Thyroid hormone differentially regulates development of beta-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney.

In mature animals, thyroid hormone produces parallel up-regulation of beta-adrenergic receptor binding sites and their linkage to adenylate cyclase; during development, these same processes may be critical in establishing the set-point for subsequent adrenergic reactivity. In the current study, we administered triiodothyronine to neonatal rats for the first five days postpartum and evaluated [125I]pindolol binding capabilities and adenylate cyclase activity in membrane preparations from heart and kidney. In the heart, hyperthyroidism elicited an initial increase in receptor density, with subsequent deficits and an eventual return to normal values by young adulthood. In contrast, the ability of isoproterenol, a beta-adrenergic agonist, to stimulate adenylate cyclase was enhanced regardless of whether receptor numbers were increased or decreased; the same effects were also present for basal adenylate cyclase activity and non-receptor-mediated stimulation by forskolin. Enhanced cyclase activity involved both increases in the magnitude of response as well as accelerated onset of the postweaning peak of enzyme activity, results which suggest a direct impact of thyroid status on the ontogenetic expression of adenylate cyclase itself. The kidney, which possesses less efficient beta-receptor coupling to adenylate cyclase in the neonate, was less drastically affected by triiodothyronine for either beta-receptor binding sites or enzyme activity. As we had previously shown that neonatal hyperthyroidism uncouples beta-receptors from growth-related enzymes, such as ornithine decarboxylase, we also evaluated whether the promotion of adenylate cyclase responses was mechanistically linked to effect on ornithine decarboxylase; administration of cyclic AMP analogs to 5 days-old rats led to inhibition of the enzyme in the heart, whereas the same treatment in 9 days-old animals was ineffective. These data suggest that thyroid hormone differentially regulates the development of beta-receptors as well as adenylate cyclase and ornithine decarboxylase, with preferential effects on tissues, such as the heart, that already possess efficient linkage of the receptors to cell transduction mechanisms at birth.     

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor-associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP-blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1-adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature.     

Role of glycosylation for beta 2-adrenoceptor function in A431 cells

A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.     

Multiple effects of N, N' dicyclohexyl carbodiimide on the beta-adrenergic receptor--adenylate cyclase system in frog erythrocytes.

Treatment of frog erythrocytes with N,N' dicyclohexylcarbodiimide (DCCD) leads to a loss of catecholamine stimulated adenylate cyclase activity without any decrease in fluoride or PGE1 stimulated cyclase. However, the concentrations of the reagent which inhibit catecholamine sensitive adenylate cyclase activity are 10 fold lower than those which inhibit specific [3H]dihydroalprenolol ([3H]DHA) beta-adrenergic receptor binding. By contrast binding of the readiolabeled beta-adrenergic agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) is considerably more sensitive than antagonist binding to the effects of DCCD. The data suggest that low concentrations of the reagent may modify the effector portion of the beta-adrenergic receptor leading to functional uncoupling of the beta-receptor adenylate cyclase system. At higher concentrations of the reagent the ligand bidning site of the beta-receptor appears also to be altered.     

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

In pigeon erythrocyte membrane, the beta-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, n-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 trinitro-phenols, indomethacin and octanoic acid did not affect the total number of beta-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.     

The relationship between beta-adrenoceptor regulation and beta-adrenergic responsiveness in hepatocytes. Studies on acquisition, desensitization and resensitization of isoproterenol-sensitive adenylate cyclase in primary culture

The role of beta-adrenoceptor regulation in the mechanisms controlling beta-adrenergic responsiveness in hepatocytes was explored, using primary monolayer cultures. When plated in vitro, these cells gradually acquire a strong catecholamine-sensitive adenylate cyclase activity and an enhanced ability to bind the beta-adrenoceptor ligand [125I]iodocyanopindolol (125ICYP). Examination of the time course showed that the increase in the number of 125ICYP binding sites was detectable within 1-2 h of culturing and slightly preceded the elevation of isoproterenol-responsive activity. Then the responsiveness rose steeply and between about 5-24 h it closely followed the increase in beta-receptor binding. Addition of isoproterenol (10 microM) to cells after 20 h of culturing caused a rapid homologous desensitization of the adenylate cyclase (50% after about 5 min). This was paralleled by a down-regulation of beta-adrenoceptors measured both in membrane particles and in total cell lysates. Removal of isoproterenol led to a resensitization of the adenylate cyclase, which was rapid and protein-synthesis-independent after a brief (10-min) desensitization, or slow and cycloheximide-sensitive after prolonged (4-h) exposure to the agonist. In both cases an up-regulation of the 125ICYP binding paralleled the recovery from refractoriness. In contrast, no concurring changes in 125ICYP binding were measured when the beta-adrenoceptor-linked adenylate cyclase activity was enhanced by pretreatment with pertussin toxin (islet-activating protein, IAP) or was desensitized by exposure of the cells to glucagon or 8-bromo-cAMP; however, these modulations of the adenylate cyclase were nonselective, since the pretreatments with IAP, glucagon or 8-bromo-cAMP affected both isoproterenol-sensitive and glucagon-sensitive activities. The results suggest that, in hepatocytes, regulation at the beta-adrenoceptor level is a major determinant for both short-term and long-term selective changes of the beta-adrenergic responsiveness.     

Promotional role for glucocorticoids in the development of intracellular signalling: enhanced cardiac and renal adenylate cyclase reactivity to beta-adrenergic and non-adrenergic stimuli after low-dose fetal dexamethasone exposure.

Fetal exposure to high doses of glucocorticoids, as used to aid lung maturation in the therapy of Respiratory Distress Syndrome, causes growth retardation and interference with development of beta-adrenergic receptor-mediated cell signalling. The current study examined whether lower levels of steroids might instead play a positive trophic role in receptor transduction. Pregnant rats were given dexamethasone at or below the threshold for growth impairment (0.05-0.2 mg/kg) on gestational days 17, 18 and 19, and the beta-receptor-mediated stimulation of adenylate cyclase was evaluated in membrane preparations from heart and kidney. The enzymatic response to isoproterenol was compared with effects on: (1) basal (unstimulated) adenylate cyclase, (2) adenylate cyclase stimulation mediated by forskolin, which bypasses the beta-receptor, and (3) development of beta-receptor binding capabilities, assessed with [125I]pindolol. In the heart, prenatal exposure to dexamethasone produced a dose-dependent enhancement of beta-receptor-mediated stimulation of adenylate cyclase activity; however, both basal and forskolin-stimulated activity were also increased and beta-receptor binding was relatively unaffected. These results suggest that enhanced responsiveness was occurring at the level of the cyclase itself, rather than by effects on receptors on their G-protein coupling to enzyme activity. Promotional effects on adenylate cyclase were detectable at the low dose of dexamethasone, without any evidence of growth impairment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prenatal terbutaline treatment: tissue-selective dissociation of perinatal changes in beta-adrenergic receptor binding from regulation of adenylate cyclase activity.

The role of prenatal beta-receptor stimulation in development of adrenergic reactivity was examined by administering the beta-agonist, terbutaline, to pregnant rats on gestational days 17, 18 and 19. On gestational day 20, liver membrane beta-receptor binding capabilities showed the depression characteristic of down-regulation, but heart and kidney receptor binding were essentially normal. Basal adenylate cyclase activity in the fetal liver membrane preparation was unchanged by terbutaline exposure and enzymatic reactivity to beta-adrenergic stimulation showed only a slight lowering; forskolin stimulation, however, was markedly increased in the terbutaline group. By postnatal day 2, receptor binding had returned to normal in the liver and remained at control levels in the other two tissues. Responsivity of adenylate cyclase to beta-receptor stimulation was markedly elevated in heart and kidney membranes; the effect represented an alteration at the level of the cyclase itself, rather than the receptor, since both basal activity and forskolin stimulation of the enzyme showed equivalent enhancement. These data thus suggest that early beta-adrenergic stimulation promotes cellular reactivity by fostering the development of membrane transduction mechanisms, rather than through effects on the receptor ligand binding site per se.     

Promotional role for glucocorticoids in the development of intracellular signalling: enhanced cardiac and renal adenylate cyclase reactivity to beta-adrenergic and non-adrenergic stimuli after low-dose fetal dexamethasone exposure.

Fetal exposure to high doses of glucocorticoids, as used to aid lung maturation in the therapy of Respiratory Distress Syndrome, causes growth retardation and interference with development of beta-adrenergic receptor-mediated cell signalling. The current study examined whether lower levels of steroids might instead play a positive trophic role in receptor transduction. Pregnant rats were given dexamethasone at or below the threshold for growth impairment (0.05-0.2 mg/kg) on gestational days 17, 18 and 19, and the beta-receptor-mediated stimulation of adenylate cyclase activity was evaluated in membrane preparations from heart and kidney. The enzymatic response to isoproterenol was compared with effects on: (1) basal (unstimulated) adenylate cyclase, (2) adenylate cyclase stimulation mediated by forskolin, which bypasses the beta-receptor, and (3) development of beta-receptor binding capabilities, assessed with [125I]pindolol. In the heart, prenatal exposure to dexamethasone produced a dose-dependent enhancement of beta-receptor-mediated stimulation of adenylate cyclase activity; however, both basal and forskolin-stimulated activity were also increased and beta-receptor binding was relatively unaffected. These results suggest that enhanced responsiveness was occurring at the level of the cyclase itself, rather than by effects on receptors or their G-protein coupling to enzyme activity. Promotional effects on adenylate cyclase were detectable at the low dose of dexamethasone, without any evidence of growth impairment. Furthermore, the effects displayed selectivity for age and tissue: adults treated with dexamethasone did not show the effect, and the heart was more sensitively affected than was the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.     

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.     

Lateral mobility of beta-receptors involved in adenylate cyclase activation

Cationized ferritin was found to inhibit the lateral mobility of intramembrane proteins in turkey erythrocyte membranes and the activation of adenylate cyclase by the (--)-epinephrine-bound beta-adrenergic receptor. It was observed that cationized ferritin has only a small direct effect on the beta-receptor and on the adenylate cyclase moiety. It is concluded that the cationized ferritin-induced inhibition of the hormone-dependent cyclase activity results from the inhibition of the lateral mobility of the receptor and therefore a decrease in the bimolecular rate of interaction between the receptor and the enzyme.     

The influence of dietary lipid supplementation on cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey

Dietary lipid supplements high in either saturated fat derived from sheep kidney fat or unsaturated fat derived from sunflower seed oil, and a low mixed fat reference diet were fed to marmoset monkeys for 20 months and the effects on cardiac membrane lipid composition, and myocardial catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor binding activity were investigated. For cardiac membranes enriched for beta-adrenergic binding activity, the dietary lipid treatment resulted in small changes in the proportion of saturated to unsaturated fatty acids and substantial changes in the (n - 6) to (n - 3) series of unsaturated fatty acids in the membrane phospholipids. The sheep kidney fat diet increased the cholesterol-to-phospholipid ratio in cardiac membranes in comparison to the other diets. This diet also significantly elevated basal and isoproterenol-, epinephrine- and norepinephrine-stimulated adenylate cyclase activity. The value of the dissociation constant (Kd) and the receptor number (Bmax) for the binding of [125I]ICYP to the beta-adrenergic receptor was significantly reduced in marmosets fed the sheep kidney fat diet. These results suggest that dietary lipids can influence the activity of the beta-adrenergic/adenylate cyclase system of the heart. Modulation of this transmembrane signalling system may be induced by changes in the properties of the associated membrane lipids, particularly by alteration in the membrane cholesterol-to-phospholipid ratio. This effect may be limited to those animal species in which the nature of the dietary fatty acid intake may be influencing cardiac membrane cholesterol homeostasis, which is in agreement with previous results in rats following dietary cholesterol supplementation (McMurchie et al. (1987) Biochim. Biophys. Acta 898, 137-153). ICYP, (-)-iodocyanopindolol.     

Inhibitors of transglutaminase prevent agonist-mediated internalization of beta-adrenergic receptors

In frog erythrocytes, desensitization of beta-adrenergic receptors is characterized by a decrease in the beta-receptor recognition sites in the plasma membrane and a concomitant increase in the number of this receptor's binding sites in the cytosol. We have documented that this redistribution of the receptor recognition sites reflects the internalization of the surface-bound beta-adrenergic receptors. The present study was addressed to examine whether transglutaminase plays a role in the agonist-mediated internalization of beta-adrenergic receptor recognition sites. Pretreatment of cells with methylamine was found to decrease the internalization and the loss of membrane-bound beta-adrenergic receptors induced by isoproterenol. Methylamine appears to be equally potent in inhibiting transglutaminase activity and in preventing internalization and the receptor loss. The effect of methylamine on soluble and on membrane-bound beta-adrenergic receptors is due to a change in Bmax rather than Kd of these binding sites. Among eight inhibitors of transglutaminase tested, the rank order potency for blocking the enzyme can be correlated with that for preventing the receptor loss and receptor internalization. Moreover, these drug effects on beta-adrenergic receptors are unrelated to the inhibition of isoproterenol-sensitive adenylate cyclase or the binding of [3H]dihydroalprenolol to beta-receptors. These result may lend credence to the view that transglutaminase participates in the internalization and the decrease of membrane-bound receptors during desensitization of beta-adrenergic receptors.     

Catecholamine-Sensitive Adenylate Cyclase in the White Perch (Roccus americanus) Retina: Evidence for β-Adrenergic and Dopamine Receptors Linked to Adenylate Cyclase

We have examined the catecholamine-sensitive adenylate cyclase in the retina of the white perch (Roccus americanus). Both dopamine and the beta-adrenergic agonist isoproterenol stimulate cyclic AMP accumulation in this retina, but serotonin, an indoleamine, and phenylephrine, an alpha-adrenergic agonist, had no effect. The stimulation of adenylate cyclase by isoproterenol is more potent and effective than that of dopamine. The effects of dopamine and isoproterenol are mediated via independent dopamine and beta-adrenergic receptors. Haloperidol, a dopamine antagonist, blocks the stimulatory effect of dopamine but not of isoproterenol. Conversely, propranolol, a beta-adrenergic antagonist, blocks the stimulatory effect of isoproterenol but not of dopamine. The effects of dopamine and isoproterenol are not additive. In fractions of purified horizontal cells we found evidence for dopamine receptors linked to adenylate cyclase but did not find evidence for the presence of cyclase coupled beta-adrenergic receptors. The cellular location of the beta-adrenergic receptors is unknown. Our findings demonstrate the existence of both beta-adrenergic and dopamine receptors coupled to adenylate cyclase in the white perch retina. However, we did not find either epinephrine or norepinephrine, endogenous ligands of the beta-receptor, to be present in retinal extracts subjected to HPLC.     

Beta-adrenergic receptors and myogenesis.

The rat myogenic cell line, L8, contains a beta-adrenergic catecholamine-sensitive adenylate cyclase. Prior to cell fusion, and continuing thereafter, beta-adrenergic sites, as determined by the stereospecific binding of (125I)-hydroxybenqylpindolol, I1(125I)IHYP] increases from 470 to 2000 sites/cell. There is also an increase in adenylate cyclase (2-5 fold) and endogenous cAMP (5-30 fold) following stimulation by catecholamine. The dissociation constant (KD) of (125I)IHYP for unfused and fused cell-homogenates, as determined by estimation with Scatchard analysis, by direct determination at receptor concentrations well below the KD, or by association (4.6 X 10(8) M-1 min-1); and dissociation (0.028 min-1) kinetics; ranged from about 40 to 70 pM. The acquisition of beta-receptors prior to fusion in L8 cells may implicate this system in the regulation of myogenesis.     

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.     

Identification of adenylate cyclase-coupled beta-adrenergic receptors in frog erythrocytes with (minus)-[3-H] alprenolol.

(minus)-Alprenolol, a potent, competitive beta-adrenergic antagonist labeled to high specific activity with tritium (17 Ci per mmol), has been used to identify binding sites in frog erythrocyte membranes having many of the characteristics to be expected of the beta-adrenergic receptors which are linked to adenylate cyclase in these membranes. The chromatographic behavior and biological activity of the labeled and native drug were essentially identical. (minus)-Alprenolol and (minus)-[3-H]alprenolol both competitively antagonize isoproterenol stimulation of frog erythrocyte membrane adenylate cyclase with a KD OF 5 TO 10 NM. (minus)-[3-H]Alprenolol binding to sites in the frog erythrocyte membranes was studied by a centrifugal assay. At 37 degrees, equilibrium binding was established within 5 min and the half-time for dissociation of bound (minus)-[3-H]alprenolol was approximately 30 s. This rapid onset and dissociation of (minus)-[3-H]alprenolol binding was in good agreement with the rapid onset of action of beta-adrenergic agonists and antagonists on the frog erythrocyte adenylate cyclase. (minus)-[3-H]Alprenolol binding was saturable. There were 0.25 to 0.35 pmol of (minus)-[3-H]alprenolol binding sites per mg of protein corresponding to 1300 to 1800 binding sites per intact frog erythrocyte. The binding sites showed half-maximal saturation at 5.0 to 10 nM (minus)-[3-H]alprenolol, which is in good agreement with the KD for alprenolol antagonism of isoproterenol stimulation of adenylate cyclase. The (minus)-[3-H]alprenolol binding sites exhibited strict stereospecificity. (minus)-Stereoisomers of beta-adrenergic antagonists or agonists were approximately 2 orders of magnitude more potent than the (+)-stereoisomers in competing for the binding sites. Comparable stereospecificity was apparent when agonists and antagonists were tested for their ability to interact with the adenylate cyclase-coupled beta-adrenergic receptors in the membranes. Potency series of 11 agonists and 13 antagonists for inhibition of binding and interaction with adenylate cyclase were identical and were characteristic of a beta2-adrenergic receptor. A variety of nonphysiologically active compounds containing a catechol moiety as well as several metabolites and cholinergic agents did not inhibit (minus)-[3-H]alprenolol binding or interact significantly as agonists or antagonists with the adenylate cyclase. The (minus)-[3-H]alprenolol binding sites studied appear to be equivalent to the beta-adrenergic receptor binding sites in the frog erythrocyte membranes.     

Regulation of postnatal beta-adrenergic receptor/adenylate cyclase development by prenatal agonist stimulation and steroids: alterations in rat kidney and lung after exposure to terbutaline or dexamethasone

Glucorticoids and adrenergic stimulation are both thought to control the development of beta-adrenergic receptors/responses. In the current study, rats were exposed to dexamethasone or terbutaline during late gestation and the development of beta-receptor binding capabilities and adenylate cyclase activity evaluated in membrane preparations from kidney and lung. Prenatal dexamethasone exposure produced postnatal adrenergic hyperreactivity of kidney adenylate cyclase; the effect resulted from increases in the enzyme itself, as both basal adenylate cyclase and forskolin-stimulation of the enzyme were also increased by dexamethasone. Similarly, prenatal terbutaline exposure evoked increases in basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase in the kidney. In the lung, dexamethasone produced an initial postnatal deficit in basal adenylate cyclase and deficient responsiveness to isoproterenol, but the deficit resolved shortly after birth. Terbutaline selectively promoted the ability of isoproterenol to stimulate lung adenylate cyclase in the first few days after birth, without alterations in basal adenylate cyclase; this was followed by a period of prolonged subsensitivity of both basal and isoproterenol-stimulated activity. Although dexamethasone and terbutaline also caused significant changes in development of beta-receptor binding capabilities, in neither tissue could these effects account for the direction or magnitude of the changes in adenylate cyclase reactivity. Thus, glucocorticoids and beta-agonists can participate in the programming of development of postsynaptic reactivity by exerting actions upon post-receptor coupling mechanisms.     

Homologous desensitization of adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor

We recently demonstrated that heterologous desensitization of adenylate cyclase in turkey erythrocytes is highly correlated with phosphorylation of the beta-adrenergic receptor. In contrast, little is known of the biochemical mechanisms underlying the homologous form of beta-adrenergic receptor desensitization, which is agonist-specific and not cAMP-mediated. Accordingly, the present studies were undertaken to examine if phosphorylation of the beta-adrenergic receptor is also associated with this form of desensitization in a well studied model system, the frog erythrocyte. Preincubation of these cells with the beta-adrenergic agonist isoproterenol leads to a 45% decline in isoproterenol-stimulated adenylate cyclase activity without significant changes in basal, prostaglandin E1-, NaF-, guanyl-5'-yl-imidodiphosphate-, forskolin-, or MnCl2-stimulated enzyme activities. There is also a 48% decline in [125I]iodocyanopindolol membrane binding sites. Conversely, preincubation of the cells with prostaglandin E1 attenuates only the prostaglandin E1-stimulated enzyme activity and does not affect [125I]iodocyanopindolol binding. Phosphorylation of the beta-adrenergic receptor was assessed by preincubating the cells with 32Pi and desensitizing them, and subsequently purifying the receptors by affinity chromatography. Under basal conditions there is about 0.62 mol of phosphate/mol of receptor whereas after desensitization with isoproterenol this increases to 1.9 mol/mol. This isoproterenol-induced receptor phosphorylation exhibits stereospecificity and is blocked by the beta-adrenergic antagonist propranolol. In addition, preincubation with prostaglandin E1 does not promote beta-adrenergic receptor phosphorylation. These data suggest that receptor phosphorylation is involved in homologous as well as heterologous forms of desensitization and may provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.