The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC C13 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 C13 cells grown and maintained in a medium containing TSH (PC C13 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC C13 starved from TSH 5 days before the experiments (PC C13 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 C13 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC C13 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 α1-adrenergic receptors in PC C13 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 C13 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 C13 5H. Conversely, treatment of PC C13 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC C13 6H and, to a lesser extent, in PC C13 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize α1-adrenergic receptor subtypes mediating the effects of norepinephrine in PC C13 cells, we used antagonists of α1A and α1Breceptors (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 C13 6H, whereas a partial inhibition of the norepinephrine response in PC C13 5H occurred; 2) WB4101, at a concentration specific for α1A receptors (0.01 μM), slightly reduced the effect of norepinephrine in PC C13 6H and reduced norepinephrine stimulation by 50% in PC C13 5H; and 3) chlorethylclonidine plus WB4101 (0.01 μM) completely abolished the noradrenergic response in both groups of cells. In line with the functional results, binding studies with [3H]prazosin showed a lower binding capacity (Bmax) for α1-binding sites in PC C13 5H than in PC C13 6H. Pretreatment of PC C13 cells with chlorethylclonidine reduced the specific binding for [3H]prazosin in both PC C13 6H and 5H. However, in PC C13 6H, chlorethylclonidine inhibition of [3H]prazosin binding was higher than that in PC C13 5H (86% and 36%, respectively, for 2.5 nM prazosin). To conclude, we found that in PC C13 thyroid cells, both α1A-and α1B-adrenergic receptors mediate the effect of NE on intracellular Ca2+and that TSH modulates the increase in intracellular Ca2+ evoked by NE through the regulation of α1B-receptor synthesis. Our data suggest that this effect of TSH is related to a cAMP/protein kinase-A-depend-ent regulatory pathway controlling the synthesis of noradrenergic receptors.
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