Selective regulation of beta-1 and beta-2 adrenergic receptors by atypical agonists

The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol. Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells. Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of [125I]iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors. Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of [125I]iodopindolol by isoproterenol and diminished the potency of the agonist. GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity. When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation. Pindolol and celiprolol, however, caused no elevation of enzyme activity. Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr. Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.     

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of food deprivation effects on ornithine decarboxylase: ornithine decarboxylase induction by alpha and beta agonists

Short-term food deprivation (FD) of preweanling rat pups causes a marked and specific suppression of liver ornithine decarboxylase (ODC) induction by alpha and beta adrenergic agonists that is mediated by postreceptor mechanisms. In the present study, we demonstrate that FD also affects the ability of adrenergic agonists to induce hepatic ODC in older animals and that these changes differ from those occurring in neonates in the duration of FD associated with changes, the subcellular mechanisms involved and the organ specificity of the effect. FD (48 hr) of 30-day-old rats caused a specific suppression of liver ODC induction by the alpha agonist phenylephrine whereas the effect of the beta agonist isoproterenol was not changed. ODC induction by vasopressin or angiotensin was unaffected, whereas the effect of aminophylline was potentiated. FD of 30-day-old rats caused a marked suppression of both alpha and beta agonist action in the heart. FD of mature (60-day-old) rats was associated with an enhanced hepatic ODC response to phenylephrine and isoproterenol. Whereas the number of receptors in heart assessed by the binding of [3H]prazosin and [125I]pindolol to alpha and beta receptors, respectively, decreased in parallel with changes in responsivity, liver ODC responses did not correlate well with receptor changes. These findings support previous findings of altered sympathetic responsivity in heart, liver and fat during FD and indicate that the sympathetic nervous system responds to FD with a complex series of changes in both pre- and postsynaptic mechanisms.     

Effects of pindolol and propranolol on beta adrenergic receptors on human lymphocytes

The cardiovascular supersensitivity observed after discontinuation of administration of beta adrenergic receptor antagonists may be explained by an increase in the density of beta adrenergic receptors. Thus, a change in the density of receptors has been observed in human lymphocytes after administration of propranolol (Aarons et al., 1980). The effects of pindolol, a beta adrenergic receptor antagonist with intrinsic sympathomimetic activity, were compared with those of propranolol or placebo. Pindolol (10 mg q.i.d.), propranolol (40 mg q.i.d.) or placebo were administered to 12 subjects for 8 days. The density of beta adrenergic receptors was determined by Scatchard analysis of the specific binding of [125I]iodopindolol on membranes prepared from human lymphocytes. Administration of pindolol resulted in a 30 to 50% decrease in the density of beta adrenergic receptors. This decrease was apparent within 1 day of beginning pindolol administration and it persisted for at least 8 days after discontinuation of drug administration. The reversibility of the decrease in receptors observed after pindolol administration was studied in 27 subjects given propranolol, pindolol or placebo for 4 days in a double-blind cross-over trial. Propranolol consistently induced a small increase in the density of beta adrenergic receptors. The density of receptors returned to predrug values within 2 days after discontinuation of propranolol administration. Pindolol induced a 30 to 50% decrease in the density of receptors which, as observed previously, persisted for at least 10 days after discontinuation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness

The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoproterenol antagonism of cardioselective beta adrenergic receptor blocking agents: a comparative study of human and guinea-pig cardiac and bronchial beta adrenergic receptors.

pA2 values against isoproterenol were determined for a number of cardioselective and noncardioselective beta adrenergic receptor blocking agents using human and guinea-pig isolated atrial and bronchial or tracheal preparations to study possible species differences. No significant differences in pA2 values for propranolol, pindolol, Ro 3-4787, acebutolol, atenolol, practolol, metoprolol, H 87/07 and tolamolol on bronchial or tracheal beta adrenergic receptors of both species were found. With respect to atrial beta adrenergic receptors, significantly lower pA2 values for human preparations, as compared to guinea-pig preparations, were found for tolamolol and CI 775. These are the only two agents in the series that derive their cardioselectivities from specific nitrogen substitutents. The different potencies of only these two compounds in antagonizing isoproterenol on atrial beta adrenergic receptors of both species suggest a difference in an accessory receptor area close to the site that interacts with the nitrogen atom of beta adrenergic agents.     

Effects of beta-2 agonist on metabolic regulation in the fetal lamb lung

To study the effects of beta-2 agonist on metabolic regulation in fetal lamb lung, ritodrine hydrochloride, a preferential beta-2 agonist, was infused i.v. at a rate of 1.3 +/- 0.4 micrograms/kg/min (mean +/- S.D.) for 24 hr into six twin chronically catheterized fetal lambs starting between 0.86 and 0.91 gestation. Lung glycogen was depleted 56% in the ritodrine-infused twins and glycogen phosphorylase a activity was increased 1.8-fold whereas glycogen synthase activity remained unchanged. Cyclic AMP-dependent protein kinase activity increased 1.7-fold, calcium-calmodulin-dependent protein kinase (phosphorylase kinase) activity increased 1.4-fold and calcium-phospholipid-dependent protein kinase (protein kinase C) activity increased 1.6-fold. In addition, the maximal binding capacity of pulmonary beta receptors decreased 49% in the ritodrine-infused twins. However, lung cyclic AMP content was unchanged after 24 hr of ritodrine infusion. We conclude that beta-2 agonist activates protein kinases, depletes glycogen and reduces the binding capacity of beta receptors in the fetal lamb lung. We speculate that these adrenergic mechanisms are involved in regulating the effects of beta-2 agonist on fetal lung liquid and surfactant production.     

Evidence for an agonist-induced, ATP-dependent change in muscarinic receptors of intact 1321N1 cells

The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate [( 3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine [( 3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cell competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H] NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.     

Interactions of flerobuterol, an antidepressant drug candidate, with beta adrenoceptors in the rat brain.

Interactions of dl-flerobuterol with central beta adrenoceptors were investigated. It inhibited the binding of [3H]CGP 12177, a selective beta adrenoceptor ligand, to membranes prepared from rat cerebral cortex, cerebellum, heart and lung. The affinity of dl-flerobuterol was very close in all tissues (Ki approximately 1 microM). In cerebral cortex, binding inhibition of [3H]CGP 12177 was stereospecific, l-flerobuterol (Ki = 483 nM) being 70-fold more potent than d-flerobuterol (Ki = 34 microM). Moreover, dl-flerobuterol (Ki = 926 nM) was 7-fold less potent than isoproterenol (Ki = 140 nM) to displace [3H]CGP 12177 binding, but 5-fold more potent than salbutamol (Ki = 4600 nM). Flerobuterol did not inhibit the radioligand binding to the other receptors at the highest concentration tested, thus leading to a very high beta adrenergic selectivity. Flerobuterol increased the concentration of cyclic AMP in slices of rat cerebral cortex in a dose-dependent manner; this effect was antagonized by atenolol and propranolol. Compared to isoproterenol or norepinephrine, which produced cyclic AMP maximal increases of 380 and 460%, respectively, it showed a weaker activity with a maximal stimulation obtained at 100 microM, corresponding to a cAMP increase of 140% over basal value (100%). These data revealed that flerobuterol possessed a beta adrenergic agonist activity. Moreover, it antagonized competitively the isoproterenol- or norepinephrine-stimulated accumulation of cAMP. At low concentrations of isoproterenol or norepinephrine, the stimulation of adenylate cyclase was only due to the action of flerobuterol, but at higher concentrations, the response of isoproterenol or norepinephrine was competitively blocked by flerobuterol. At 10 microM, isoproterenol surmounted fully this antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroanatomically selective down-regulation of beta adrenergic receptors by chronic imipramine treatment: relationships to the topography of [3H]imipramine and [3H] desipramine binding sites

The down-regulation of beta adrenergic receptors by chronic imipramine treatment was investigated with high resolution autoradiography of [125I]pindolol binding to brain sections. Neuroanatomically selective down-regulation of [125I]pindolol binding was found after chronic imipramine treatment. Subdivisions of the amygdala and hippocampus and discrete cortical regions were differentially affected. In the hippocampus, reduction of [125I]pindolol binding was observed in imipramine-treated rats in the CA-1 stratum radiatum and dentate molecular layer, but not in the CA-3 stratum radiatum. In the amygdala, the basolateral nucleus exhibited reduced [125I]pindolol binding after imipramine treatment but the central and medial nuclei were not affected. Chronic imipramine treatment was also associated with reduced [125I]pindolol binding in layer 1 of the cingulate cortex and layer 3 of the piriform cortex. In contrast, no effect on [125I]pindolol binding was apparent in the ventrolateral thalamic nucleus, caudate-putamen, lateral hypothalamus or layers 2 and 3 of the somatosensory cortex. In order to determine if regional variation in binding sites for imipramine, or its pharmacologically active metabolite desipramine, was responsible for the observed neuroanatomically selective reduction in [125I]pindolol binding, the binding of [3H]imipramine and [3H]desipramine was investigated. In some brain regions that exhibited high densities of [3H]imipramine and [3H]desipramine binding sites, [125I]pindolol binding was reduced after chronic treatment with imipramine. However, other regions that contained high densities of binding sites for antidepressant drugs did not show a reduction in [125I]pindolol binding after chronic imipramine treatment. Thus, regional binding of [3H]imipramine or [3H]desipramine cannot fully explain the neuroanatomical specificity of imipramine-induced beta adrenergic receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta Adrenergic Receptors of Polymorphonuclear Particulates in Bronchial Asthma

We have tested the beta adrenergic receptor theory of bronchial asthma by determining the number and affinity of binding sites of the beta adrenergic radioligand [(3)H]dihydroalprenolol (DHA) and the activity of adenylate cyclase in broken cell preparations of polymorphonuclear leukocytes (PMN). We studied 31 control subjects (group 1), 30 asthmatics receiving no systemic adrenergic medication (group 2), and 17 asthmatics receiving adrenergic agonists systemically (group 3). Control subjects and asthmatics taking no adrenergic drugs bound similar amounts of DHA at 0.5 nM and 30 nM DHA and had about 900 binding sites per PMN. In contrast, asthmatics receiving adrenergic agonists had a >70% decrease in their number of DHA binding sites per PMN (254+/-57). In a subset of our three groups of subjects (eight from group 1, six from group 2, and five from group 3) we measured DHA binding at several DHA concentrations and found similar values (0.4-0.7 nM) for the dissociation constant of DHA among these subjects.In further studies we examined the interaction of the agonist (-)-isoproterenol with beta adrenergic receptors in 8 normal subjects and 10 asthmatics not receiving adrenergic medication. We tested the ability of isoproterenol to compete for DHA binding sites and to stimulate adenylate cyclase in sonicates prepared from PMN and examined under identical conditions. The dissociation constants for the competition of isoproterenol for DHA binding sites in normal and asthmatic subjects were virtually identical ( approximately 1.0 muM). In addition, the (activation constant) values for stimulation of adenylate cyclase were similar (0.16-0.19 muM) in the two groups of subjects.Thus, these data suggest that asthma per se is not associated with alteration in either the number or affinity of beta adrenergic receptors in PMN. Our findings indicate that previous reports of abnormal beta adrenergic receptor function in asthmatic patients may in part be explained by prior treatment of such patients with adrenergic agonists. Because the asthmatics who received adrenergic agonists in our study tended to be more ill and to receive additional medication compared to subjects in group 2, we cannot rule out unequivocally that severe asthma may be associated with decreased binding to beta adrenergic receptors. Nevertheless, we conclude that beta adrenergic receptors on PMN from asthmatics are relatively normal unless such patients are treated with adrenergic agonists.     

Subtype-selective regulation of beta adrenergic receptor-adenylyl cyclase coupling by phorbol esters in 3T3-L1 fibroblasts

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-induced changes in beta adrenergic receptor density and receptor-mediated responsiveness in slices of rat cerebral cortex.

Incubation of slices of rat cerebral cortex with the beta adrenergic receptor agonist (-)-isoproterenol led to a 30 to 50% decrease in the number of binding sites for [125I]iodohydroxybenzylpindolol and to a 60 to 80% decrease in isoproterenol-stimulated cyclic AMP accumulation. The density of beta adrenergic receptors was also decreased following incubation with (-)-norepinephrine but not with (+)-isoproterenol or dopamine and the decrease in receptor density was blocked by co-incubation with the beta adrenergic receptor antagonist sotalol. The half-time for loss of receptors was approximately 3 min and recovery was observed during a 1 hr reincubation of tissue slices or following exposure to guanine nucleotides. A decrease in beta adrenergic receptor density was also observed following chronic treatment with desmethylimipramine which blocks norepinephrine reuptake and thus potentiates the effects of neurally released norepinephrine at adrenergic receptors. The loss of receptors induced in vitro could be reversed by reincubation or by exposure to guanine nucleotides. In contrast, the loss of receptors induced in vivo was not affected by these procedures.     

Impaired beta adrenergic receptor binding and function in cystic fibrosis neutrophils.

Cystic fibrosis (CF), a genetic disease characterized by abnormalities of exocrine gland and mucociliary function, has recently been shown to be associated with abnormal adrenergic and cholinergic physiologic responses in addition to decreased beta adrenergic-induced cyclic AMP generation in human leukocytes. In this study we have attempted to elucidate the nature of this hyporesponsiveness by assessing beta adrenergic receptor number and affinity (KD) in the intact neutrophil using the antagonist ligand [3H] dihydroalprenolol and cyclic AMP responses to isoproterenol in addition to histamine, and prostaglandin E1 in CF subjects, CF obligate heterozygotes (CFH), and normal control subjects. CF patients had significantly less (p less than 0.025) cyclic AMP stimulation above basals levels with isoproterenol (0.1 microM to 0.1 mM), compared with control values, but no consistent differences between groups were noted with histamine or PGE1. CF neutrophils had significantly fewer (p less than 0.005) beta adrenergic receptors per neutrophil (398.0 +/- 54.2 vs. 819.4 +/- 67.2) compared with control neutrophils, but the KD (0.740 +/- 0.11 vs. 0.630 +/- 0.05 nM) did not differ significantly (p greater than 0.05). There was no correlation between clinical severity and either cyclic AMP generation or dihydroalprenolol binding (r = 0.27 and 0.24, respectively, p greater than 0.05). The CFH group had approximately 50% of the cyclic AMP stimulation compared with controls, but the number (909.8 +/- 89.3) and KD (0.710 +/- 0.09 nM) of their beta adrenergic receptors were indistinguishable from control subjects. These findings suggest "down regulation" of the beta receptor in the CF patient. The cause of this remains unknown. Although the etiology of the decreased cyclic AMP responses in CFH was not due to decreased beta adrenergic receptors as assessed by antagonist ligand binding, further studies inthe CFH group to include agonist binding, receptor-adenylate cyclase coupling, intrinsic adenylate cyclase activity, and catecholamine metabolism may help determine the basic cause of beta adrenergic hyperesposiveness in both CFH and CF.     

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors

In tissues with two classes of binding sites for a drug, it is common to estimate the proportion of each class of binding site by inhibiting the binding of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of binding site, however, will be obtained only if the radioligand is nonselective or used at a concentration that saturates both classes of binding sites. A method of simultaneous regression analysis of multiple inhibition curves, using the program MLAB on the PROPHET system, was used to quantify the selectivity of radioligands for beta-1 or beta-2 adrenergic receptors. The selectivity of [125I]iodopindolol, [125I]iodocyanopindolol, [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol for beta-1 and beta-2 adrenergic receptors was assessed by inhibiting the binding of each radioligand with the beta-1-selective unlabeled ligand ICI 89,406 at increasing concentrations of the radioligand, using membranes prepared from C6 glioma cells, which have both beta-1 and beta-2 adrenergic receptors. Scatchard plots for all four radioligands were linear, with correlation coefficients greater than 0.95. [125I]Iodopindolol and [125I]iodocyanopindolol were 3.2- and 2-fold selective, respectively, and [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol were 5.8- and 2.3-fold selective, respectively, for beta-2 adrenergic receptors. Values obtained for the densities of beta-1 and beta-2 adrenergic receptors and the affinities of the receptors for ICI 89,406 were independent of the radioligand used.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro Desensitization of Beta Adrenergic Receptors in Human Neutrophils. ATTENUATION BY CORTICOSTEROIDS

The receptor alterations involved in catecholamine-induced desensitization of adenylate cyclase in human neutrophils have been investigated as has the ability of hydrocortisone to modify such alterations. Incubation of human neutrophils with isoproterenol for 3 h in vitro resulted in an 86% reduction in the ability of isoproterenol to stimulate cyclic AMP accumulation in the cells. Two types of receptor alterations were documented. There was a 40% reduction in the number of beta adrenergic receptors (42 vs. 25 fmol/mg protein, P < 0.005) present after desensitization as assessed by [³H]dihydroalprenolol ([³H]DHA) binding. In addition the receptors appeared to be relatively uncoupled from adenylate cyclase. This uncoupling was assessed by examining the ability of the agonist isoproterenol to stabilize a high-affinity form of the receptor, detected by computer modelling of competition curves for [³H]DHA binding. Desensitized receptors were characterized by rightward-shifted agonist competition curves. When hydrocortisone was added to the desensitizing incubations (combined treatment) there was a statistically significant attenuation in the desensitization process as assessed by the ability of isoproterenol to increase cyclic AMP levels in the cells. Although combined treatment did not prevent the decline in receptor number, it did attenuate the uncoupling of the receptors. Combined treatment resulted in competition curves intermediate between the control and the rightward-shifted desensitization curves. Prednisolone was similar to hydrocortisone in attenuating isoproterenol-induced uncoupling. Thus, steroids appeared to attenuate agonist-induced desensitization of the beta adrenergic receptor-adenylate cyclase system by dampening the ability of agonists to uncouple receptors without modifying their ability to promote down-regulation of beta adrenergic receptors.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Effects of ethanol administration and withdrawal on neurotransmitter receptor systems in C57 mice

C57BL/6 mice were treated with 7% (v/v) ethanol in a Bio-Serve liquid diet for 7 days. Some animals were then allowed to withdraw from ethanol for a period of 24 hr. The severity of the ethanol withdrawal was assessed by monitoring behavioral changes and by quantitating the decrease in body temperature that occurred during the first 16 hr of withdrawal. Animals withdrawn from ethanol for 24 hr showed a decreased hypothermic response to apomorphine suggesting that changes in dopaminergic systems had occurred. This possibility was further examined in homogenates of striatum by measuring dopamine-stimulated adenylate cyclase activity and the binding of [3H]spiroperidol. However, there were no changes observed in either basal- or dopamine-stimulated adenylate cyclase activity or in the density or affinity or receptors for [3H]spiroperidol. The affinity of apomorphine for the dopamine receptor was also unchanged. In other experiments, alpha and beta adrenergic receptor-mediated increases in cyclic AMP accumulation were assessed in slices of cerebral cortex. There was no change in cyclic AMP accumulation due to either alpha or beta adrenergic receptors. There was, however, a significant decrease in the density of beta adrenergic receptors in both the ethanol-treated mice and in the withdrawn mice. This decrease was restricted to the beta-2 receptor subtype with no change being observed in the density of beta-1 adrenergic receptors. Ethanol administration was also associated with a significant increase in the density of muscarinic cholinergic receptors in the hippocampus and cerebral cortex. The effect was not observed in animals allowed to withdraw for 24 hr.