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Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and the effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP.

Rat thyroid cells in primary culture augment cAMP production when challenged with beta-adrenergic agonists; at 10(-5) M the potency is isoproterenol greater than norepinephrine greater than epinephrine. In analogy with human thyroid cells, rat thyroid primary cultures display alpha-adrenergic-stimulated cGMP production which inhibits TSH and norepinephrine stimulation of cAMP. Adrenergic regulation of cyclic nucelotide production is lost in the cloned thyroid cell line of rat origin known as FRTL-5. Also the potentiating effect of phentolamine on TSH stimulation of cAMP production in thyroid primary culture becomes an inhibitory one in the FRTL-5 cells. Neither 'soluble factors' nor contamination of other cell populations could account for the different behaviour of the primary culture and the cell line toward adrenergic regulation. The reported activation by norepinephrine of iodide efflux in FRTL-5 cells rules out the loss of specific adrenergic receptors in the FRTL-5 cells. It is proposed that the cloning of FRTL-5 cells from primary cultures causes an 'alteration' in the coupling of adrenergic receptors to the adenylate cyclase system. This alteration does no affect those mechanism of message transduction that do not involve cAMP as the signal.

Forskolin, at 10(-11) M, stimulates guanylate cyclase activity in primary human thyroid cell cultures, but does not modify cAMP accumulation. At a 10-fold higher concentration it still stimulates guanylate cyclase activity and becomes an inhibitor of cAMP production. Above 10(-9) M, forskolin stimulation of cGMP decreases, while it also becomes a stimulator of cAMP production. There is an additive effect of TSH and forskolin on cAMP production at concentrations of the diterpene which are stimulatory. Concentrations of forskolin which are inhibitory for cAMP, but stimulatory for cGMP, are inhibitory for TSH stimulation of cAMP. The addition of 8-bromo-cGMP duplicates the forskolin effect at low concentrations.

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)