Dataset Information

Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

ABSTRACT: Glucocorticoids are known to increase the cyclic AMP response to parathyroid hormone (PTH) in cultured bone organs or bone cells. Using the osteoblast-like cell line ROS 17/2.8, which possesses receptors for both PTH and glucocorticoids, we investigated which component of the complex hormone receptor-guanine nucleotide regulatory unit--adenylate cyclase was affected by dexamethasone treatment. In response to PTH, isoproterenol or forskolin, a compound that is supposed to act directly on the catalytic unit, cyclic AMP production by intact cells and adenylate cyclase activity in purified plasma membrane were markedly increased by dexamethasone. Whereas NaF, guanosine 5'-[beta gamma-imido]triphosphate and Mn/ stimulated adenylate cyclase activity were similarly enhanced in membranes isolated from glucocorticoid-treated cells, the activity of the stimulatory guanine nucleotide regulatory unit, as assessed by reconstitution into membranes from the CYC- clone, which is genetically devoid of this component, was not altered. Thus in osteoblast-like cells dexamethasone appears to increase cyclic AMP synthesis by influencing the catalytic unit. Moreover, since it has been reported that glucocorticoids may produce changes in cell calcium metabolism, we evaluated cytoplasmic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ stores mobilizable by the bivalent-cationophore ionomycin, by using the intracellular fluorescent indicator Quin-2. The results indicated that dexamethasone treatment did not influence [Ca2+]i but markedly decreased ionomycin-releasable Ca2+ stores.

SUBMITTER: Rizzoli R

PROVIDER: S-EPMC1147005 | biostudies-other | 1986 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.

Project description:The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate cyclase.

Dataset Information

Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets