Desensitization of β-adrenergic receptor-coupled adenylate cyclase activity (original) (raw)
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Pharmacological Research Communications, 1985
The mechanism(s) of agonist-induced desensitization of the beta adrenergic receptor-coupled adenylate cyclase has been investigated in the L8 skeletal muscle cell line. Different beta agonists stimulate cAMP accumulation in intact L8 cells with the following potency: Isoproterenol greater than Procaterol = Epinephrine greater than Norepinephrine greater than Phenylephrine. Preincubation of intact cells for 30 min at 37 degrees C with the same agonists induces a dose-dependent desensitization of beta receptor function. The degree of desensitization induced is correlated with the potency of agonists in stimulating cAMP accumulation. The same agonists induced internalization of the adrenergic beta receptor, with potency similar to that inducing desensitization and cAMP accumulation. Thus it is possible to conclude that in L8 cells beta receptor are of the beta 2 subtype and that agonist-induced beta adrenergic receptor desensitization proceeds via internalization of the beta adrenergic recognition site.
Archives of Biochemistry and Biophysics, 1978
Subcellular fractions of rat skeletal muscle enriched in plasma membrane (sarcolemma) were used to characterize properties of the fl-adrenergie receptor and adenylate cyelase. The equilibrium binding of (-)-[:~H]dihydroalprenolol to the sarcolemmal membrane was characterized by a high affinity, K = 3.6 • 10 ~ liters/tool and 1.2 • 10 ~ mol/g of membrane protein of homogeneous noninteracting sites. The binding was reversible, saturable, and stereospecific. Displacement of labeled dihydroalprenolol by adrenergic ligands revealed an order of potency corresponding to the fie-type response: (-)-isoproterenol > (-)-epinephrine > norepinephrine. The (+)-stereoisomers and the a-adrenergic antagonist phentolamine showed little ability to compete with (-)-[:~H]dihydroalprenolol for occupancy of the binding sites. In the membrane fractions, a substantial basal activity of adenylate cyclase activity was demonstrated (35-50 pmol of cyclic AMP/mg/5 min). This activity was stimulated 40-to 50-fold by sodium fluoride, 7-to 8-fold by isoproterenol, and 5-to 6-fold by guanylyl 5'-imidodiphosphate [Gpp(NH)p]. Maximum activation depended upon optimal Mg ~+ ion concentration (I0 mM). In the presence of 0.1 mM Gpp(NH)p, the isoproterenol activation of adenylate cyclase was increased twofold. In addition, the concentration of hormone required for half-maximal stimulation of enzyme activity was shifted from 2.2 • 10-~ M in the absence of Gpp(NH)p to 2.5 • I0 ~ M in the presence of Gpp(NH)p. Gpp(NH)p also stimulated epinephrine and norepinephrine activation without altering the fl2-type potency series for the enzyme. Gpp(NH)p had no effect on either the number or the affinity of (-)-[:JH]dihydroalprenolol binding sites, but it did increase the dissociation constant for isoproterenol by approximately fivefold. Deceased. This paper represents the last investigations carried out by Dr. Stuart P. Grefrath. His warmth, friendship, and critical acumen will be missed.
Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y-D2L cells
FEBS Letters, 2001
Adenylate cyclase activity in NS20Y cells expressing D 2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D 2 receptors. Furthermore, chronic (18 h) activation of D 2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (s 10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D 2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of GK K s or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Characterization of β-adrenoceptors on rat skeletal muscle cells grown in vitro
Biochemical Pharmacology, 1990
The binding properties of an hydrophilic Badrenergic receptor radio&and, (-)['H](4-(3tert-butylamino-2-hydroxypropoxy)-benzimidazolo-2-one); ([3H]CGP-12177), were investigated in rat skeletal muscle cells in culture. The binding of ['H]CGP-12177 at 25" was saturable, reversible and of high affinity (& = 1.3 2 0.3 nM). The maximal number of [3H]CGP-12177 binding sites was 30.6 f 3.2 fmol/dish (34 ? 3.5 fmol/mg protein). BAdrenergic agonists and antagonists inhibited [3H]CGP-12177 binding. The competing ligand inhibition binding is a typical one for &adrenoceptors. The increase in &adrenoceptors was independent of cell fusion. Amiodarone (10m5 M) decreased the @adrenoceptor number in skeletal muscle cells differentiated in vifro by 48%, while the affinity for (3H]CGP-12177 was not affected. Catecholamines have long been known to act on skeletal muscle and to modify its response to both direct and indirect (nerve) stimulation.
Proceedings of the National Academy of Sciences, 1984
Only part of the ,B-adrenergic receptors can undergo functional coupling to the adenylate cyclase regulatory unit. This receptor subpopulation shows an increased affinity for agonists in the presence of Mg2' and undergoes rapid "inactivation" (locking-in of the agonist) by the alkylating reagent N-ethylmaleimide in the presence of agonists. Several experimental conditions, known to modify the total receptor concentration without alteration of the other components of the acenylate cyclase system, do not affect the percentage of receptors that can undergo functional coupling: (i) homologous regulation of B13 receptors in rat brain by noradrenaline (through antidepressive drug or reserpine injections); (it) upand down-regulation of the 182 receptors in Friend erythroleukemia cells by, respectively, sodium butyrate and cinnarizine treatment; and (iiW) dithiothreitol-mediated inactivation of receptors in turkey erythrocytes, Friend erythroleukemia cells, and rat brain. Our findings argue against a stoichiometric limitation in the number of regulatory components, genetically different receptor subpopulations, bound guanine nucleotides, or reduced accessibility of part of the receptors to the agonists
Journal of Autonomic Pharmacology, 1993
Using agonists (adrenaline, noradrenaline, isoprenaline, dopamine, epinine, procaterol) and antagonists (CGP 20712A and ICI 118,551) in radioligand binding and adenylate cyclase-stimulation studies we demonstrate that the human neuroblastoma cell line SK-N-MC contains a homogeneous population of P,-adrenoceptors coupling to stimulation of adenylate cyclase. 2 A 34 h treatment with isoprenaline reduced P,-adrenoceptor density, concentration-dependently, maximally by 69-+ 1 %. Functional PI-adrenoceptor desensitization was comparatively small. 3 Although 100 nM isoprenaline caused significantly greater receptor downregulation than 1 O,UM isoprenaline (47 * 2%), desensitization of CAMP accumulation in intact cells or of adenylate cyclase stimulation in cell membranes was attenuated similarly or even more with 1 0 ,~~ isoprenaline. 4 While both agonist concentrations did not significantly alter immunodetectable a-subunits for Gi or Go or G-protein P-subunits, 1 0 ,~~ (but not 1 0 0 n~) isoprenaline decreased immunodetectable Gsa. Accordingly, only treatment with 1 O,UM isoprenaline reduced receptor-independent adenylate cyclase stimulation by GTP, NaF or forskolin. Neither agonist concentration desensitized adenylate cyclase activation by MnCl,. 5 We conclude that isoprenaline produces a concentration-dependent regulation of P1-adrenoceptor number in SK-N-MC cells, which is weaker at higher than at intermediate agonist concentrations, and is accompanied by only minor desensitization of adenylate cyclase activity; whether this involves alterations of postreceptor events depends on the agonist concentration used.
Biology of Reproduction, 1980
Pretreatment of ratswith thespecific (3-adrenergic catecholamine agonist isoproterenol resulted in loss of adenylate cyclase sensitivity to the hormone in a subsequently prepared uterine smooth muscle particulate fraction. This agonist-mediated enzyme desensitization was accompanied by a rapidreductionin the smooth muscle cellconcentration of 3-adrenergic catecholaminereceptors determined by specific binding of the radioactive antagonist (â€")-(3U)-dihydroalprenolol. Decrease in receptor number was not accompanied by a change in high affinity binding of the radioligand (Kd<5 nM) as determined by Scatchard analysis. Similar reductions in receptor concentration could be achieved by incubating muscle strips with isoproterenol in vitro at agonist doses <1.0 .tM. Receptor down-regulation was blocked by the potent 3-adrenergic catecholamine antagonist (â€")alprenolol and reversed when the receptor-bearing microsomal fraction was exposed to guanylyl nucleotide. These results are more consistent with an agonist-induced change in receptor properties than with irreversible loss. Agonist-dependent down-regulation of the 3-adrenergic receptor ac counts for adenylate cyclase desensitization in the myometrium and implies that these hormones have important selfregulating roles in the control of smooth muscle cell sensitivity and uterine motility.