==================================BSR28================================== 28. Effects of guanonucleotides (GTP, GppNHp) on beta-adrenergic receptors (binding) in the brain. 1 UI - 86300649 AU - Feder D ; Im MJ ; Klein HW ; Hekman M ; Holzh:ofer A ; Dees C ; Levitzki A ; Helmreich EJ ; Pfeuffer T TI - Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components. AB - In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone-dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl-polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol-induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS) MH - Adenyl Cyclase/*BLOOD/ISOLATION & PURIFICATION ; Animal ; Brain/ METABOLISM ; Cattle ; Cell Membrane/METABOLISM ; Detergents ; Enzyme Activation ; Erythrocyte Membrane/*METABOLISM ; Guanine Nucleotide Regulatory Protein/METABOLISM ; Guanosine Triphosphate/ANALOGS & DERIVATIVES/METABOLISM/PHARMACODYNAMICS ; Liver/METABOLISM ; Myocardium/ METABOLISM ; Rabbits ; Receptors, Adrenergic, Beta/ISOLATION & PURIFICATION/*METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Thionucleotides/PHARMACODYNAMICS ; Turkeys SO - EMBO J 1986 Jul;5(7):1509-14 2 UI - 86280486 AU - Vanecek J ; Sugden D ; Weller JL ; Klein DC TI - See-saw signal processing in pinealocytes involves reciprocal changes in the alpha 1-adrenergic component of the cyclic GMP response and the beta-adrenergic component of the cyclic AMP response. AB - Pineal cyclic AMP and cyclic GMP are regulated by norepinephrine (NE) acting through alpha 1- and beta-adrenoceptors. beta-Adrenergic stimulation appears to be an absolute requirement and alpha 1-adrenergic activation amplifies beta-adrenergic stimulation of the cyclic AMP response 10-fold and the cyclic GMP response 100-fold, respectively. Chronic deprivation of adrenergic stimulation, due to exposure to constant light (LL) or by surgical denervation, enhances the cyclic AMP response and diminishes the cyclic GMP response as compared to control animals in a 10:14 light/dark (LD) cycle. This phenomenon is termed see-saw signal processing. In the current study we find these changes do not reflect shifts in the time course or Ka of these responses. Dose-response studies indicate the beta-adrenergic component of cyclic AMP stimulation is enhanced and the alpha 1-adrenergic component of cyclic GMP stimulation is diminished in LL pinealocytes. Several observations indicate these changes may reflect alterations in Ca2+-sensitive postreceptor mechanisms. MH - Adenosine Cyclic Monophosphate/*PHYSIOLOGY ; Animal ; Calcium/ PHARMACODYNAMICS ; Guanosine Cyclic Monophosphate/*PHYSIOLOGY ; Isoproterenol/PHARMACODYNAMICS ; Kinetics ; Light ; Male ; Nitroprusside/ PHARMACODYNAMICS ; Norepinephrine/PHARMACODYNAMICS ; Periodicity ; Phenylephrine/PHARMACODYNAMICS ; Pineal Body/DRUG EFFECTS/*PHYSIOLOGY/ RADIATION EFFECTS ; Quinacrine/PHARMACODYNAMICS ; Rats ; Rats, Inbred Strains ; Receptors, Adrenergic, Alpha/*PHYSIOLOGY ; Receptors, Adrenergic, Beta/*PHYSIOLOGY ; Tetradecanoylphorbol Acetate/ PHARMACODYNAMICS SO - J Neurochem 1986 Sep;47(3):678-86 3 UI - 86280454 AU - Okada F ; Tokumitsu Y ; Ui M TI - Desensitization of beta-adrenergic receptor-coupled adenylate cyclase in cerebral cortex after in vivo treatment of rats with desipramine. AB - 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. MH - Adenyl Cyclase/*METABOLISM ; Animal ; Cerebral Cortex/DRUG EFFECTS/ *ENZYMOLOGY ; Desipramine/*PHARMACODYNAMICS ; Drug Tolerance ; Enzyme Activation/DRUG EFFECTS ; Guanosine Triphosphate/PHARMACODYNAMICS ; Guanylyl Imidodiphosphate/PHARMACODYNAMICS ; Isoproterenol/ PHARMACODYNAMICS ; Kinetics ; Male ; Pindolol/ANALOGS & DERIVATIVES/ METABOLISM ; Rats ; Rats, Inbred Strains ; Receptors, Adrenergic, Beta/ DRUG EFFECTS/*PHYSIOLOGY SO - J Neurochem 1986 Aug;47(2):454-9 4 UI - 86112488 AU - Green RM ; Stiles GL TI - Chronic caffeine ingestion sensitizes the A1 adenosine receptor-adenylate cyclase system in rat cerebral cortex. AB - Caffeine consumption causes significant physiologic effects due to its antagonism of adenosine receptors. The A1 adenosine receptor is coupled in an inhibitory manner to adenylate cyclase. To study the effects of chronic caffeine ingestion, rats were provided with 0.1% caffeine drinking solution for 28 d. The A1 adenosine receptor agonist radioligand [3H]phenylisopropyladenosine identifies two affinity states in control rat cerebral cortex membranes with a high affinity dissociation constant (KH) of 0.40 +/- 0.08 nM and low affinity dissociation constant (KL) of 13.7 +/- 3.9 nM, with 33% of the receptors in the high affinity state. In membranes from caffeine-treated animals, all of the A1 receptors are shifted to the high affinity state with a dissociation constant (KD) of 0.59 +/- 0.06 nM. Guanylyl-imidodiphosphate (10(-4) M) decreases binding by 43% in control membrane, with no change in KH or KL, while membrane binding in caffeine-treated animals decreases by 45% with a threefold shift in KD to 1.5 +/- 0.3 nM. Concomitant with the enhanced high affinity A1 receptor state and increased sensitivity to guanine nucleotides, membranes from treated animals show a 35% enhancement in (-)-N6-(R-phenylisopropyl)adenosine-mediated inhibition of adenylate cyclase compared with controls (P less than 0.03). Photoaffinity crosslinking the receptors with [125I]N6-2-(3-iodo-4-aminophenyl)ethyladenosine reveals that A1 receptors from both groups migrate as Mr 38,000 proteins. beta-adrenergic receptor binding with [125I]iodocyanopindolol shows a decrease in the number of beta-receptors from 233 +/- 7 fmol/mg protein in control membranes to 190 +/- 10 fmol/mg protein in treated membranes (P = 0.01). These data indicate that the adenosine receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system and downregulation of beta-adrenergic receptors, and provides a molecular mechanism for the caffeine withdrawal syndrome. MH - Adenosine/*METABOLISM ; Adenyl Cyclase/*METABOLISM ; Animal ; Caffeine/ *ADMINISTRATION & DOSAGE/TOXICITY ; Cerebral Cortex/ENZYMOLOGY/ *METABOLISM ; Cross-Linking Reagents ; Guanine Nucleotides/ PHARMACODYNAMICS ; Kinetics ; Male ; Phenylisopropyladenosine/ PHARMACODYNAMICS ; Rats ; Rats, Inbred Strains ; Receptors, Adrenergic, Beta/DRUG EFFECTS ; Receptors, Endogenous Substances/ANALYSIS/*DRUG EFFECTS ; Substance Withdrawal Syndrome/CHEMICALLY INDUCED/METABOLISM ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Time Factors SO - J Clin Invest 1986 Jan;77(1):222-7