution of a transmembrane protein into supported planar lipid membranes

In hepatocytes from control rats, the ureogenic action of epinephrine is mainly mediated through alpha 1-adrenoceptors and the effect is independent of the presence of extracellular calcium. In hepatocytes from adrenalectomized rats, both alpha 1- and beta-adrenoceptors are involved in the action of epinephrine. Furthermore, the alpha 1-adrenergic-mediated stimulation of ureogenesis in these cells is dependent on the presence of extracellular calcium. Our results indicate that glucocorticoids modulate the calcium dependency of alpha 1-adrenergic effects and are consistent with our suggestion that two pathways are involved in the transduction of the alpha 1-adrenergic signal.