Adenylate cyclase cell culture

Goldberg H, dayman P, Skorecki K Mechanism of U inhibition on vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells. Am J Physiol 24 F995-F1002, 1988... 

Brunton, L.L., Wiklund, R.A., VanArsdale, P.M. and Gilman, A.G. (1976). Binding of [ Hlprostaglandin to putative receptors linked to adenylate cyclase of cultured cell clones. J. Biol Chem., 251, 3037-3044... 

Kassis, S., Olasmaa, M., Sullivan, M. and Fishman, P.H. (1986). Desensitization of the j5-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells Receptor sequestration versus receptor function. ]. Biol Chem., 261, 12233-12237... 

These results suggest that synthesis and degradation of cAMP in cultured carrot cells are both controlled and switched on/off according to the concentration of Ca2+ in carrot cytoplasm. Adenylate cyclase activity is induced in the cells only in the resting state, and the enzyme activity is... 

Some cellular responses occur too rapidly following steroid hormone exposure to involve the multi-step process of nuclear receptor activation. For example, 17/6-estradiol can rapidly stimulate adenylate cyclase and cause a near-instantaneous increase in intracellular cAMP in cultured prostate cells. These effects are mediated by the interaction of steroid hormones with cell surface proteins. 

The role of cyclic AMP as modulator of prolactin secretion was first suggested by the finding of a stimulatory effect of cyclic AMP derivatives (17-22) and inhibitors of cyclic nucleotide phosphodiesterase activity such as theophylline and IBMX (22-26) on the secretion of this hormone. More convincing evidence supporting a role of cyclic AMP in the action of dopamine on prolactin secretion had to be obtained, however, by measurement of adenohypophysial adenylate cyclase activity or cyclic AMP accumulation under the influence of the catecholamine. As illustrated in Fig. 1, addition of 100 nM dopamine to male rat hemipituitaries led to a rapid inhibition of cyclic AMP accumulation, a maximal effect (30% inhibition) being already obtained 5 min after addition of the catecholamine. Thus, while dopamine is well known to stimulate adenylate cyclase activity in the striatum (27, 28), its effect at the adenohypophysial level in intact cells is inhibitory. Dopamine has also been found to exert parallel inhibitory effects on cyclic AMP levels and prolactin release in ovine adenohypophysial cells in culture (29) and purified rat mammotrophs (30). Using paired hemipituitaries obtained from female rats, Ray and Wallis (22) have found a rapid inhibitory effect of dopamine on cyclic AMP accumulation to approximately 75% of control. 

The above-described data show that CRF added to cells of the rat Intermediate lobe In culture causes a rapid stimulation of oe-MSH release and cyclic AMP accumulation, thus demonstrating a direct action of the peptide on pars intermedia cells (15). It is however difficult, using intact cells, to dissociate between increases in cyclic AMP levels due to stimulation of adenylate cyclase activity or to Inhibition of cyclic nucleotide phosphodiesterase or to a combination of both effects. Definitive proof of the role of adenylate cyclase In the action of CRF In the intermediate lobe of the pituitary gland is provided by the following findings of a CRF-lnduced stimulation of adenylate cyclase activity in homogenate of rat and bovine pars Intermedia cells. 

The present data clearly demonstrate that the 41-amino acid ovine CRF Is a potent stimulator of adenylate cyclase activity In rat and bovine pars Intermedia tissue. Our previous data have shown that CRF causes a rapid and marked stimulation of cyclic AMP accumulation in rat pars Intermedia cells in culture (15). The final proof of the role of adenylate cyclase in the observed changes of cyclic AMP levels had to be obtained by direct measurement of adenylate cyclase activity. 

Although earlier studies failed to find consistent effects of DA on cAMP levels or adenylate cyclase activity in anterior pituitary cells [13], intact pituitary gland [14,15] or homogenates [13,16], a functional connection between the two is now supported by many experimental approaches. DA and DA agonists inhibit cAMP levels in cultured rat pituitary cells at concentrations in the nanomolar range, comparable to those which inhibit PRL release [17-21], DA also inhibits cAMP accumulation stimulated by VIP or TRH [20]. Inhibition is also seen in human prolactinoma cells [22]. 

Unlike adrenal and hepatic tissue, vascular smooth muscle apparently possesses only a single receptor type. The observed affinity of this receptor varies among different muscle preparations from a Kd of 15-50 nM in aorta [1] to a Kd of 2-5.5 nM in mesenteric artery and cultured vascular cells [9], Nonetheless, it appears that, in at least some smooth muscle cell types, All alters the activity of both phospholipase C and adenylate cyclase, presumably via a single receptor type. 

Anandamide parallels d9-THC in competing with the binding of [3H]HU-243 (6) to the brain cannabinoid receptor [33]. However, under most experimental conditions an amidase blocker has to be used in order to prevent anandamide hydrolysis during the binding experiments [38], Using neuroblastoma cells that express this receptor naturally, as well as cultured cell lines transfected with this receptor, it was shown that, like d9-THC, anandamide inhibits A-type voltage-dependent calcium channels [39] and adenylate cyclase [40, 41],... 

Several processes in the immune response are affected by lithium in vivo and in vitro 139). The proliferative responses of hamster lymphoid cells to concanavalin A or phytohemagglutinin, which stimulate mitosis in T cells, were enhanced by lithium in a serum-free culture system. Proliferative stimulation also was obtained with lithium using the B cell mitogen lipopolysaccharide, but the B cell mitogens dextran sulfate and trypsin had no effect 140-143). Lithium increased the effects of suboptimal concentrations of stimulants, but had smaller effects on stimulation by optimal concentrations. With concanavalin A, the response to optimal stimulatory concentrations was inhibited 140). Paradoxical results such as these may be due to inhibitory effects of lithium on adenylate cyclase, or to effects on membrane transport systems 141). Most of these experiments used very high concentrations of lithium, considerably in excess of normal therapeutic doses (maximal inhibitory concentrations were 10 mM with hamster cells and 5 mM with human lymphocytes). At therapeutic levels of lithium, increased incorporation of [ H]thymidine was seen in human peripheral blood mononuclear cells. 

Previous reports that some TCA and non-TCA, like Iprindol and mianserin, were inhibitors of histamine-sensitive adenylate cyclase from mammalian brain Iri vitro were confirmed for a wide range of antidepressants.22 xhe effect appeared to be mediated by H2-receptors but neither monoamine oxidase inhibitors (MAOI) nor selective serotonin (5-HT) uptake inhibitors were very effective. However, TCA also blocked histamine-stimulated cyclic GMP formation in cultured nerve cells by a mechanism which involved Hx-receptors.23 The relevance of these findings, and indeed of the role of histamine in the central nervous sytem, has still to be defined. 

The exact mechanism(s) responsible for fluoride s nephrotoxicity remain to be defined. The fluoride ion interferes with normal cell function on several levels. Fluoride is an inhibitor of several cellular enzyme systems and dirninishes tissue respiration and anaerobic glycolysis [89]. In the kidney, fluoride interferes with transport of sodium in the proximal convoluted tubule. It also inhibits adenylate cyclase in the collecting system and dirninishes the action of antidiuretic hormone. Experimental evidence in rats indicates that the chloride dependent pump, in the thick ascending part of Henle s loop, also is inhibited [90]. In human collecting duct cell cultures, exposure to fluoride ions inhib-... 

Bakardjieva, A., Galla, H. J., and Helmreich, E. J. M., Modulation of the /8-receptor adenylate cyclase interactions in cultured change liver cells by phospholipid enrichment. Biochem. 18, 3016 (1979). 

Maus, M.P., Bertrand, S., Drouva, R., Rasolonjanahary, C., Kordon, J., Glowinski, J., Fremont, J. and Enjalbert, A. (1989) Differential modulation of D and D2 dopamine-sensitive adenylate cyclases by 17/ -estradiol in cultured striatal neurons and anterior pituitary cells. J. Neurochem. 52 410-418. 

The use of in vivo bioassays (e.g., hyperglycemia) as a measure of insulin in an intact animal or organ is largely historic because the hormones can be measured directly, but in vitro bioassays or bioassays using endocrine responsive tissue cell cultures can be useful in screening compounds. These assays can measure changes of adenylate cyclase, intracellular calcium, and phosphoinositol metabolites. 

Activation of Cyclic AMP Generation in Intact Cells - Forskolin activates adenylate cyclase in Intact cells and tissues with similar characteristics as those observed for activation of the enzyme in membranes and solubilized preparations. These Include preparations of rat35 and human36 adipocytes, human platelets,37 tissue slices from brain and other peripheral tissues,3° and various endocrine and secretory tissues.39 Forskolin stimulates adenylate cyclase in S49 lymphoma cells,25 32 j-at astrocytomas.rat pheochromocytoma cells, 2 cultured pituitary cells, 3-48 cardiomyocytes, >30 cultured leydlg cells,31 and cultured kidney cells.32,53 Forskolin increases intracellular cyclic AMP rapidly with an EC50 of about lOpM, and results in elevations of cyclic AMP over basal levels which range between two and fifty-fold, depending on cell type and tissue. 

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