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.     

Uncoupling of the beta-adrenergic receptor as a mechanism of in vitro neutrophil desensitization

Human leukocytes have been useful in studying desensitization phenomena to beta-adrenergic agonists in a number of clinical conditions. For example, we have previously shown that oral terbutaline causes a time-dependent decrease in neutrophil (PMN) beta receptor number, using the beta antagonist ligand [3H]dihydroalprenolol (DHA), in conjunction with a significant loss of isoproterenol-induced adenylate cyclase activity. In the present in vitro study we have explored the mechanism for beta-adrenergic desensitization and have compared conditions for homologous and heterologous desensitization, using the intact PMN model. PMN preincubated with isoproterenol (10(-4)M), washed thoroughly, then restimulated, desensitize rapidly so that within 10 min 80% of control isoproterenol-induced cyclic AMP stimulation is lost. Cells washed free of isoproterenol recover full responsiveness in 1 to 2 hr. The estimated isoproterenol desensitization EC50 in cells washed and then restimulated is 1 X 10(-5)M, and the EC50 in unwashed cells that are restimulated is 9 X 10(-8)M. Rank-order potency studies of catecholamine desensitization show isoproterenol greater than epinephrine greater than norepinephrine, a beta-2 pattern. Isoproterenol-induced desensitization results in a small reduction in [3H]DHA binding sites, which becomes statistically significant (p less than 0.05) from control values at 1 hr (67% of control) and 3 hr (64%). Since the change in number of beta receptors did not explain the profound, rapid loss of beta agonist-induced cyclic AMP responsiveness, we explored the possibility of an uncoupling phenomenon. In the absence of GTP, isoproterenol binding is characterized by an EC50 of 6.6 +/- 2.6 X 10(-7)M, which is significantly different (p less than 0.05) from the EC50 of 38.1 +/- 9.1 X 10(-7)M found when cells are previously desensitized with isoproterenol for 10 min. GTP does not affect the EC50 of desensitized cells. These findings are consistent with the uncoupled receptor state fitting the model described by Su et al. Finally, prolonged (3 hr) isoproterenol preincubation results in a small but significant (p less than 0.05) loss of cyclic AMP responsiveness to histamine (67.7% +/- 11.7 of control) and PGE1 (59.3% +/- 7.4), suggesting heterologous desensitization. These studies suggest that the human PMN is a suitable model to study both homologous and heterologous desensitization in vitro.     

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.     

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)     

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)     

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)     

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.     

Pineal beta adrenergic receptor: correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase.

3H-l-Alprenolol, a potent competitive beta adrenergic antagonist, binds to sites in rat pineal gland membranes. The properties of these binding sites were compared to those of the receptors which mediate the beta adrenergic activation of pineal adenylate cyclase. Both sites are highly stereospecific. The l-stereoisomers of alprenolol and propranolol were at least two orders of magnitude more potent than the d-stereoisomers in inhibiting isoproterenol-stimulated adenylate cyclase or 3H-l-alprenolol binding. The dissociation constants (Kd) of the l-stereoisomers of both alprenolol and propranolol were 10 to 22 nM as determined by competition for binding sites or by inhibition of isoproternol-stimulated adenylate cyclase. Beta adrenergic agonists which stimulated adenylate cyclase also competitively inhibited the binding of 3H-l-alprenolol. They showed the same order of potency (isoproterenol greater than norepinephrine greater than or equal to epinephrine) and the same individual affinities in the two systems. Alpha adrenergic blockers were ineffective in inhibiting either adenylate cyclase stimulation or 3H-l-alprenolol binding. Isoproternol stimulation of adenylate cyclase acrivity, and 3H-l-alprenolol binding, were rapid and rapidly reversible. The 3H-l-alprenolol binding sites were saturable and bound 0.6 pmol of ligand per mg of added protein. The data suggest that the binding of 3H-l-alprenolol occurs at sites indistinguishable from the pineal beta adrenergic receptor.     

Effects of clenbuterol and antidepressant drugs on beta adrenergic receptor/N-protein coupling in the cerebral cortex of the rat

Repeated administration of the centrally acting beta adrenergic agonist clenbuterol to rats reduced the ability of isoproterenol to increase levels of cyclic AMP in slices of cerebral cortex. This lessened response to isoproterenol was not due to a reduction in beta receptor density but appeared to be due to diminished receptor/N-protein coupling. This was determined by measuring the ability of isoproterenol to inhibit the binding of the beta adrenergic antagonist [125I]iodopindolol to membranes prepared from cerebral cortex. Using membranes prepared from vehicle-treated rats, isoproterenol, in the absence of GTP, inhibited the binding of [125I]iodopindolol with an IC50 value of 85 nM and a Hill coefficient of 0.65. GTP (250 microM) increased the IC50 value to 290 nM and the Hill coefficient to 0.98. After repeated administration of 10mg/kg of clenbuterol to rats for 8 days, isoproterenol inhibited the binding of [125I]iodopindolol with an IC50 value of 125 nM and a Hill coefficient of 0.90; GTP increased the IC50 value to 170 nM and the Hill coefficient to 0.98. It was inferred from the results of modeling of the isoproterenol competition curves that the repeated administration of clenbuterol reduced or eliminated the high-affinity component of isoproterenol binding. These effects of clenbuterol were found to depend on dose and duration of treatment and were reversible. Repeated administration of the antidepressant drugs desipramine, imipramine, phenelzine, zimelidine and mianserin twice daily for 21 days, by contrast, did not affect receptor/N-protein coupling.(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.     

Characterization of high-affinity beta2-adrenergic receptor binding of (-)-[3H]-dihydroalprenolol to human polymorphonuclear cell particulates.

Human PMNs have well-described responses to beta-adrenergic catecholamines; these include elevation of cellular levels of cyclic AMP and inhibition of the release of lysosomal contents. Using the radioactive beta-adrenergic antagonist (-)-[3H]DHA in direct ligand-binding studies, we have identified and characterized beta-adrenergic receptors on particulate preparations of PMNs. These particulates bind DHA rapidly (t1/2 less than 1 min) and reversibly (t1/2 = 8 to 9 min). DHA binding is saturable and of high affinity (dissociation constant = 1 to 5 nM) and low capacity (870 +/- 128 receptors/cell, mean +/- S.D.) to a single class of binding sites. Competition for DHA binding sites by both beta-adrenergic agonists and antagonists is stereoselective [(-)-isomers more potent that (+)-isomers]. The rank order of potency of adrenergic agents in such competition studies indicates that these receptors are of the beta2 type. Since PMNs can be obtained in high purity with relative ease, the combined use of pharmacologic and ligand-binding studies in PMNs provide a useful system for studying beta-adrenergic receptors and their function in human subjects.     

Ethanol enhancement of isoproterenol-stimulated melatonin and cyclic AMP release from cultured pineal glands

Norepinephrine stimulates the synthesis of melatonin in the pineal gland. The action of norepinephrine is believed to be mediated primarily by beta adrenergic receptors, and involves activation of adenylate cyclase. Ethanol, 25 to 50 mM, added to cultured pineal glands in vitro, enhanced isoproterenol-induced stimulation of cyclic AMP and melatonin production. The action of ethanol was observed only at doses of isoproterenol that produced a submaximal effect, and ethanol alone had no effect on cyclic AMP or melatonin release. Butanol, at a concentration of 2 mM, was as effective as 50 mM ethanol in increasing isoproterenol-stimulated cyclic AMP and melatonin release, indicating that the response to alcohols was not due simply to changes in osmolarity, and may reflect a hydrophobic interaction of the alcohols with the cell membrane. The effects of ethanol on pineal cyclic AMP and melatonin release were reversible after a 15-min preincubation, but not after a 2-hr preincubation, suggesting that, over a long incubation period, ethanol may sensitize the pineal beta adrenergic receptor-coupled adenylate cyclase system to isoproterenol. The findings in this study are consistent with earlier work showing that ethanol increases cerebral cortical beta adrenergic receptor-coupled adenylate cyclase activity, and demonstrate that the effect of ethanol on the receptor-effector system can result in an endocrinological response.     

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)     

Adenylate cyclase and beta adrenergic receptor development in the mouse heart

We have demonstrated previously a postnatal peak for the beta adrenergic receptor in the heart and detected the appearance of a beta adrenergic receptor before an (-)-isoproterenol inducible increase in heart rate. The present study examined 1) agonist displaceable [3H] dihydroalprenolol (DHA) binding in the neonatal and adult mouse heart and 2) adenylate cyclase in fetal, neonatal and adult mouse heart. 3[H]DHA binding displaceable by (-)-isoproterenol gave a similar Ki from 1 day neonate through adult. Similar to the result found for antagonist displacement binding, there was a dramatic increase in the agonist displaceable [3H] DHA binding postnatally. The maximum was achieved in 2 weeks and then gradually declined to adult level. Cyclase activity (basal, (-)-isoproterenol- and NaF- stimulated) paralleled beta adrenergic receptor increases before birth. However, no early postnatal peak was present. In the 13 day fetal mouse heart, there is no (-)-isoproterenol increase in heart rate, but beta adrenergic receptor (13 +/- 4% of adult) and (-)-isoproterenol-stimulated adenylate cyclase activity (15 +/- 5% of adult) are present. It is concluded that 1) no significant difference exists between the agonist and antagonist displaceable [3H] DHA binding during development, 2) adenylate cyclase activity increases significantly during the last third of pregnancy in parallel with the beta adrenergic receptor, 3) both the beta adrenergic receptor and adenylate cyclase activity can be detected before the heart rate responses and 4) total adenylate cyclase activity does not increase in parallel with the early postnatal beta adrenergic receptor peak.     

3,3′,5-Triiodothyronine Administration In Vivo Modulates the Hormone-sensitive Adenylate Cyclase System of Rat Hepatocytes

The ability of 10 muM epinephrine or isoproterenol to stimulate cyclic AMP accumulation was decreased in hepatocytes isolated from hyperthyroid (triiodothyronine treated) as compared to euthyroid rats. In the presence of methylisobutylxanthine, epinephrine or isoproterenol-stimulated cyclic AMP accumulation was approximately 65% lower in hyperthyroid as compared with euthyroid rat hepatocytes. The ability of glucagon to stimulate a cyclic AMP response was also decreased in the hyperthyroid state, when assayed in either the absence or presence of a methyl xanthine. The character of the catecholamine-stimulated cyclic AMP response was beta adrenergic in both the hyperand euthyroid states. No evidence for an alpha(2) adrenergic mediated component of catecholamine action on cyclic AMP levels was noted. Cyclic AMP phosphodiesterase activity of hepatocyte homogenates was not altered in the hyperthyroid state. Hormone-stimulated, guanine nucleotide- and fluoride-activatable adenylate cyclase activity was reduced in subcellular fractions obtained from hyperthyroid as compared with euthyroid rat hepatocytes. Beta adrenergic receptor binding was reduced approximately 35% and glucagon receptor binding reduced approximately 50% in the hyperthyroid as compared with euthyroid rat hepatocyte membrane fractions. The status of the regulatory components of adenylate cyclase were examined by in vitro treatment of subcellular fractions with cholera toxin. The ability of cholera toxin to modulate adenylate cyclase was not altered by hyperthyroidism. Cholera toxin catalyzed AD[(32)P]ribosylation of hyperthyroid and euthyroid rat hepatocyte proteins separated electrophoretically displayed nearly identical autoradiograms. Studies of the reconstitution of adenylate cyclase activity of S49 mouse lymphoma cyc(-) mutant membranes by detergent extracts from rat hepatocyte membranes, indicated that hyperthyroidism was associated with a reduced capacity of regulatory components to confer fluoride, but not guanine nucleotide activatability to catalytic cyclase. Thyroid hormones regulate the hormone-sensitive adenylate cyclase system of rat hepatocytes at several distinct loci of the system.     

Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors.Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.     

Desensitization of beta adrenergic receptors linked to adrenocorticotropin secretion

Stimulation of beta adrenergic receptors on AtT-20 cells increases intracellular cyclic AMP levels and adrenocorticotropin hormone (ACTH) release. Pretreatment of these cells with catecholamines reduces the ability of (-)-isoproterenol to stimulate both cyclic AMP formation and ACTH secretion. This beta receptor desensitization is time- and dose-dependent and is reversible. Various beta adrenergic agonists can induce this desensitization with a rank order of potency of salmefamol greater than or equal to (-)-isoproterenol greater than or equal to epinephrine greater than or equal to norepinephrine greater than or equal to (+)-isoproterenol. (+/-)-Propranolol but not practolol can block the (-)-isoproterenol-induced beta receptor desensitization. Long-term treatment of AtT-20 cells with (-)-isoproterenol reduces the density of beta receptors but does not affect the affinity of these sites for [3H]dihydroalprenolol. In addition to desensitizing beta receptors, (-)-isoproterenol pretreatment enhances basal ACTH secretion. This effect was dose-dependent and blocked by (+/-)-propranolol. Forskolin-stimulated cyclic AMP formation and ACTH secretion was not altered by (-)-isoproterenol treatment indicating that the desensitization of beta receptors on AtT-20 cells is the result of receptor-adenylate cyclase uncoupling. No cross-desensitization of corticotropin releasing factor or vasoactive intestinal peptide receptors occurred as (-)-isoproterenol treatment did not alter the effect of these peptides on cyclic AMP synthesis or ACTH secretion.     

Effect of imipramine and adrenocorticotropin administration on the rat brain norepinephrine-coupled cyclic nucleotide generating system: alterations in alpha and beta adrenergic components

Continuous treatment (1-3 weeks) with imipramine or adrenocorticotropin (ACTH) decreases the responsiveness of the norepinephrine-coupled cyclic nucleotide generating system in rat brain cerebral cortex. Experiments were undertaken to determine which component of the second messenger system is influenced by the hormone and antidepressant. Neither treatment modified the amount or function of extractable stimulatory guanine nucleotide binding protein or the activities of adenylate cyclase or phosphodiesterase. While both imipramine and ACTH treatment decreased the cyclic AMP response to norepinephrine, only imipramine administration influenced the response to isoproterenol. ACTH treatment was found to reduce the alpha adrenergic potentiation of isoproterenol- and 2-chloroadenosine-stimulated cyclic AMP production, as well as reduce the sensitivity of the norepinephrine response to prazosin. These findings indicate that imipramine and ACTH treatments decrease the responsiveness of the rat brain norepinephrine-stimulated cyclic AMP generating system through actions on the alpha and beta adrenergic receptor components. The results suggest that noradrenergic receptor activity may be under the control of adrenal and/or pituitary hormones.     

Changes in mechanical events and adenosine 3',5'-monophosphate levels induced by enantiomers of isoproterenol in isolated rat atria and uteri.

Beta adrenergic receptors of rat atria and uteri were examined with the use of enantiomers of isoproterenol as agonists and mechanical responses and adenosine 3',5'-monophosphate (cyclic AMP) levels as measured effects. Assuming that stereoselectivity reflects the unique asymmetry of receptors, potency differences between the enantiomers are expected to provide a sensitive indication of ligand binding. All effects in each tissue were investigated under similar experimental conditions. Both isomers produced the same maximum effect on all measured responses. Enantiomeric potency differences (in log units) for positive chronotropic and inotropic responses and increases in cyclic AMP levels in atria were 3.31, 3.51 and 3.48, respectively. In uteri, the values for reduction of spontaneous contractile amplitude and increases in cyclic AMP were 2.90 and 2.79 log units, respectively. Even though these absolute values varied slightly with the experimental conditions, they were consistently smaller in uteri than in atria. In both tissues, dose-response curves for production of mechanical effects were greater than 2 log units to the left of those for increases in cyclic AMP levels. Regardless of the interpretation of this phenomenon, the results show the following. 1) The stereoselectivity for isoproterenol-induced effects is different between the two tissues at both levels of response. Therefore, it is suggested that this reflects dissimilar beta adrenergic receptor types in rat atrium vs. rat uterus. 2) The stereochemical selectivity for isoproterenol-induced mechanical effects and increases in cyclic AMP is the same in rat atrium and in rat uterus. Therefore, the data support the postulate that cyclic AMP is formed from interaction of isoproterenol with a receptor that is similar to the one activated to produce a mechanical effect.     

Norepinephrine and BRL 37344 stimulate adenylate cyclase by different receptors in rat brown adipose tissue

The beta adrenergic activation of adenylate cyclase was examined in membrane homogenates of rat interscapular brown adipose tissue (IBAT). In control membranes, isoproterenol and norepinephrine (NE) stimulated adenylate cyclase with activation constants of about 20 and 300 nM, respectively. Exposure of rats to 4 degrees C for 3 days increased the maximal stimulation of adenylate cyclase to these agonists but did not alter the respective activation constants. The beta 1-selective antagonist 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethylamino-2-pr opa nol blocked isoproterenol stimulation of adenylate cyclase in control and cold-exposed membranes at a concentration 100 times lower than did the beta 2-selective antagonist erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobuta n-2-ol. These data indicate that typical adrenergic agonists stimulate IBAT adenylate cyclase via beta 1 receptors. (R*,R*)-4-[2-[2 [9 3-chlorophenyl)-2-hydroxyethyl]amino)propyl) phenyl]phenoxyacetic acid (BRL 37344), an atypical agonist with activity at the beta 3 receptor, stimulated adenylate cyclase in control membranes with an activation constant of approximately 700 nM. Membranes of cold-exposed rats exhibited a high affinity response to BRL 37344 similar to that seen in control membranes and, in addition, a low affinity response. BRL 37344 stimulation of adenylate cyclase was unaffected by 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethyl-amino-2-prop anol, whereas stimulation by NE or epinephrine was potently blocked.(ABSTRACT TRUNCATED AT 250 WORDS)