Regulation of beta 2-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells: effects of agonist, forskolin, and protein synthesis inhibition.

Incubation of rat C6 glioma cells with beta-adrenergic receptor (beta AR) agonist or with agents that increase cAMP levels results in down-regulation of the beta 2AR, as measured by the loss of radioligand binding sites. In the present study, the role of beta 2AR mRNA expression and stability in the down-regulation of beta 2AR sites in C6 cells was examined. Isoproterenol or forskolin treatment decreased beta 2AR mRNA levels in a time-dependent manner, with maximal loss of approximately 50% being observed after 2 hr. Pretreatment of the cells with a potent protein synthesis inhibitor, Pseudomonas exotoxin A, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 2AR mRNA. Exposure to agonist did not significantly influence the half-life of beta 2AR mRNA, which was approximately 60 min. In contrast, isoproterenol treatment for 2 hr significantly decreased the rate of beta 2AR gene transcription, as determined by nuclear run-on analysis. Based on these results, we propose that agonist regulation of beta 2AR mRNA in C6 cells is mediated by activation of the cAMP system and occurs at the level of beta 2AR gene transcription, not mRNA stability. In addition, the observed requirement for protein synthesis indicates that down-regulation of beta 2AR mRNA may be mediated by expression of a repressor of beta 2AR gene transcription.

Agonist and cyclic AMP-mediated regulation of beta 1-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells.

Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down-regulation of beta 1-adrenergic receptors (beta 1 AR) as measured by loss of radioligand binding sites. The present study examines the influence of isoproterenol and forskolin treatment on levels of beta 1 AR mRNA, mRNA stability, and gene transcription rate. Isoproterenol treatment of C6 cells altered beta 1 AR mRNA levels in a biphasic manner; i.e., short-term exposure (30-60 min) increased by 50%, whereas longer exposure (2-6 h) decreased by 50% the levels of beta 1 AR mRNA. The extent of both the up- and down-regulation was dependent on agonist concentration. Similar regulation of beta 1 AR mRNA was observed in forskolin-treated cells. Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 1 AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6-h time course. The mechanisms underlying beta 1 AR mRNA down-regulation were examined. The half-life of beta 1 AR mRNA was slightly increased (from 61 to 77 min) after a 2-h exposure of the cells to either isoproterenol or forskolin. Nuclear run-on analysis demonstrated that the rate of beta 1 AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90-240 min, consistent with the time course for up- and down-regulation of beta 1 AR mRNA. Isoproterenol treatment (120 min) also decreased the level of beta 1 AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4-thiouridine-pulsed cells. The results demonstrate that beta 1 AR mRNA has a relatively short half-life and that agonist regulation of beta 1 AR mRNA is mediated by activation of the cyclic AMP system. Moreover, the results indicate that agonist regulation of beta 1 AR mRNA occurs at the level of beta 1 AR gene transcription, not mRNA stability. Finally, the observed requirement for protein synthesis indicates that beta 1 AR mRNA down-regulation may be mediated by the induction of a repressor of the beta 1 AR gene.

Independent and coordinate regulation of beta 1- and beta 2-adrenergic receptors in rat C6 glioma cells.

Rat C6 glioma cells have both beta 1- and beta 2-adrenergic receptors in approximately 7:3 ratio. When the cells were exposed to the beta-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [3H] CGP-12177 to intact cells. Using the beta 1-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both beta 1 and beta 2 receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of approximately 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [125I]iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous beta 1-and beta 2-adrenergic receptors in the same cell line.

beta(1)-Adrenergic and D(1) Dopaminergic Receptors in Human Neurotumor Cells: Differences in Spare Receptors and Desensitization of Adenylyl Cyclase.

Haman SK-N-MC neurotumor cells express both beta(1)-adrenergic and D(1) dopaminergic receptors. Although there were sevenfold more beta(1) than D(1) receptors, maximum stimulation of adenylyl cyclase activity by isoproterenol was only threefold more than that by dopamine. We had shown previously that the pattern of agonist-mediated densensitization was different for the two receptors. To compare the efficiency of the two receptors to couple to the same adenylyl cyclase and to explore the possible role of spare receptors, the cells were exposed to N -ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which irreversibly alkylates and inactivates catecholamine receptors. Loss of receptor binding activity was dependent on the concentration of EEDQ, D(1) receptors being more sensitive to EEDQ than beta(1) receptors with IC(50) values of 1 and 30 muM, respectively. beta(1) receptors were more sensitive to the irreversible beta-adrenergic antagonist N(8) -bromoacetyl-N(1)-(3'-(4-indolyloxy)-2'-hydroxy-propyl)-[Z]-1,8-diamino-p-menthane (BIM) with an IC(50) of 1 nM. Inactivation of D(1) receptors parallelled the reduction in dopamine-stimulated adenylyl cyclase activity without a shift in K(act), indicating the absence of spare D(1 receptors in SK-N-MC cells. By contrast, there appeared to be spare beta1) receptors as the K(act) shifted before any reduction in isoproterenol-stimulated activity occurred in EEDQ- or BIM-treated cells. Inactivation of 70% of the beta(1) receptors resulted in only a 20% decrease in isoproterenol-stimulated adenylyl cyclase activity, and the remaining beta(1) receptors were as efficient as D(1) receptors in stimulating adenylyl cyclase. In addition, inactivation of the spare beta(1) receptors did not alter the unique pattern of agonist-mediated desensitization. We conclude that human beta(1) and D(1) receptors are equally efficient at stimulating adenylyl cyclase but differ in their mechanisms of agonist-mediated desensitization.

Spinal cord metabolic changes in murine retrovirus-induced motor neuron disease.

Decreased cerebrospinal fluid concentrations of cyclic nucleotides in human motor neuron disease and decreased spinal cord concentrations of cyclic nucleotides in murine (Wobbler) motor neuron disease suggest that an abnormality in cyclic nucleotide metabolism may play a role in motor neuron degeneration. Retroviruses cause decreased cellular levels of cyclic nucleotides in infected cells. We induced a motor neuron degeneration with a neurotropic retrovirus, but not with a non-neurotropic retrovirus. In paralyzed mice, mean cAMP was decreased 21% in posterior horn segments and 34% in anterior horn segments compared to controls. The proportion of spinal cord phosphorylase a decreased 24% in paralyzed mice compared to controls. The content of cGMP decreased 48% in the cerebellum and 25% in both anterior and posterior horn segments of the spinal cords of paralyzed mice compared to controls. White matter content of these chemicals did not decrease in the posterior column of affected animals. Spinal cord content of ATP increased 20-22% in all three compartments, but the spinal cord content of phosphocreatine increased dramatically in white matter (46%), posterior horn gray matter (69%), and anterior horn gray matter (103%) compared to controls. Changes in high-energy phosphate intermediate and cyclic nucleotide metabolites occurred only in topographical regions showing neuronal and astrocyte pathological changes, but did not occur in the cerebral cortex.

Distribution of cyclic nucleotides in layers of the cerebellum after decapitation.

The distribution of the cyclic nucleotides was examined in layers of the mouse cerebellum following decapitation. Cyclic AMP increased and cyclic GMP decreased in all three layers of the cerebellum examined. The increase in cyclic AMP in the granular layer was far greater than in either the molecular or white layers. In the cerebellum from control mice, the cyclic GMP concentration was highest in the molecular layer and lowest in the white layer. Even after decapitation, this cyclic GMP gradient in the cerebellum was maintained.

Sparing of metabolic stress in Purkinje cells after maximal electroshock.

Experimental seizures were induced in mice by application of 50 mA for 0.2 sec via corneal electrodes. The reproducible conclusive behavior was characterized by a sequence of 2 sec of tonic flexion, 13 sec of tonic extension, and 8 sec of clonus followed by a postictal depressive stage. The animals were frozen and tissues were prepared for analysis according to Lowry and Passonneau [Lowry, O. H. & Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis (Academic, New York)]. Freeze-dried samples (1-10 ng) of pyramidal cell bodies and adjacent neuropil from the parietal cortex and of Purkinje cell bodies and adjacent neuropil from the cerebellum were analyzed for glucose, ATP, and P-creatine (0.01-0.05 pmol). There were marked decreases in these energy stores after the maximal electroshock in three of the areas examined. In the Purkinje cell bodies, however, the metabolic stress was dampened; glucose concentrations decreased, but the levels of ATP were maintained and, to a lesser extent, those of P-creatine. The results indicate that the output from the Purkinje cells is less than in the other regions examined in the excitable stages of the convulsion. The lesser energy debt probably reflects lower energy demand as well as a lower discharge intensity. The fact that Purkinje cells are spared from the metabolic stress imposed on other regions may be a partial explanation of the seizure activity. A diminished output from the Purkinje cells could be a situation that permits cortical convulsive activity.