Alpha(1a)-adrenergic and Alpha(1b)-adrenergic Receptors Mediate the Effect of Norepinephrine On Cytosolic Calcium Levels In Rat Pc C-13 Thyroid-cells - Thyrotropin Modulation of Alpha(1b)-linked Response Via A Adenosine 3',5'-monophosphate-protein Kinase-a-dependent Pathway

The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC Cl3 rat thyroid cells and identify the molecular mechanisms by which TSH regulates the noradrenergic response. We studied TSH regulation of norepinephrine-induced cytosolic calcium increase by means of the fluorescent probe fura-2. In PC Cl3 cells grown and maintained in a medium containing TSH (PC Cl3 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC Cl3 starved from TSH 5 days before the experiments (PC Cl3 5H). In both group of cells the calcium response to norepinephrine was concentration dependent and reduced by the removal of extracellular calcium ions. Reintroduction of TSH in the culture medium of the PC Cl3 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC Cl3 6H. This effect was complete after a 48-h incubation period and was abolished by the simultaneous treatment of the cells with the protein synthesis inhibitor cycloheximide, suggesting that TSH may stimulate the synthesis of alpha(1)-adrenergic receptors in PG Cl3 cells. Because in these cells we found that TSH increased cAMP levels as well as inositol phosphate production, we tested whether the activation of a protein kinase-A and/or protein kinase-C was involved in TSH regulation of the adrenergic response. We found that the treatment of PC Cl3 5H cells with forskolin restored the effect of norepinephrine on the calcium level, and that KT5720, an inhibitor of the protein kinase-A, was able to prevent the recovery of the noradrenergic response induced by the readdition of TSH to the culture medium of PC Cl3 5H. Conversely, treatment of PC Cl3 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC Cl3 6H and, to a lesser extent, in PC Cl3 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize alpha(1)-adrenergic receptor subtypes mediating the effects of norepinephrine in PC Cl3 cells, we used antagonists of alpha(1A) and ale receptors (WB4101 and chlorethylclonidine respectively). Under these experimental conditions we found that 1) chlorethylclonidine caused an almost complete inhibition of the norepinephrine-induced calcium increase in PC Cl3 6H, whereas a partial inhibition of the norepinephrine response in PC Cl3 5H occurred; 2) WB4101, at a concentration specific for alpha(1A) receptors (0.01 CIM), slightly reduced the effect of norepinephrine in PC Cl3 6H and reduced norepinephrine stimulation by 50% in PC Cl3 5H; and 3) chlorethylclonidine plus WB4101 (0.01 mu M) completely abolished the noradrenergic response in both groups of cells. In line with the functional results, binding studies with [H-3]prazosin showed a lower binding capacity (B-max) for alpha(1)-binding sites in PC Cl3 5H than in PC Cl3 6H. Pretreatment of PC Cl3 cells with chlorethylclonidine reduced the specific binding for [H-3]prazosin in both PC Cl3 6H and 5H. However, in PC Cl3 6H, chlorethylclonidine inhibition of [H-3]prazosin binding was higher than that in PC Cl3 5H (86% and 36%, respectively, for 2.5 nM prazosin)