Effects of thyrotropin and cholera toxin on the thyroidal adenylate cyclase-adenosine 3',5'-monophosphate system

The present experiments examined the relationship between cholera toxin and TSH stimulation of the adenylate cyclase system in bovine thyroid tissue. Preincubation of thyroid slices for 20 min at 4 C with a maximal concentration of cholera toxin (100 microgram/ml) did not impair the subsequent stimulation of cAMP by submaximal amounts of TSH (1 mU/ml) during a 5-min incubation at 37 C. Incubation of cholera toxin or TSH with mixed gangliosides, followed by the addition of thyroid slices resulted in inhibition of the cholera toxin but not the TSH stimulation of cAMP formation. Previous exposure of thyroid slices to TSH induced refractoriness to subsequent stimulation of cAMP formation by TSH, but the response to cholera toxin was unchanged. NAD is necessary for cholera toxin, but not TSH, stimulation of adenylate cyclase. In the absence of NAD, cholera toxin inhibited the effect of maximal concentrations of TSH and prostaglandin E1 on adenylate cyclase activity but had no effect on NaF stimulation. In the presence of NAD, the stimulation of adenylate cyclase activity of bovine thyroid plasma membranes by a maximal amount of TSH was not influeced by maximal amounts of cholera toxin. Cholera toxin had a biphasic action on the binding of [125I]iodo-TSH, with low concentrations enhancing and high concentrations inhibiting binding. TSH augmented the binding of [125I]iodo-cholera toxin over the range of 1-100 mU/tube. Cholera toxin at 10 microgram/ml maximally inhibited binding. In addition to the requirement for ribosylation of adenylate cyclase, the present results indicate that the mechanisms of action of TSH and cholera toxin on cAMP formation are different.     

Stimulation by forskolin of the thyroid adenylate cyclase, cyclic AMP accumulation and iodine metabolism

Forskolin, a diterpene hypotensive drug, activates adenylate cyclase in brain and in some other tissues (Seamon et al., 1981). Forskolin activated adenylate cyclase in particulate preparations and enhanced cyclic AMP accumulation in slices of dog thyroid. These effects were maximal within minutes and remained constant afterwards. The action of forskolin on intact cells disappeared rapidly after washing. It reproduced two known cyclic AMP-mediated TSH effects: the activation of secretion and of protein iodination. Forskolin thus provides a very convenient tool for the study of the action of defined elevations of cyclic AMP level in thyroid cells. The activation by forskolin of adenylate cyclase was not reduced by Mn2+ which uncouples TSH and PGE1 action. This suggests that in the thyroid also, forskolin acts beyond the receptor level. The effect of forskolin on cyclic AMP accumulation was inhibited by the known negative regulators of this system in the thyroid, acetylcholine, iodide, norepinephrine, PGF1 alpha and adenosine. On the other hand, forskolin potentiated the effects of TSH, PGE1 and cholera toxin. These data show that, though it does not require the receptors for its action, forskolin does not uncouple them from the catalytic unit of adenylate cyclase.     

Effect of thyrotropin-induced desensitization of bovine thyroid adenylate cyclase on the nucleotide regulatory protein

The stimulation of adenylate cyclase by TSH was decreased 50-60% in crude membranes prepared from homogenates of bovine thyroid slices that had previously been incubated for 2 h with the hormone. The diminished response was not associated with any significant change in the binding capacity or affinity for 125I-labeled TSH. The apparent affinities of the desensitized adenylate cyclase for TSH or GTP were not different from those of the enzyme prepared from thyroid slices that had been incubated without TSH. Decreased adenylate cyclase responses to NaF, cholera toxin, or guanyl-5'-yl-imidodiphosphate were also observed in the desensitized membrane, whereas the enzyme responses to prostaglandin E1, GTP, or forskolin were not decreased. However, desensitization caused no decrease in the cholera toxin-catalyzed ADP ribosylation of the 40,000 mol wt polypeptide guanine nucleotide-binding component of the adenylate cyclase. The desensitized membranes showed basal adenylate cyclase activity similar to that of the control membranes using adenyl-5'-yl-imidodiphosphate as substrate in the absence of a nucleotide-regenerating system. These results suggest that the in vitro TSH-induced desensitization of thyroid adenylate cyclase reflects an alteration in the activation processes of the nucleotide regulatory protein.     

Inhibition of cyclic AMP formation by iodide in suspension cultures of porcine thyroid follicle cells

In the present study porcine thyroid cells in suspension cultures were employed to investigate the suppressive effect of iodide on adenylate cyclase under basal conditions and following incubation with TSH, PGE1, cholera toxin and forskolin. Within 30 min of incubation with iodide (half-maximal effect 10(-5) M), inhibition was established and remained unchanged up to 40 h of culture. The inhibitory action was abolished by methimazole. TSH, PGE1, cholera toxin and forskolin stimulated cAMP accumulation 10-, 3-, 24- and 22-fold, respectively. Iodide pretreatment reduced basal cAMP levels and also made the cells less sensitive to stimulation by the various agents. High concentrations of TSH or PGE1 could not overcome the suppressive influence of iodide, whereas with high concentrations of cholera toxin and forskolin the reduction in cAMP levels in iodide-treated cultures was less pronounced. Membranes isolated from iodide-treated cultures produced significantly lower amounts of cAMP compared to control membranes. Furthermore, iodide did not inhibit basal or forskolin-stimulated cAMP production in human fibroblasts. The results demonstrate that iodide via an iodination-dependent mechanism influences cAMP generation in thyroid cells. It is suggested that the inhibitory activity, which has a long half-life, involves stable modification of the membrane-localized catalytic unit of adenylate cyclase such that its activation by the regulatory unit is rendered less efficient.     

Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.     

Desensitization of the thyroid cyclic AMP response to thyroid stimulating immunoglobulin: comparison with TSH

Studies were conducted to examine the characteristics of thyroid cell cAMP stimulation by thyroid stimulating immunoglobulins (TSI) and to compare the cAMP response to TSI and TSH in desensitized human thyroid cells. In terms of cAMP production, preexposure (eight hours) of the cells to TSI induced a desensitization very similar to TSH-induced desensitization: both TSH- and TSI-desensitized cells showed a normal response to cholera toxin and forskolin stimulation; TSH and TSI desensitization was interchangeable in that desensitization by either stimulator affected the action of the other; the time of recovery from either TSH and TSH desensitization was identical; the cycloheximide (10(-4) mol/L) prevented both TSI- and TSH-induced desensitization; preexposure of the cells to iodine, which affects mainly the adenylate cyclase catalytic unit, or to epinephrine, which activate the inhibitory regulatory protein Ni by the alpha 2-adrenergic stimulation, induced a similar inhibition of the subsequent stimulation by both TSH or TSI. The remarkable similarities between TSH and TSI in stimulating and desensitizing thyroid cells strongly support the concept that TSI activates thyroid adenylate cyclase by interacting with the TSH receptor and not through an allosteric mechanism.     

Alteration of the functional activity of Gs protein in thyrotropin-desensitized pig thyroid cells.

Changes in the sensitivity of adenylyl cyclase observed in pig thyroid cells cultured 2 days in the presence of thyroid-stimulating hormone (TSH) or forskolin were assessed by examining the properties of Gs protein. Chronic treatment of thyroid cells with various concentrations of TSH (0.01-1 mU/ml) or forskolin (0.1-10 microM) increased the response of adenylyl cyclase to a further stimulation by forskolin or NaF + AlCl3 ([AlF4]-). In contrast, the enzyme activation promoted by guanosine 5'-(beta,gamma-imido) triphosphate (Gpp(NH)p) was markedly affected. There was a significant increase in adenylyl cyclase activation by Gpp(NH)p in membranes from cells treated with low concentrations of TSH (less than or equal to 0.1 mU/ml) or forskolin (less than or equal to 1 microM) but a significant decrease in membranes from cells cultured with a higher concentration of TSH (1 mU/ml) or forskolin (10 microM). This decrease in Gpp(NH)p-stimulated adenylyl cyclase activity was mimicked by 8-bromo-cAMP but not by 1,9-dideoxyforskolin, a forskolin analogue which has lost its ability to activate adenylyl cyclase. There was a good correlation with the ability of Gs protein to be ADP-ribosylated by cholera toxin: labeling of Gs protein decreased following chronic treatment of thyroid cells with TSH (1 mU/ml) or forskolin (10 microM). In contrast, under the same experimental culture conditions a slight but significant increase in the quantity of Gs subunits was observed by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of growth in cultured rat thyroid cells

The purpose of this study was to determine the role of cAMP in the growth of FRTL and FRTL5 cells, 2 continuous cultured thyroid lines. TSH, at concentrations similar to those reported to induce growth in primary dog thyroid cultures, played an essential role for growth. Stimulators of adenylate cyclase, cholera toxin and forskolin, and cAMP analogues, dibutyryl cAMP and 8-bromo cAMP, mimicked the effect of TSH in both groups of cultured cells. The present data confirm the role of TSH in controlling growth of both cell lines and suggest that cAMP is an essential intracellular mediator of TSH action.     

Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells

Porcine thyroid follicle cells in monolayer cultures were shown to contain one single class of high-affinity EGF receptors with Kd = 4.5 X 10(-10) M and approximately 20 000-25 000 receptors per cell. Suspension cultures of aggregated follicle cells, exposed to TSH for 3 days, showed a 3-fold increase in [125I]EGF binding. Scatchard analysis demonstrated that this was due to an increase in receptor number. Other cAMP-elevating agents (cholera toxin, dibutyryl cAMP, forskolin) induced a similar effect. In suspension cultures, preincubation with TSH or cholera toxin for 2 days reduced the subsequent [3H]thymidine incorporation. This inhibition was overcome by a low concentration of EGF (0.1 ng/ml). At higher concentrations of EGF (1-10 ng/ml) the incorporation of [3H]thymidine was potentiated 2-3-fold in cultures preexposed to TSH or cholera toxin. The results demonstrate the presence of a high-affinity EGF receptor in porcine thyroid follicle cells. Receptor expression, as well as responsiveness to the mitogenic action of EGF, is modulated in vitro by TSH, through a cAMP-dependent process.     

Forskolin stimulation of adenylate cyclase in human thyroid membranes

Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with half-maximal stimulation occurring at 5-10 microM. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p- (above 1 microM). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.     

Influences of cholera toxin on thyroid stimulation by thyrotropin and thyroid-stimulating antibody

Thyroid-stimulating antibody (TSAb) is an immunoglobulin G (IgG) occurring in the blood in hyperthyroid Graves' disease patients; it stimulates the thyroid in a manner analogous to the action of TSH, i.e. by activation of adenylate cyclase. Since cholera toxin is also known to stimulate thyroid adenylate cyclase, we studied possible interaction of the enterotoxin on effects of TSAb and TSH using slices of canine thyroid in vitro and an increase in the concentration of cAMP as endpoint. Normal human IgG, known to inhibit the binding of [125I]-iodo-TSH to thyroid membranes, decreased stimulation of thyroid slices by TSH; this inhibitory effect occurred also with preparations of TSAb (inevitably comprised mainly of normal IgG) that were themselves stimulatory. The cholera toxin effect was not prevented by normal IgG and, by factorial analysis of variance, was shown to potentiate the action of subsequently added TSAb or TSH. There was also positive interaction of the effect of TSAb with the combination of TSH and cholera toxin. The data indicate that responses of thyroid tissue to TSAb and TSH are readily influenced by effects of other membrane-active agents (in the present context, normal IgG and cholera toxin).     

Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.     

Insulin-like growth factor-I potentiates thyrotropin stimulation of adenylyl cyclase in FRTL-5 cells

We reported that TSH and insulin-like growth factor-I (IGF-I), which were known to synergistically stimulate DNA synthesis, synergize to elevate the 1,2-diacylglycerol content of FRTL-5 thyroid cells. We presented evidence that cAMP is the proximal mediator of these actions of TSH. To further define the mechanism of this interaction, we investigated the effects of IGF-I on TSH stimulation of adenylyl cyclase. Long and short term effects of IGF-I or high doses of insulin were studied in FRTL-5 cells that were maintained in serum-, hormone-, and growth factor-free medium for 4-7 days (basal cells). When cells were incubated with high doses of insulin for 7 days and acutely stimulated, a 10-fold increase in sensitivity and a 2-fold increase in maximal responsiveness of cAMP accumulation to TSH were observed. To study shorter term effects, cells were preincubated with insulin for 3 h and then exposed to TSH, cholera toxin, or forskolin. Incubation with high doses of insulin for 3 h caused 30-300% increases in cAMP accumulation at high doses of TSH (greater than or equal to 1 mU/ml), cholera toxin (greater than 0.1 microM), and forskolin, but did not affect the EC50 for TSH. Dose-response studies were consistent with insulin acting via receptors for IGF-I, and IGF-I caused a similar effect. There was a 45% increase in adenylyl cyclase activity stimulated by TSH in membranes isolated from cells incubated with high doses of insulin for 3 h. Pretreatment of FRTL-5 cells with pertussis toxin, which ADP-ribosylates the inhibitory G-protein Gi, or adenosine, which we show inhibits cAMP accumulation by interacting with Gi, did not affect insulin/IGF-I enhancement of cAMP accumulation. We suggest that synergism of actions of TSH and IGF-I may in part be due to IGF-I enhancement of TSH stimulation of adenylyl cyclase.     

Influence of thyroglobulin on basal and stimulated human thyroid adenylate cyclase activity

The interaction of thyroglobulin (Tg), thyroid-stimulating immunoglobulins (TSI), and TSH on human thyroid plasma membranes from nontoxic goiter was studied in vitro by an adenylate cyclase assay system using human thyroid homogenate. Purified Tg [3 X 10(-10) M (0.2 micrograms/ml) to 3 X 10(-8) M (20 micrograms/ml)] exerted a dose- and time-dependent inhibitory influence on basal adenylate cyclase activity. The inhibition was prevented by preincubation with Tg antibody in excess. Tg (3 X 10(-8) M) caused a significant reduction in the TSH- and TSI-stimulated adenylate cyclase activities, but did not influence stimulation with NaF (8 mM). Fractions of thyroid homogenates were obtained by differential centrifugation, and the maximal inhibitory influence of Tg was located in the 5000 X g fraction. Thus, Tg is an efficient inhibitor of basal and TSH- or TSI- stimulated adenylate cyclase activities, and might be involved in a short loop counterregulation of thyroid adenylate cyclase sensitivity in vivo.     

Chronic and acute effects of forskolin on isolated thyroid cell metabolism

The chronic treatment (2 days or more) of cultured thyroid cells with 1-10 microM forskolin (forskolin-treated cells) sensitizes the response of adenylate cyclase to further acute stimulation by 100 microM forskolin or 10 mU/ml thyrotropin (TSH). This positive regulation, similar to that produced by 0.1 mU/ml TSH (TSH-treated cells), is obtained between 2 and 3 days of culture. The acute response to TSH or forskolin of cells treated for 4 days with forskolin increases with the concentration of forskolin present during the chronic treatment. This result is different from that obtained after a chronic treatment with TSH which induces refractoriness beyond 0.1 mU/ml. These cells are then desensitized to TSH but not to forskolin. When both agonists are mixed together, their acute effect is additive on control, TSH- and forskolin-treated cells. The chronic treatment of cultured thyroid cells with 1-10 microM forskolin produces, just like 0.1 mU/ml TSH, a chronic phospholipid effect characterized by enhanced incorporation of 32Pi into phosphatidylinositol (PI) and phosphatidic acid. The acute challenge of these cells with 100 microM forskolin evokes a reverse phospholipid effect, i.e. a decreased incorporation of 32Pi into PI. The acute stimulation of TSH-treated cells with TSH produces a reverse phospholipid effect whereas the acute stimulation of forskolin-treated cells with TSH gives a normal phospholipid effect as it does on control cells. These results show that the observed effects of TSH on cAMP accumulation and phospholipid turnover are not independent and are regulated in an inverse reciprocal pattern.     

Stimulation of thyroid adenylate cyclase activity by sera from patients with non-thyroidal illness

We have previously shown that sera from many hypothyroid patients stimulated adenylate cyclase activity as measured by serum bioactive TSH concentrations produced by FRTL-5 cell line. This TSH-stimulating activity did not correlate with serum immunoreactive TSH. IgG fractions of these sera did not stimulate FRTL-5 cells. The present study was, therefore, undertaken to investigate the thyroid stimulating activity of sera from patients with non-thyroidal illness. Studies were performed in 36 patients with various non-thyroidal illness. In these patients, serum concentrations of T4, free thyroxine, T3, and TSH were determined. In addition, sera were incubated with FRTL-5 cells or porcine thyroid cells in primary culture in the presence of 0.4 mM MIBX, and medium cAMP concentrations were determined by radioimmunoassay. Sera obtained from some patients with various non-thyroidal illness increased cAMP concentrations in culture media of FRTL-5 cells as well as that of porcine thyroid cells. The thyroid stimulating effects of sera were not disease specific and significantly correlated inversely with serum T3 and T4 concentrations. Serum TSH concentrations in these patients were within the normal range even by the newly developed ultrasensitive assay. Although the nature of substance(s) present in sera of patients with low T3 syndrome which stimulates thyroid adenylate cyclase is not entirely known, it is conceivable that there exist mechanisms independent of TSH to compensate the decreased serum T3 levels in low T3 syndrome.     

Thyroglobulin gene expression as a differentiation marker in primary cultures of calf thyroid cells

A system of calf thyroid follicular cells in primary cultures has been developed to investigate the control of thyroglobulin gene expression in normal cells in vitro. In low (0.1%) serum conditions, the cells remained quiescent and formed dense aggregates surrounded by slowly spreading cells. High expression of thyroid-specific differentiation markers such as thyroglobulin (Tg) mRNA accumulation and iodide transport required the continuous exposure of cells to thyrotropin (TSH) or other adenylate cyclase activators (cholera toxin and forskolin). In the absence of TSH, Tg mRNA decreased to low but still detectable levels. Addition of TSH, forskolin or cholera toxin restored high Tg gene expression. Hydrocortisone moderately stimulated basal Tg mRNA accumulation and strongly potentiated the effect of TSH. Growth promoters including serum (1-10%), epidermal growth factor (EGF), fibroblast growth factor (FGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) induced calf thyroid cells to develop as a monolayer and inhibited both basal and TSH-stimulated expression of specialized functions. Moreover, only a partial restoration of this expression was achieved after addition of TSH or forskolin to well spread-out cells that had proliferated in response to EGF or serum. The results show that in calf thyroid cells, iodide transport and Tg gene expression are regulated by TSH through cyclic AMP; hydrocortisone potentiates this effect on Tg gene expression, while all growth promoting factors inhibit the expression of these differentiated functions.     

The biological activity of bovine and human thyrotropin is differently affected by trypsin treatment of human thyroid cells: thyroid-stimulating antibody is related to human thyrotropin.

Pretreatment of cultured human thyroid cells with trypsin decreased the cAMP response to bovine TSH (bTSH) (by 50-60%). In striking contrast, in trypsin treated cells the cAMP stimulation by both human TSH (hTSH) and thyroid-stimulating antibodies (TSab) was unimpaired, indicating a similar behavior for these two stimulators. The effect of trypsin on inhibiting cAMP stimulation by bTSH was: 1) dose dependent; 2) present at a trypsin concentration as low as 3.3 mg/L; 3) fully reversible within 24 h after removal of the enzyme. In accordance with the altered biological activity in human thyroid cells exposed to trypsin the binding of labeled bTSH was reduced (about 40%). On the contrary, in the same cells, the binding of labeled human TSH was enhanced (about 3-fold). The cAMP response to cholera toxin and forskolin was unaffected in trypsin treated cells, indicating that the tryptic treatment did not alter any other component of the adenylate cyclase complex. The medium obtained from trypsin-treated human thyroid cells was able to neutralize the biological activity of bTSH but not that of hTSH or TSab. Our study demonstrates that in human thyroid cells: 1) trypsin impaires bovine, but not human TSH or TSab biological activity; 2) bovine and human TSH may bind to different components of the TSH receptor.     

Antilipolytic effects of insulin and adenylate cyclase inhibitors on isolated human fat cells

Antilipolysis induced by insulin by adenylate cyclase inhibitors was compared in isolated human fat cells when lipolysis was activated at well-defined steps in the cyclic AMP system. The latter was achieved with isoprenaline (beta-adrenoreceptor agonist), cholera toxin and pertussis toxin (acting on the GTP-sensitive coupling proteins), forskolin (stimulating the catalytic component of adenylate cyclase), enprofylline (selective phosphodiesterase inhibitor) and N6-monobutyryl-cyclic-AMP or 8-bromo cyclic-AMP (cyclic AMP analogues which are resistant or sensitive to phosphodiesterase, respectively). Clonidine (alpha 2-adrenoreceptor agonist), prostaglandin E2 and N6-(phenylisopropyl) adenosine (adenosine analogue) failed to inhibit lipolysis stimulated by cholera toxin or pertussis toxin, but were effective under all other conditions. Insulin failed to inhibit lipolysis stimulated by enprofylline or N6-monobutyryl cyclic AMP, but was effective under all other circumstances. In conclusion, insulin and adenylate cyclase inhibitors are antilipolytic in human fat cells through different mechanisms. Adenylate cyclase inhibitors act predominantly on the GTP-sensitive coupling proteins and, to a minor extent, at some yet unidentified distal step in the lipolytic machinery. As regards insulin, the major site of the antilipolytic action is phosphodiesterase.     

Effects of proteolytic enzymes and protease inhibitors on bovine thyroid adenylate cyclase activity

Trypsin, chymotrypsin, and papain stimulate basal adenylate cyclase activity in bovine thyroid plasma membranes in a dose-related, albeit biphasic, fashion. Each of the proteases enhanced TSH-stimulated adenylate cyclase activity over basal activity. The proteases also enhanced GTP-, guanosine 5'-(beta, gamma-imidotriphosphate)-, prostaglandin E1-, and cholera toxin-stimulated adenylate cyclase to varying degrees. Fluoride-stimulated activity was enhanced by chymotrypsin and papain, but not by trypsin. When Mn++ was substituted for Mg++ in the adenylate cyclase assay, no stimulation by the proteases were observed. To see if endogenous membrane proteases are required for optimal thyroid adenylate cyclase response to TSH and other stimulators, studies were performed using the protease inhibitors tosylamide 2-phenylethyl-chloromethyl ketone (TPCK) and p-tosyl-L-arginine methyl ester (TAME), inhibitors of chymotrypsin and trypsin, respectively. TPCK (0.15 mM) had no effect on basal adenylate cyclase activity, but did inhibit TSH-, trypsin-, and chymotrypsin-stimulated activities by approximately 90%. Guanosine 5'-(beta, gamma-imido) triphosphate- as well as cholera toxin-stimulated activities were inhibited by approximately 50%, whereas prostaglandin E1- and fluoride-stimulated activities were inhibited by approximately 25%. TAME (6 mM) produced similar results, except that no effect on fluoride activity was seen, while basal activity was inhibited by approximately 20%. Thus, various serine proteases augment both basal and hormone-stimulated adenylate cyclase in bovine thyroid. Since both trypsin- and chymotrypsin-stimulated as well as TSH-induced enzyme activities were inhibited by TPCK and TAME, it would appear that augmentation of thyroid adenylate cyclase activity may, in part, result from stimulation of endogenous proteases.