Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Inhibition of hormone-stimulated adenylate cyclase activity after altering turkey erythrocyte phospholipid composition with a nonspecific lipid transfer protein. Phosphatidylinositol uncouples catecholamine binding from adenylate cyclase activation

The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization.     

Down-regulation of protein kinase C in rat glioma C6 cells: effects on the beta-adrenergic receptor-coupled adenylate cyclase

Continuous exposure of rat glioma C6 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a time and dose dependent loss of [3H]phorbol dibutyrate binding sites and protein kinase C activity. Thus, by 24 h, the cells were essentially depleted of protein kinase C activity. In agreement with previous studies, TPA treatment caused a reduction in isoproterenol-stimulated adenylate cyclase activity and a sequestration of beta-adrenergic receptors. Cells were treated with TPA for 24-48 h to completely down-regulate protein kinase C and then exposed to isoproterenol. Agonist-mediated desensitization of adenylate cyclase and sequestration of beta-adrenergic receptors occurred at similar rates in control and TPA-treated cells. In addition, agonist-mediated down-regulation of beta-adrenergic receptors was not impaired by the absence of protein kinase C activity. Although both agonists and phorbol esters cause desensitization of the beta-adrenergic receptor-coupled adenylate cyclase, agonist-mediated events can occur independently of protein kinase C.     

Isoproterenol-induced desensitization of adenylate cyclase in human astrocytoma cells. Relation of loss of hormonal responsiveness and decrement in beta-adrenergic receptors.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.     

Reduced sensitivity to catecholamine in Werner's syndrome fibroblasts

The beta-adrenergic receptor-coupled adenylate cyclase system has been investigated in normal and Werner's syndrome fibroblasts. The basal levels of cAMP in Werner and normal control cells were similar, whereas the isoproterenol-induced increase in cAMP levels was far less for Werner cells than for control cells. In the broken cell preparations isoproterenol stimulated the adenylate cyclase of only control cells, not of Werner cells, although NaF or prostaglandin E1 stimulated the enzyme of both cells to the same extent. The beta-adrenergic receptor concentrations analyzed with hydrophilic radioligand were nearly equal in Werner and in control cells. A reduction of functional activity of the beta-adrenergic receptor in Werner cells is thus suggested.     

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.     

Expression of rat mRNA coding for hormone-stimulated adenylate cyclase in Xenopus oocytes

Beta-Adrenergic agonist-stimulated hyperpolarization, whole-cell cAMP accumulation, and activity of isoproterenol-stimulated membrane-bound adenylate cyclase (EC 4.6.1.1) in Xenopus laevis ovarian oocytes are entirely dependent on the presence of nascent follicle cells. A method was developed to remove rapidly and completely all extra-oocyte cell types to yield defolliculated oocytes that exhibited normal viability and resting membrane potentials yet lacked beta-adrenergic receptor (beta AR)-stimulated responses. Purified follicle membranes contained beta AR-stimulated adenylate cyclase activity, whereas oocyte cell membranes did not. Purified oocyte membrane preparations from X. laevis oocytes previously microinjected with C6-2B rat astrocytoma mRNA, and subsequently defolliculated, exhibited novel beta AR and forskolin-stimulated adenylate cyclase activity. These experiments demonstrate that oocytes expressed rat C6-2B mRNA coding for the beta-adrenergic receptor and the components necessary for forskolin-stimulated adenylate cyclase activity.     

Influence of thyroid hormone on glycogen metabolism in rat liver.

The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glycose levels were not changed in any thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The "enzyme activity" ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the "enzyme activity" ratio and the ratio of the "energy charge" ratio of cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.     

Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.     

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.     

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.     

Modulation by islet-activating protein of adenylate cyclase activity in C6 glioma cells

The cAMP content of intact cells as well as adenylate cyclase of the membrane-rich particulate fractions was studied with C6 glioma cells that had been exposed to the culture medium supplemented with islet-activating protein (IAP), one of the pertussis toxins. Both the increase in the cellular cAMP content in response to a beta-adrenergic agonist and the stimulation of membrane adenylate cyclase by the beta-agonist and/or GTP were markedly enhanced by the IAP treatment of C6 cells, but no change was induced in affinities of the agonist (or an antagonist) or GTP for their respective sites of action (or binding). The concentration of IAP required for the half-maximal enhancement was as low as 1 pg/ml, when the time of cell exposure to the toxin was prolonged to 18 h. No enhancement was observed for the basal cAMP content or basal enzyme activity, nor was activation of adenylate cyclase by Gpp(NH)p (or NaF) affected by IAP treatment. The Vmax value of a specific and low Km GTPase was significantly smaller in the membranes of IAP-treated cells than in those of control cells. Cholera toxin treatment of cells activated adenylate cyclase without exerting any influence on these IAP actions. Thus, IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.     

Hormone action at the membrane level. VIII. Adrenergic receptors in rat heart and adipocytes and their modulation by thyroxine.

The regulation of adrenergic receptors in rat heart was measured in rats made hyperthyroid by injection with thyroxine and made hypothyroid by addition of propylthiouracil to the drinking water. Hyperthyroid rats display cardiac hypertrophy and a decrease in epididymal fat pad weight. The maximal beta-receptor level of ventricular membranes, as determined by (-)-[3H]dihydroalprenolol binding, was increased 60% by thyroxine treatment and decreased about 30% by propylthiouracil treatment. The affinity of the beta receptor was unchanged after thyroxine or propylthiouracil treatment. The maximal activity of the isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) varied with thyroid state in a manner parallel to the increase in beta-adrenergic binding sites. Thyroxine treatment also increases by 2-fold the beta receptors in isolated rat fat cells. Propylthiouracil treatment lowered the level of alpha receptors in heart by 30% as measured by [3H]dihydroergocryptine binding, but increased the affinity about 2.5-fold. The highest level of alpha receptors was seen in control hearts. These studies indicate that thyroxine may control the turnover of beta-adrenergic receptors in heart and fat cells and regulate physiological responses in these tissues via a hormone-hormone interplay system. Thyroxine treatment reduced the activity of the membrane-bound Mg2+-ATPase (EC 3.6.1.3) and 5'-mononucleotidase (EC 3.1.3.5) but appears to increase the activity of the (Na+ + K+)ATPase (EC 3.6.1.4).     

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.     

Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4 alpha-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. The catecholamine isoproterenol induced a 51% desensitization. Incubation of 32Pi labeled erythrocytes with TPA promoted a 3- to 4-fold increase in phosphorylation of the beta-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating beta-adrenergic receptor function.     

Hormone action at the membrane level VIII. Adrenergic receptors in rat heart and adipocytes and their modulation by thyroxine

The regulation of adrenergic receptors in rat heart was measured in rats made hyperthyroid by injection with thyroxine and made hypothyroid by addition of propylthiouracil to the drinking water. Hyperthyroid rats displayed cardiac hypertrophy and a decrease in epididymal gat pad weight. The maximal beta-receptor level of ventricular membranes, as determined by (?)-[³H]dihydroalprenolol binding, was increased 60% by thyroxine treatment and decreased about 30% by propylthiouracil treatment. The affinity of the beta receptor was unchanged after thyroxine or propylthiouracil treatment. The maximal activity of the isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) varied with thyroid state in a manner parallel to the increase in beta-adrenergic binding sites. Thyroxine treatment also increases by 2-fold the beta receptors in isolated rat fat cells.Propylthiouracil treatment lowered the level of alpha receptors in heart by 30% as measured by [³H]dihydroergocryptine binding, but increased the affinity about 2.5 fold. The highest level of alpha receptors was seen in control hearts. These studies indicate that thyroxine may control the turnover of beta-adrenergic receptors in heart and fat cells and regulate physiological responses in these tissues via a hormone-hormone interplay system.Thyroxine treatment reduced the activity of the membrane-bound Mg²⁺-ATPase (EC 3.6.1.3) and 5′-mononucleotidase (EC 3.1.3.5) but appears to increase the activity of the (Na⁺ + K⁺)ATPase (EC 3.6.1.4).     

Skeletal muscle beta-receptors and isoproterenol-stimulated vasodilation in canine heart failure

To investigate whether heart failure alters beta-adrenergic receptors on skeletal muscle and its associated vasculature, the density of beta-adrenergic receptors, isoproterenol-stimulated adenylate cyclase activity, and coupling of the guanine nucleotide-binding regulatory protein were compared in 18 control dogs and 16 dogs with heart failure induced by 5-8 wk of ventricular pacing at 260 beats/min. Hindlimb vascular responses to isoproterenol were compared in eight controls and eight of the dogs with heart failure. In dogs with heart failure, the density of beta-receptors on skeletal muscle was reduced in both gastrocnemius (control: 50 +/- 5; heart failure: 33 +/- 8 fmol/mg of protein) and semitendinosus muscle (control: 43 +/- 9; heart failure: 27 +/- 9 fmol/mg of protein, both P less than 0.05). Receptor coupling to the ternary complex, as determined by isoproterenol competition curves with and without guanosine 5'-triphosphate (GTP), was unchanged. Isoproterenol-stimulated adenylate cyclase activity was significantly decreased in semitendinosus muscle (control: 52.4 +/- 4.6; heart failure: 36.5 +/- 9.5 pmol.mg-1.min-1; P less than 0.05) and tended to be decreased in gastrocnemius muscle (control: 40.1 +/- 8.5; heart failure: 33.5 +/- 4.5 pmol.mg-1.min-1; P = NS). Isoproterenol-induced hindlimb vasodilation was not significantly different in controls and in dogs with heart failure. These findings suggest that heart failure causes downregulation of skeletal muscle beta-adrenergic receptors, probably due to receptor exposure to elevated catecholamine levels, but does not reduce beta-receptor-mediated vasodilation in muscle.     

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.     

Involvement of Dopamine Neurons in the Regulation of β-Adrenergic Receptor Sensitivity in Rat Prefrontal Cortex

The contribution of dopamine (DA) afferents to the regulation of beta-adrenergic receptor sensitivity (isoproterenol-stimulated adenylate cyclase activity) in the rat prefrontal cortex was investigated by comparing the effects of lesions affecting either both DA and noradrenaline (NA) or NA fibers alone. Bilateral 6-hydroxydopamine (6-OHDA) lesions made in the ventral tegmental area destroyed ascending DA and to a variable extent ascending NA fibers innervating the prefrontal cortex. Two opposite effects were observed depending on the extent of cortical NA denervation: (a) When NA denervation was complete (less than 4% of controls), a marked increase in the isoproterenol-sensitive adenylate cyclase activity (+78%) was found. The amplitude of this denervation supersensitivity was similar to that occurring following complete and selective destruction of NA innervation induced by bilateral 6-OHDA injections made into the pedunculus cerebellaris superior. (b) When 6-OHDA injections into the ventral tegmental area led to a partial destruction of cortical NA afferents (10-40% of control values), a hyposensitivity of the isoproterenol-induced adenylate cyclase activity (-30%) was observed. This effect contrasted with the moderate supersensitivity seen in rats with partial, but selective, destruction of NA innervation (pedunculus cerebellaris superior lesions). The hyposensitivity of beta-adrenergic receptors obtained in rats with partial lesions of cortical NA fibers, but devoid of cortical DA innervation, suggests that DA neurons may regulate, under certain conditions, the denervation supersensitivity of beta-adrenergic receptors.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.