Human calcitonin increases both cyclic AMP and cyclic GMP accumulation in human kidney cells.

Salmon calcitonin stimulates both adenylate cyclase and guanylate cyclase systems in human kidney cortical cells but does not modify the cyclic nucleotide levels of medullary cells. In order to compare the effect of mammalian calcitonin and of calcitonin from the ultimobranchial body on cyclic nucleotides, the action of both salmon and human calcitonin was compared in intact kidney cells. The role of the regulatory unit in relation to the stimulation exerted by both calcitonins on the adenylate cyclase activity of kidney plasma membranes was also studied. Low concentrations of human calcitonin produced a significant increase of cyclic GMP accumulation in human kidney cortical cells. Higher hormone doses, active in stimulating the adenylate cyclase system, resulted in a progressive decline of the response. In kidney medullary cells human calcitonin was a more efficacious and potent stimulator of cyclic AMP than of cyclic GMP accumulation. Neither of the two calcitonins stimulated kidney cortical or medullary plasma membrane adenylate cyclase in the absence of guanylyl 5'-imidodiphosphate. However, in the presence of guanylyl 5'-imidodiphosphate, both calcitonins stimulated the cortical adenylate cyclase system. Under the same conditions, medullary adenylate cyclase activity was stimulated only by human calcitonin. These observations suggest that human calcitonin stimulates cyclic nucleotide accumulation in human kidney cortex and medulla, while salmon calcitonin is active only at the cortical level. This phenomenon could be explained on the basis of hormone-receptor binding.

Vasoactive intestinal peptide stimulation of human thyroid cell function.

Neurons containing vasoactive intestinal peptide (VIP) have been found to reach thyroid follicular cells in man and other mammals. Human thyroid cells in culture were exposed to different concentrations of VIP and TSH, and thyroid cell activation was assessed by measurements of cAMP accumulation. VIP exerted a dose-dependent activation, and this thyroid-stimulating effect was more rapid and short-acting than that of TSH. At low concentrations of the two agents, their effects were additive, but they seemed to initiate their effects via different receptors, since polyphloretin phosphate inhibited the effect of TSH but not that of VIP. Moreover, antiadrenergic and anticholinergic agents did not influence thyroid activation by VIP. In addition to cAMP activation in cell cultures, VIP stimulated T4 release from human thyroid slices. It is possible that, in addition to sympathetic-adrenergic and parasympathetic-cholinergic nerves, VIPergic nerves may participate in the autonomic nervous control of thyroid function.

Evidence for alpha-adrenergic receptors acting through the guanylate cyclase system in human cultured thyroid cells.

In order to investigate the presence of alpha-adrenergic receptors in human thyroid, we have studied the effect of alpha-adrenergic agonists and antagonists on cGMP cellular content of human thyroid cells in primary culture. Epinephrine as well as TSH were not able to modify the cGMP cellular levels, while norepinephrine significantly increased cGMP accumulation already at 10 nM, a dose inactive on cAMP accumulation. A non selective alpha-adrenergic antagonist, phentolamine, significantly inhibited cGMP accumulation induced by norepinephrine. Norepinephrine-induced cGMP accumulation was unaffected by prazosin, an alpha 1-adrenergic antagonist, but was abolished by yohimbine, an alpha 2-adrenergic antagonist. Phenylephrine, an alpha-adrenergic agonist, produced an increase of cellular cGMP levels without modifying cAMP content. In the presence of TSH, the cGMP response to norepinephrine was not modified; however, the increase of cAMP levels was inhibited by norepinephrine at doses inactive on cAMP accumulation, but active on cGMP levels. The present results demonstrate the existence in human thyroid cells of alpha 2-adrenergic receptors, regulating the guanylate cyclase system. It may be postulated that the counter-regulation exerted by alpha-adrenergic agonists on the response to TSH operates on the TSH-dependent adenylate cyclase.

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding.

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.

Studies of catecholamine effect on cyclic AMP in human cultured thyroid cells: their interaction with thyrotrophin receptor.

Even though adrenergic nerve terminals between and around thyroid follicles and catecholamine stimulation of thyroid adenylate cyclase have been reported, there is no uniform concept on catecholamine interaction with thyrotrophin (TSH) receptors. Therefore, the effect of catecholamines on TSH-stimulated cyclic AMP (cAMP) accumulation in human follicular thyroid cells has been investigated, to thus eliminating the extrathyroidal actions of catecholamines. Epinephrine, norepinephrine and isoproterenol appeared to be rapid and potent stimulators of intracellular cAMP accumulation, the half maximum increase doses being 4 X 10(-7)M, 1 X 10(-5)M and 5 X 10(-7)M, respectively. While propranolol (1 X 10(-5)M) prevented the stimulatory effect of catecholamines and failed to inhibit the effect of bovine TSH, phentolamine (1 X 10(-5)M) enhanced the potency of norepinephrine and bovine TSH, leaving that of epinephrine unchanged. The effects of epinephrine (2 X 10(-8)M) and isoproterenol (2 X 10(-8)M) were additive to that of bovine TSH (0.5 mU/ml), but the effect of simultaneous stimulation with norepinephrine (5 X 10(-7)M) and bovine TSH (0.5 mU/ml) was lower than expected. Prenalterol, a selective beta 1-agonist, did not stimulate cAMP accumulation, while terbutaline, a selective beta 2-agonist, exerted a potent stimulation. Metoprolol, a selective beta 1-adrenergic blocker, did not affect the response of thyroid follicular cells to isoproterenol. These results demonstrate the existence of beta-adrenergic receptors in human thyroid follicular cells, mainly of the type beta 2, apparently not correlated with TSH receptor. The existence of alpha-adrenergic receptors which counter-regulate TSH functional responses in human thyroid follicular cells is suggested.

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Monoclonal antibodies to the thyrotropin receptor: the identification of blocking and stimulating antibodies.

Monoclonal antibodies to the thyrotropin (TSH) receptor have been obtained from fusions of mouse myeloma cells with spleen cells immunized with solubilized thyroid membrane preparations. Two monoclonal antibodies which inhibit 125I-TSH binding and are reactive with the glycoprotein component of the bovine TSH receptor (11E8 and 13D11), are shown to inhibit basal and TSH stimulated adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. Both antibodies also inhibit 125I-TSH binding in vitro, whether binding is measured at pH 6.0 in low salts and at 0-4 C or at pH 7.4 in 50 mM NaCl and at 37 C. The glycoprotein component is thus a portion of the physiologic TSH receptor in vivo and 125I-TSH binding studies apparently measure the high affinity glycoprotein component under nonphysiologic conditions and conditions more representative of the physiologic milieu. A third monoclonal antibody whose interaction with thyroid membranes is prevented by TSH is shown to stimulate adenylate cyclase activity in bovine thyroid membranes and human thyroid cells. This stimulating antibody only weakly inhibits 125I-TSH binding to thyroid membranes or to the glycoprotein component of the TSH receptor. The 22A6 antibody does, however, immunoprecipitate mixed brain gangliosides, in distinct contrast to the monoclonal antibodies to the glycoprotein receptor component, i.e., 11E8 and 13D11. The results support the speculation that autoimmune antibodies which inhibit TSH binding to thyroid membranes are not necessarily identical to antibodies which stimulate function; that antibodies directed at the high affinity initial site of TSH interaction with a cell can behave as blocking rather than stimulating antibodies and that a possible relationship exists between stimulating antibodies and the low affinity TSH binding sites (gangliosides) on thyroid membranes.

Reduced mitogenic action of insulin evaluated as 3H-thymidine uptake in diabetic cultured fibroblasts.

As cultured diabetic fibroblasts have a shorter lifespan than normal subjects, the mitogenic activity of foetal calf serum (FCS) and insulin have been studied by taking skin biopsies from 5 patients with juvenile onset diabetes (JODM), 8 with adult onset diabetes (AODM) and 5 with chemical (latent) diabetes (CDM). In controls a high dose of insulin (20 microgram/ml) produced a tritiated thymidine uptake lower than 10% FCS. At lower doses (0.1 to 1.0 microgram/ml) a dose-dependent uptake was observed. In diabetic fibroblasts 10% FCS produced a lower 3H-thymidine uptake than in controls, with p less than 0.005 in JODM, p less than 0.01 in AODM and p less than 0.01 in CDM. A high dose of insulin produced a lower uptake in patients with JODM (p less than 0.01), AODM (p less than 0.01) and CDM (p less than 0.05) when compared with controls. However, when lower doses of insulin were used, 2 out 5 cases of JODM and 4 out of 8 cases of AODM showed a similar response to controls, while 3H-thymidine uptake was low or absent in the remaining cases, including all those with CDM. This study indicates that a reduced mitogenic action of FCS and insulin exists in many patients affected by clinical or chemical diabetes. Our results suggest, however, that the sensitivity to the two mitogenic factors seems to be dissociated: in fact, while a reduced response to FCS is present in all patients, the response to insulin seems to be correlated to the patient's equilibrium at the time of skin biopsy.

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue.

Since Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI2 on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI2 caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE2. Two optically active isomers of 5,6-dihydro-PGI2, i.e. stable synthetic analogs of PGI2, had qualitatively similar effects to PGI2. The relative potency ratio between the alpha- and beta- isomer as well as their potency compared to PGI2 were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI2 and its stable analogs had a greater effect than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI2 concentrations. TSH had no detectable effect on thyroidal PGI2 synthesis and release. In cultured thyrocytes the effects of PGI2 and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE2. These findings suggest the existence of a specific PGI2-responsive adenylate cyclase system in human thyroid cells other than thyrocytes, of possible physiologic significance.

Thyrotrophin-responsive adenylate cyclase activity in thyroid toxic adenoma.

The adenylate cyclase system was studied in hyperfunctioning autonomous nodules in comparison with normal thyroid tissue. The basal, TSH- and NaF-stimulated adenylate cyclase activities were tested in purified plasma membrane preparations. Basal enzyme activity in membranes from hyperfunctioning nodules was variable and the response to TSH was either normal, low or absent. The present study demonstrates that an intact adenylate cyclase activity, hyporesponsive to TSH, may exist in the cell membrane of the adenoma.

TSH-responsive adenylate cyclase activity in thyroid tissue from patients with Graves' disease.

Basal adenylate cyclase activity of thyroid plasma membranes obtained from six patients with Graves' disease was slightly but not significantly lower than normal (83.3 +/- 13.9 pmol cAMP/10 min/mg of protein versus 120.9 +/- 19.5 pmol cAMP/10 min/mg of protein). In five of these patients the adenylate cyclase activity was stimulated by bovine TSH with an apparent Km value similar to that of normal thyroid (3.1 +/- 0.5 X 10-9 M versus 3.4 +/- 0.6 X 10-9 M). The response to prostaglandin E2 was also normal. In the sixth patient adenylate cyclase activity was stimulated by prostaglandin E2 but not by bovine TSH. The distribution of basal adenylate cyclase activity in various gradient layers was studied in two TSH-responsive patients. A relative increase of this activity was found in the denser layer when compared to normal thyroid tissue. This could be the expression of an altered ratio between the protein and lipid components of the plasma membranes in patients with Graves' disease.