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C M Rotella

Publications and source records attributed to C M Rotella.

At least 91 records · Page 5Linked to original sources
PubMed1986

Loss of adrenergic regulation of cAMP production in the FRTL-5 cell line.

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.

Adrenergic alpha-Agonists↗
PubMed1986

Transient lack of response to TSH of human cultured thyroid cells obtained from hyperfunctioning tissue.

TSH-induced cAMP accumulation in cells obtained from normal and pathological thyroid tissue was studied during the first 12 days of primary culture. In normal thyroid tissue cultures (N = 7), the response of cAMP to TSH was present from the second day of culture and reached its maximum after 8 days. A similar behaviour was observed in cultures obtained from euthyroid sporadic goitres (N = 8), even if the rate of response was slightly lower than that of normal tissue. Similarly, cultured cells from euthyroid 'autonomous' nodules (N = 8) appeared to be responsive to TSH during the period of study, but the rate of response was also lower than in the controls. On the contrary, in cultures obtained from toxic adenomas (N = 5) and from diffuse toxic goitres (N = 5) the response to TSH was absent during the first 4 days of culture. The cells became sensitive to TSH from 6 and 6 day onwards, with the rate of response increasing progressively and reaching its maximum on day 12. Finally, in cultured cells obtained from different areas of multinodular toxic goitres (N = 4), the response to TSH was similar to that of euthyroid goitres in cells prepared from 'cold' areas, and to that of toxic adenomas in cells obtained from 'hot' areas. The present data demonstrate the existence of an inhibitory action of unknown factors, possibly iodothyronines or thyroglobulin, on the TSH effect in short-term cultures obtained from thyrotoxic tissues. A normal TSH responsiveness can be restored when the culture is prolonged.

Adenoma↗
PubMed1985

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.

Adenylyl Cyclases↗
PubMed1985

Catecholamine, vasoactive intestinal peptide and thyrotrophin-dependent cAMP levels display a different sensitivity to iodothyronines in both normal and pathological human thyroid cells in culture.

As the interactions of iodothyronines on adrenergic and vipergic receptors are not clear, the effect of exogenous T3 and T4 on catecholamine- and VIP-induced cAMP accumulation in human normal thyroid cells after eight days of primary culture has been investigated. To evaluate the effect of endogenous iodothyronines, the response of the adenylate cyclase system to isoprenaline, adrenaline, VIP, and TSH was studied during a 10 d period. T3 and T4 were unable to modify the catecholamine- and VIP-induced cAMP accumulation in human normal thyroid cells after 6-8 days of culture, while the response to TSH was significantly inhibited. In cells cultured from thyrotoxic tissue, the response of the adenylate cyclase system to catecholamines and VIP, during a 10 d primary culture, showed a behaviour similar to controls. TSH responsiveness was negligible up to the fourth day of culture, while in normal cells a response to all the agonists was present from the beginning. In view of the lack of effect of iodothyronines on catecholamine- and VIP-induced cAMP accumulation, and of the superimposable behaviour of the response to catecholamines and VIP in normal and hyperthyroid cells during the first days of culture, we can conclude that iodothyronines do not directly modify the response of the adenylate cyclase system to adrenergic and vipergic stimulation in human thyroid follicular cells. The lack of responsiveness to TSH of cells obtained from hyperthyroid tissue during the first 4 d of culture, associated with normal responsiveness to catecholamines and VIP, points to a possible involvement of biogenic amines and neuropeptides in sustaining such hyperthyroid states.

Adult↗
PubMed1984

Forskolin perturbs cGMP as well as cAMP levels in human thyroid cells.

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.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies.

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.

Adult↗
PubMed1983

Specificity of thyroglobulin interactions with thyroid cells and membranes.

Homologous species specificity is demonstrated with bovine and human thyroglobulin in which the two terminal sugars of the B carbohydrate chain, sialic acid and galactose have been removed by enzymatic hydrolysis. The species specificity is demonstrated by measuring the ability of the deglycosylated thyroglobulin derivatives to inhibit thyrotropin-induced increases in cAMP in human, rat and bovine thyroid cells in culture. Thus human-human or bovine-bovine interactions have higher activity coefficients by at least an order of magnitude than their heterologous counterparts. The homologous interactions are confirmed in binding studies and shown to be associated with negligible degradation of the bound ligand over a 24 hour period.

Animals↗
PubMed1983

Structural changes caused by thyrotropin in thyroid cells and in liposomes containing reconstituted thyrotropin receptor.

Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.

Animals↗
PubMed1983

Salmon calcitonin and cGMP production by human kidney: studies in vivo and in vitro.

In order to evaluate whether or not the action of salmon calcitonin (sCT) at the kidney level could be mediated through specific receptors for the hormone, we have studied the effects of sCT infusions on urinary excretion of cyclic nucleotides in humans. Parallel in vitro studies have been conducted by evaluating the effects of sCT on cyclic nucleotide levels in primary cultures of cortical and medullary human kidney cells. In vivo experiments showed that sCT induced an increase in cGMP in human urine, which was rapid and short-lasting, being superimposable on the increase of urinary excretion of calcium and magnesium. The increase of inorganic phosphate urinary excretion was delayed and appeared to parallel that of urinary cAMP. On the other hand, our in vitro experiments showed that sCT stimulated the guanylate cyclase-cGMP system of human kidney cortical cells at nanomolar concentrations, while higher concentrations of the hormone were required to activate the adenylate cyclase-cAMP system. In addition, sCT was not able to significantly modify the cellular levels of either nucleotide in human kidney medullary cells. Present data demonstrated a direct effect of sCT on human kidney cortical cGMP production, while the efficacy of sCT on the kidney cortex adenylate cyclase-cAMP system appears to be delayed and/or reduced.

Aged↗
PubMed1983

Effect of prostaglandin E2 on adenylate cyclase system in human kidney cell cultures: interactions with parathormone and arginine-vasopressin.

We have used human kidney cortical and medullary cells in primary culture as an experimental model, in order to study the effect of prostaglandin E2 on the adenylate cyclase-cyclic AMP system at renal level, in comparison with that of parathormone and desamino 1-D-arginine 8-vasopressin. Prostaglandin E2 is a more potent stimulator of cyclic AMP accumulation in cortical cells than in medullary ones, the minimal effective dose being 3 nM in the cortex and 20 microM in the medulla. The maximal response was obtained with 30 microM in cortical cells and with 0.3 mM in medullary ones. The effects of combining submaximal and maximal active doses of the three hormones were always additive when tested on cortical cells. On the contrary, prostaglandin E2, at doses effective on the medullary cells adenylate cyclase system, produced an increase of cyclic AMP accumulation significantly lower than expected when incubated with submaximal and maximal amounts of desamino 1-D-arginine 8-vasopressin. Present data provide evidence for the existence of distinct receptors for parathormone, arginine-vasopressin and prostaglandin E2 at cortical level; the possible role of prostaglandin E2 in the counter-regulation of arginine-vasopressin action on medullary cells in unlikely, in view of the high prostaglandin E2 concentration which is necessary to exert this effect.

Adenylyl Cyclases↗
PubMed1983

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.

Cells, Cultured↗
PubMed1983

Characterization of the optimal stimulatory effects of graves' monoclonal and serum immunoglobulin G on adenosine 3',5'-monophosphate production in fRTL-5 thyroid cells: a potential clinical assay.

Immunoglobulin G (IgG) preparations derived from the sera of patients with hyperthyroidism due to Graves' disease (TSAb) as well as a monoclonal IgG derived from heterohybridoma fusions of Graves' lymphocytes augmented cAMP levels in a continuous strain of functioning rat thyroid cells (clone FRTL-5) in culture. Optimal stimulation was the same for both types of IgG preparations when measured after 2 h of incubation with 5 X 10(4) cells/well and using cells maintained in a nongrowth, TSH-deficient medium for 7 days. At low IgG concentrations, the stimulatory activities of both preparations exhibited a linear dependence on concentration and similar Ka values (approximately 4 X 10(-8) M) despite the fact that 65% of the Graves' serum IgG preparations had a significantly better ability to inhibit TSH binding to membrane preparations. The Ka value for TSH in the same assay was about 5 X 10(-12) M. Using this cell assay, 90% of a series of hyperthyroid Graves' IgG preparations exhibited stimulating activity, a value comparable to the frequency of positive results found by ourselves and others using human thyroid cell and slice systems. In contrast, only 10% of patients who were euthyroid 1 yr after antithyroid drug withdrawal (n = 21) exhibited stimulating activity, and no stimulating activity was detected in patients with nontoxic nodular goiter (n = 11), toxic adenoma (n = 5), or thyroid carcinoma (n = 6). The studies suggest that an optimized rat FRTL-5 thyroid cell system is a clinically useful and convenient alternative to human thyroid cell and slice systems for detecting TSAbs.

Animals↗
PubMed1983

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.

Adrenergic beta-Agonists↗
PubMed1983

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.

Adrenergic alpha-Agonists↗
PubMed1982

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.

Antibodies, Monoclonal↗
PubMed1982

Effects of TSH on cAMP levels and thyroid hormone release in human thyroid 'autonomous' nodules: relationship with iodothyronine and iodine content in thyroglobulin.

The effect of TSH on the adenylate cyclase-cAMP system and in vitro iodothyronine release, together with the iodothyronine and iodine content of 19s thyroglobulin, were studied in seven clinically euthyroid patients with autonomous thyroid nodules. Basal cAMP and cGMP content together with phosphodiesterase and protein-kinase activities were normal in nodular, and suppressed in extranodular tissue. TSH-dependent cAMP accumulation was reduced in nodular tissue, but normal in the suppressed extranodular tissue. In vitro TSH-dependent iodothyronine release from nodular and extranodular tissue was absent. Thyroxine and iodine content of thyroglobulin extracted from nodular tissue was reduced, while triiodothyronine content was normal but with a low T4/T3 ratio. In extranodular tissue T3, T4 and iodine contents were reduced. In conclusion, autonomous thyroid nodules produced a poorly iodinated thyroglobulin leading to preferential T3 secretion with increased circulating free thyroid hormones even in clinically euthyroid patients.

Adult↗