Effect of repeated restraint stress, desmethylimipramine or adrenocorticotropin on the alpha and beta adrenergic components of the cyclic AMP response to norepinephrine in rat brain slices

The cyclic AMP response to catecholamines in rat cortical slices is mediated by a beta adrenergic receptor which is coupled to adenylate cyclase and an alpha adrenergic receptor which potentiates the response to beta receptor stimulation. The present studies examined the effects of repeated restraint stress, adrenocorticotropin or desmethylimipramine administration on the beta and alpha adrenergic components of this response. Restraint was found to produce a small nonsignificant decrease of the beta receptor response accompanied by a significant reduction of the alpha receptor-induced potentiation of the beta response. Desmethylimipramine was found to lower the cyclic AMP response to beta receptor stimulation but not to alter the alpha-induced potentiation of the beta response. Adrenocorticotropin, like restraint stress, was found to reduce only the alpha-induced potentiation of the beta response. Experiments with adenosine and histamine showed that restraint stress lowered the alpha-induced potentiation of cyclic AMP responses to these neurohormones also. It is concluded that restraint stress acts primarily to reduce the response to stimulation of central alpha adrenergic receptors whereas desmethylimipramine acts primarily to reduce the response to stimulation of beta adrenergic receptors. Adrenocorticotropin has the same effect as restraint stress suggesting that pituitary adrenal hormones mediate the stress effect.     

Interaction of beta adrenergic agonists and antagonists with brain beta adrenergic receptors in vivo

There is interest in knowing whether beta adrenergic antagonists or agonists, when administered systemically, can enter the brain to interact with central beta adrenergic receptors. To study this, the reduction in the radioactive content in the brain of rats after administration of (-)-[125I]iodopindolol (IPIN) by systemically administered beta agonists or antagonists was measured. Previous studies show that after the i.v. administration of IPIN the binding in vivo to various areas of the central nervous system has the characteristics expected of binding to beta adrenergic receptors. Of the antagonists tested, pindolol and butylpindolol showed potent interactions with beta receptors in both cortex and cerebellum whereas atenolol and practolol did not interact at doses up to 30 mg/kg. CGP-12177 showed moderate potency in inhibiting IPIN binding in vivo. We have shown previously that propranolol and alprenolol inhibit IPIN binding with high potency in cortex and cerebellum. At high doses, butoxamine, a beta-2 antagonist, reduced the binding of IPIN in the cerebellum but not in the cortex. Of the agonists tested, clenbuterol and prenalterol caused a significant dose-dependent reduction of the binding of IPIN, with clenbuterol being more potent. Isoproterenol, salbutamol, salmefamol and dobutamine had no effect. With the exception of CGP-12177, the affinity of the drugs for central beta adrenergic receptors measured in vitro was correlated significantly with their ability to inhibit IPIN binding in vivo whereas their degree of lipophilicity was not correlated significantly with potency in vivo. The inhibition of IPIN binding in vivo from brain areas can be used to evaluate whether drugs penetrate into brain and interact with central beta adrenergic receptors.     

Agonist interactions with beta adrenergic receptors in rat brain

Agonist interactions with beta adrenergic receptors on membranes prepared from rat brain were examined by measuring agonist inhibition of [125I]iodopindolol binding in the absence or presence of GTP. When rat cerebral cortical membranes were prepared with 1 mM EDTA in the homogenization medium and 2.5 mM MgCl2 was included in the binding reaction, then 250 microM GTP increased the Hill coefficient for isoproterenol from 0.77 to 0.99 and increased the IC50 from 88 to 213 nM. By contrast, I-propranolol competition curves were steep (Hill coefficient = 0.98) and were not affected by GTP. It was inferred from the results of computer-modeling that, in the absence of GTP, isoproterenol bound to two states of the receptor; GTP converted isoproterenol binding to a single low-affinity state. I-Propranolol bound to a single state in the absence or presence of GTP. The effect of GTP on I-epinephrine inhibition of [125I]iodopindolol binding was essentially identical to its effect on isoproterenol inhibition. GTP and GDP were the most potent of all the nucleotides tested. Guanylylimidodiphosphate (1 mM) produced only partial shifts in the isoproterenol competition curves and GMP and ATP were inactive. In membranes prepared from rat hippocampus and hypothalamus, isoproterenol competition curves and GTP effects were qualitatively similar to those observed in cerebral cortex. However, GTP produced only partial shifts of I-isoproterenol competition curves in cerebellum and neostratium. It appears that agonists, but not antagonists, can stabilize a high-affinity ternary complex with the beta adrenergic receptor and the guanine nucleotide binding regulatory protein in membranes prepared from various regions of the rat brain.     

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

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

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.     

Alterations in beta adrenergic physiological response characteristics after long-term treatment with desmethylimipramine: interaction between norepinephrine and gamma-aminobutyric acid in rat cerebellum

Changes in beta adrenergically mediated responses of cerebellar Purkinje cells to norepinephrine (NE) were assessed in rats chronically treated with desmethylimipramine (DMI). A decreased efficacy of NE action after DMI pretreatment was manifested in both a higher mean iontophoretic current dose of the catecholamine required to induce threshold depressions of Purkinje cell spontaneous activity and a markedly diminished ability of NE to enhance inhibitory responses to gamma-aminobutyric acid. No significant differences were found in the responsiveness of Purkinje cells to gamma-aminobutyric acid alone between control and DMI-pretreated animals. However, in a significant portion of the Purkinje cells tested in the DMI-pretreated group, concurrent NE application resulted in an antagonism rather than an enhanced responsiveness to gamma-aminobutyric acid. The results of this electrophysiological study provide evidence for a "modulatory subsensitivity" toward noradrenergic actions in the cerebellum after a decrease in beta receptor number induced by prolonged treatment with DMI.     

Changes in beta adrenergic receptors in submaxillary glands of chronically reserpine- or isoproterenol-treated rats

Submaxillary glands of rats, chronically treated with isoproterenol or reserpine undergo morphological and functional alterations. These changes have been described to resemble those seen in human cystic fibrosis. Since it has been proposed that the beta adrenergic-mediated response is altered in exocrine glands of cystic fibrosis patients, we have examined whether the drug-induced alterations in rat salivary glands were accompanied by changes in the numbers and affinities of beta adrenergic receptor sites. Beta receptor characteristics were determined by means of direct binding studies with the beta adrenergic antagonist [3H]dihydroalprenolol. Compared to controls, specific binding capacities of [3H]dihydroalprenolol per unit of protein increased by 110 +/- 14% after reserpine treatment and decreased by 34 +/- 11% after isoproterenol administration (P less than .001). The difference in the number of receptor sites remained statistically significant whether expressed per gram of fresh weight or per unit of the membrane marker 5'-nucleotidase activity. Dissociation constants of the binding were not significantly different between the treatment groups. The observed changes in the number of beta receptors showed an inverse relationship to the drug-induced presumed changes of catecholamine concentrations at the receptor sites. This suggests the existence of a feedback system which maintains the balance within the autonomous nervous system. We speculate that in cystic fibrosis this adaptive system is genetically abnormal.     

Relative importance of alpha and beta adrenergic receptors during resuscitation.

Successful resuscitation from cardiac arrest in the asphyxiated dog model has been ascribed to the use of artificial ventilation, closed chest cardiac massage, and administration of a vasopressor. Controversy remains over whether the most commonly employed vasopressor, epinephrine, exerts its effects primarily by elevating diastolic pressure and reestablishing coronary flow, or by exciting cardiac pacemaker cells and enhancing myocardial contractility. To observe pure alpha and beta adrenergic receptor influences during resuscitation, three groups (alpha-blocked, beta-blocked, unblocked) of dogs were studied. beta-blocked dogs resuscitated with phenylephrine and unblocked dogs resuscitated with epinephrine experienced 100% successful resumption of spontaneous circulation after 5 min of asphyxia-induced arrest. Only 27% of alpha-blocked animals resuscitated with isoproterenol were successfully revived. The appearance of the ECG during cardiac arrest and resuscitation could in no way be used to predict the outcome of resuscitation attempts. Results suggest that, initially, alpha receptor stimulation with concomitant diastolic pressure elevation is more important to the success of resuscitation than beta receptor stimulation.     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. II. Agonist binding to soluble beta adrenergic receptors

The thermodynamic parameters associated with the interactions of agonists and antagonists with digitonin-solubilized beta adrenergic receptors were determined. A rapid method for measuring the binding of [125I]iodopindolol to soluble receptors using glass-fiber filters was developed. The binding of [125I]iodopindolol, an antagonist with intrinsic sympathomimetic activity, to soluble receptors was temperature-sensitive as is the binding of the ligand to membrane-bound receptors. The interactions of propranolol and timolol with soluble receptors were independent of temperature. In contrast, the binding of agonists to soluble receptors was sensitive to temperature, although insensitive to GTP. Thermodynamically, the interactions of the antagonists timolol and propranolol with soluble beta adrenergic receptors were entropy-driven, with little contribution from changes in enthalpy. This is consistent with a hydrophobic interaction between the receptor and the antagonist. The binding of [125I]iodopindolol was enthalpy-driven. The binding of full agonists with soluble receptors was described thermodynamically by changes in enthalpy and entropy that were negative relative to the values for propranolol and timolol, suggesting that the guanine nucleotide-binding protein required for stimulation of adenylate cyclase activity and an intact lipid environment are not involved in the thermodynamics of formation of the low-affinity component of agonist binding. These results are consistent with an agonist-induced change in the conformation of the receptor.     

Comparison of iprindole, imipramine and mianserin action on brain serotonergic and beta adrenergic receptors

Iprindole in vitro displaces mianserin from specific recognition sites (IC50 3 microM). Daily doses (5 mg/kg i.p.) of iprindole repeated for 3 weeks attenuate the norepinephrine stimulation of adenylate cyclase studied in brain slices. Whereas the attenuation of noradrenergic receptor function elicited by imipramine can be antagonized by lesions of serotonergic axons, the inhibition of norepinephrine-induced stimulation of adenylate cyclase elicited by iprindole or mianserin is not inhibited by serotonin axon lesions. Iprindole given daily repeatedly reduces the maximal number of binding sites of [3H]mianserin and [3H]ketanserin; both actions are unchanged by lesions of central serotonergic axons.     

Perinatal changes in the coupling of beta 1- and beta 3 adrenergic receptors to brown fat adenylyl cyclase.

The stimulation of adenylyl cyclase by catecholamines in neonatal brown adipose tissue (BAT) is markedly biphasic, suggesting the existence of receptors that have both high and low affinities for catecholamines. The identities of these receptors were examined by comparing responses in neonatal BAT membranes to those of Chinese hamster ovary cells which had been transfected to express the cloned rat beta 1 and beta 3 receptors. The results from these experiments indicate that high-affinity stimulation of adenylyl cyclase by catecholamines in BAT is mediated by beta 1 receptors, as evidenced by the potencies of norepinephrine and isoproterenol at this receptor and the potent blockade of the receptor by alprenolol. The low-affinity catecholamine receptor appears to be the beta 3 receptor, as indicated by the low potency of catecholamine agonists and the inability of low concentrations of alprenolol to block this activity. Furthermore, this receptor, like the cloned rat beta 3 receptor, was antagonized by (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP 12177) and was stimulated by (R',R')-4-(2-[(2[(3-chlorophenyl)-2- hydroxyethyl]amino)propyl]phenyl)phenoxyacetic acid (BRL 37344). These results indicate that both beta 1 and beta 3 receptors couple to adenylyl cyclase in BAT and that activation of adenylyl cyclase in neonatal BAT is mediated primarily by beta 3 receptors. Beta 3 receptors were also clearly detected in weanling BAT with the beta 3-selective agonist BRL 37344. However, when catecholamines were used to stimulate activity, the activation of adenylyl cyclase by beta 1 receptors, which occurred at low concentrations of catecholamines, obscured the activation of adenylyl cyclase by beta 3 receptors, which occurred only at high concentrations.     

Effect of beta adrenergic blockade on renin response to renal nerve stimulation.

The ability of d,l-propranolol to block renin secretion in response to various extrarenal stimuli, such as hemorrhage and hypoglycemia, has been interpreted to indicate the presence of an intrarenal beta receptor regulating renin release. However, two problems complicate this interpretation: (a) the stimuli have effects outside the kidney, and (b) d,l-propranolol has a local anesthetic, as well as a beta adrenergic blocking, action. In the present study, the effects of a purely intrarenal stimulus, in the form of renal nerve stimulation (RNS), on renin secretion was examined. The effects of d,l-propranolol (anesthetic and beta-blocking activity), l-propranolol (beta-blocking activity only), and d-propranolol (local anesthetic activity only) on the renin response to RNS were examined. In a control group of animals, two sequential RNS increased mean renin secretion from 401 to 1,255 U/min (P less than 0.25) and from 220 to 2,179 U/min (P less than 0.01). In a second group the first RNS increased renin secretion from 201 to 1,181 U/min (P less than 0.01), but after d,l-propranolol was given RNS did not significantly alter renin secretion (33 to 55 U/min). In a third group the initial RNS increased renin secretion from 378 to 1,802 U/min (P less than 0.025), but after l-propranolol was given RNS had no significant effect on renin secretion (84 to 51 U/min). A fourth group of dogs showed a rise in renin secretion from 205 to 880 U/min (P less than 0.001) in response to the first RNS, while the second RNS, given after an infusion of d-propranolol, caused a rise in renin secretion from 80 to 482 (P less than 0.005). The nature of the electrical stimulus was consistent in all groups and caused no detectable changes in renal or systemic hemodynamics or in urinary electrolyte excretion. The results, therefore, indicate that renin secretion can be stimulated through intrarenal beta receptors independent of changes in systemic or renal hemodynamics or in tubular sodium reabsorption. Hence the effect of beta stimulation on renin secretion would appear to result from a direct action on the renin-secreting cells of the juxtaglomerular apparatus.     

Influence of fasting on lipolytic response to adrenergic agonists and on adrenergic receptors in subcutaneous adipocytes

The influence of 1 week's total fasting on the lipolytic effect of adrenergic agonists and on the binding of adrenergic antagonists was examined in isolated adipocytes of subcutaneous specimens removed from the hypogastric and the femoral sites in seventeen obese women. In the femoral adipocytes the lipolytic sensitivity to isopropyl noradrenaline decreased 30-fold (P less than 0.01) during fasting. The specific binding of the radioligands (-)-[3H]-dihydroalprenolol and (-)-[125I]-cyanopindolol decreased significantly during fasting, essentially owing to a reduction in the receptor density. In adipocytes from the hypogastric region no such changes were found. For both tissue regions fasting induced a right-ward shift in the dose-response curve for the inhibitory effect of the alpha 2 agonist, clonidine, on theophylline-induced lipolysis, corresponding to a 10-fold decrease in sensitivity. There was also a significant decrease of about 20% in the alpha 2-adrenoceptor density, as estimated with the radioligand [3H]-yohimbine. The results suggest that the regulation of the lipid mobilization in man by the sympathetic nervous system during fasting occurs not only through an increase in the level of circulating noradrenaline but also through changes in the adrenergic receptor density of the adipocytes.     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. I. High- and low-affinity states of agonist binding to membrane-bound beta adrenergic receptors

The properties associated with ligand interactions with membrane-bound beta adrenergic receptors prepared from L6 myoblasts were examined at five temperatures between 10 degrees and 30 degrees C. The interactions of antagonists with membrane-bound receptors were insensitive to temperature, whereas the interactions of agonists were temperature-dependent. The affinity constants for the low-affinity binding states of agonists (KL) decreased slightly with decreasing assay temperature. The small temperature-dependent changes in KL values were similar to the changes in Kd values observed in studies of the binding of agonists in the presence of GTP. The high-affinity dissociation constants (KH) for binding of full agonists to membrane-bound receptors in the absence of GTP were approximately 50-fold lower at 10 degrees than at 30 degrees C. The KH values for partial agonists also decreased with decreasing temperature, but the changes were smaller in magnitude. Thermodynamically, the binding of antagonists was primarily entropy-driven, whereas the binding of agonists was enthalpy-driven. The energetics of the low-affinity component of agonist binding to membrane-bound receptors were similar to the energetics for binding of agonists to membrane-bound receptors in the presence of GTP. Under these conditions, standard enthalpy change (delta H degree) and standard entropy change (delta S degree) values for binding of agonists were more negative than the corresponding values for binding of antagonists, possibly reflecting a conformational change in the receptor or an increased ordering of the lipids surrounding the receptor. The interaction of the receptor with the guanine nucleotide-binding protein to form the high-affinity component of agonist binding was thermodynamically described by larger negative changes in enthalpy and entropy than the values for formation of the low-affinity component of agonist binding. There was a correlation between the efficacies of ligands in activating adenylate cyclase and the delta H degree and delta S degree values for high-affinity binding of agonists. Thus, the extent or nature of the interaction between the guanine nucleotide-binding protein and the receptor may determine the efficacies of ligands.     

Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland.

Denervation resulted in a marked increase in the beta adrenergic response and isoproterenol-stimulated cyclic adenosine 3':5'-monophosphate accumulation of dispersed cells prepared from a rat submandibular gland. This increase in beta adrenergic response was paralleled by an increase in the density of beta adrenergic receptors in membranes prepared from these glands. Denervation also produced a moderate increase in alpha adrenergic receptor density in membranes prepared from whole glands. However, the alpha adrenergic response in cells, epinephrine-induced release of potassium, appeared unaltered by denervation. Furthermore, membranes prepared from denervated dispersed cells did not show the increase in alpha adrenergic receptor density seen in membranes from an intact denervated gland. These data demonstrate that removal of noradrenergic nerve terminals affects alpha and beta adrenergic receptors differently. While it is clear that beta adrenergic membrane receptors participate in denervation-induced supersensitivity, the changes in alpha adrenergic membrane receptors are more complex and may not contribute to the supersensitivity seen after denervation. On the basis of competitive binding studies, the adrenergic receptors in membranes from intact submandibular glands were subclassified as beta-1 and alpha-2. Denervation did not alter the binding characteristics of the alpha-2 receptors in this gland, demonstrating that alpha-2 adrenergic membrane receptors can be postsynaptic in this adrenergically innervated tissue.     

Reduced beta adrenergic responsiveness in rats treated with estrogenic agents.

Chronic treatment with ethynyl estradiol alone (48-50 mug/kg/day) and in combination with several doses of norethynodrel either attenuated or prevented the increase in tail skin temperature accompanying sC. administration of isoproterenol (200 mug/kg b.wt.) to male and female rats. Dietary administration of an oral contraceptive containing norethynodrel and mestranol (7.5 mg/kg of food) was also accompanied by an attenuated response to isoproterenol. Attenuation of the tail skin temperature response was present in all groups after 3 to 5 weeks of treatment but was detectable in the oral contraceptive-treated group after 1 week of treatment. All steroids and combinations, except norethynodrel administered alone (154 mug/kg/day), reduced body weight gain.