Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T₃) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T₃ (1·10^?6) and cyclic AMP (2·10^?4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[¹⁴C]isobutyric acid as well as net alanine uptake and urea production were elevated by T₃ and by cyclic AMP. T₃ increased the oligomycin-sensitive O₂ consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T₃ on hepatic gluconeogenesis was independent of exogenous Ca²⁺ concentration. T₃ by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese (v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phosphoenolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phosphoenolpyruvate. Cyclic AMP action was not affected by the further addition of T₃. Glucagon (1·10^?8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T₃ stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.     

Effects of eicosatetraynoic acid (ETYA) on cultured pig thyroid cells. Relationships between the inhibition of the phosphatidate-phosphatidyl inositol cycle, the iodination and the cyclic AMP responsiveness to thyrotropin

The arachidonate inhibition of the adenylate-cyclase system of cultured pig thyroid cells was not mediated by cyclooxygenase, lipoxygenase or peroxidase metabolites. Indeed ETYA, an inhibitor of cyclooxygenase and lipoxygenase, and methimazole, an inhibitor of peroxidase and iodination were without effect on the arachidonate inhibition. Moreover the effect of arachidonate was amplified by a combination with ETYA. In 32P incorporation experiments we observed a modification of the labelling of individual phospholipids of cultured pig thyroid cells resulting in a decrease into phosphatidylinositol (PI) and an increase into phosphatidate (PA) of arachidonate and ETYA-treated cells. These results may be explained by an inhibition of CDP-diacylglycerol: inositol transferase and conversely a stimulation of PI specific phospholipase C yielding a decrease in PI and an increase in PA, which inhibits in turn adenylate cyclase activity possibly by Ca2+ translocation.     

Production of cyclic AMP from extracellular ATP by intact LM cells

Intact LM cells, a line of cultured mouse fibroblasts, exhibited and adenylate cyclase (APT pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence exogenous [α-³²]ATP which was 20–30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E₁ stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an endenylate cyclase activity whic is accessible to extracellular ATP.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2–3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low K_m (0.17 μM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study. Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H₂ utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H₂ uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H₂ uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H₂ metabolism.     

Identification of an intracellular pathway of thyroxine synthesis by dispersed thyroid cells

This study deals with the identification of the biochemical events involved in the metabolic sequence leading from the synthesis to the release of thyroxine in the dispersed thyroid cell system. (1) Using an experimental model allowing the differentiation between intracellular and extracellular sites of iodination, it is shown that thyroxine is synthesized inside the cells by an iodinating system sensitive to thyrotropin stimulation. (2) The secretion of thyroxine synthesized inside the cells is not mediated by an exocytotic-endocytotic phenomenon. Colchicine, vinblastine, fluoride, propanolol and chlorpromazine, at concentrations equal to or 10–100-times higher than those required to inhibit hormone release in follicular-organized thyroid tissue have no effect on thyrotropin-stimulated thyroxine secretion. (3) The secretion involves the intracellular proteolysis of hormone-containing iodoprotein(s) which, in addition to free thyroxine, generates free mono- and diiodotyrosines. Free thyroxine is released into the incubation medium and iodotyrosines are deiodinated under normal conditions and accumulate in the presence of an inhibitor of iodotyrosine deiodinase: 3,5-dinitrotyrosine. This proteolysis is inhibited by 5 mM chlorpromazine. These data indicate that the complete metabolic sequence leading from the uptake of iodide to the release of free thyroxine into the incubation medium can be described as an ‘intracellular metabolic sequence for thyroxine synthesis’.     

Infinite cis influx of cyclic AMP into human erythrocyte ghosts

Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ at 30°C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments.Lowering the temperature reduces $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ from 0.69 mM at 30°C to 0.478 mM at 20°C, 0.108 mM at 10°C and 0.072 mM at 4°C ($\text{Q}{}_{10}\text{= 2.4}$). The $\text{Q}{}_{10}$ for activation of influx permeability of 10^?5 M cyclic AMP is 1.55.     

Hyperoxic-induced alterations in lung cell structure and function: Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 ± 115 fmol/μg DNA in cells exposed to p_O2 677 Torr for 96 h compared to 136 ± 17 fmol/μg DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.     

Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung cancer cells and elevates cyclic AMP levels

Vasoactive intestinal polypeptide (VIP) receptors were characterized on non-small cell lung cancer (NSCLC) cells. ¹²⁵I-VIP bound specifically to membranes derived from 6 NSCLC cell lines. Specific ¹²⁵I-VIP was time dependent and a linear function of EPLC-65H membrane concentration. ¹²⁵I-VIP bound with high (K_d=0.2 nM) and moderate (K_d=39 nM) affinity to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP > rGHRH > PHI = helodermin > secretin > glucagon. Also VIP elevated the cAMP levels 10-fold using cell line ADLC-5M2. These data indicate that functional VIP receptors are present on NSCLC cells.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte: Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K⁺ concentration on both [³H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between β-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [³H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K⁺ concentrations, and at each K⁺ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K⁺ concentrations. In contrast to basal potassium influx, which is 50–70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K⁺, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to β-adrenergic receptor occupancy over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy β-adrenergic receptor sites function independently of each other.Analysis of the relation of catecholamine-dependent potassium transport to the number of β-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

Effect of chronic treatment of ohmefentanyl stereoisomers on cyclic AMP formation in Sf9 insect cells expressing human mu-opioid receptors

The binding affinity of ohmefentanyl stereoisomers for mu-opioid receptors and the effect of chronic ohmefentanyl stereoisomers pretreatments on intracellular cAMP formation were investigated in Sf9 insect cells expressing human mu-opioid receptors (Sf9-mu cells). Competitive assay of [3H]ohmefentanyl binding revealed that these isomers had high affinity for micro-opioid receptors in Sf9-mu cells. Isomer F9204 had the highest affinity for mu-opioid receptors with the Ki value of 1.66 +/- 0.28 nM. After pretreated Sf9-mu cells with increasing concentrations of these isomers for 6 h, addition of naloxone (1 microM) precipitated an overshoot of foskolin-stimulated cAMP accumulation. The ability of these isomers to induce cAMP overshoot differed greatly with the order of F9202>F9205>F9208>F9206>F9204>F9207. Of these isomers, F9202 was 2.7-fold less potent than F9204 in receptor binding affinity, but 71.5-fold more potent in ability to induce cAMP overshoot. These results suggested that there was a significant stereo-structural difference among ohmefentanyl stereoisomers in ability to induce naloxone-precipitated cAMP overshoot in Sf9-mu cells.     

Rapid, transient methylation of four proteins in aggregative amoebae of Dictyostelium discoideum as a response to stimulation with cyclic AMP

In Dictyostelium discoideum, extracellular cAMP induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells are shown to respond to a 10(-6) M cAMP-pulse with increased methylation of 4 proteins with app. Mr 110000, 46000, 28000 and 16000. The Mr 110000 and 28000 proteins show a triphasic response with maxima 15, 60 and 150-180 s after stimulation. The responses of the Mr 46000 and 16000 proteins are monophasic, maxima being reached 3 and 15 s after stimulation, respectively. Optimal responses of methylation are observed over 10(-7) - 10(-6) M cAMP. The methylation reaction may be involved in the processing of the chemotactic signal.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells

The effects of sulfonylureas and a biguanide on membrane-bound low K_m cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 μM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 μM). Tolbutamide (0.5–10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. K_m, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 μM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells

We uncovered a new regulation of thyrocyte function by bone morphogenetic protein (BMP) under the influence of thyrotropin (TSH) using primary culture of porcine thyrocytes. The BMP type I receptors, ALK-2 (ActRIA), -3 (BMPRIA), and -6 (BMPRIB), were expressed in porcine thyrocytes, while ALK-6 was not detected in human thyroid. Treatment with BMP-2, -4, -6, -7, and TGF-beta1 exhibited a dose-dependent suppression of DNA synthesis by porcine thyrocytes. BMP-2, -4, -6, -7, and TGF-beta1 suppressed TSH receptor mRNA expression on thyrocytes, which was consistent with their suppressive effect on TSH-induced cAMP synthesis and TSH-induced insulin-like growth factor-1 expression. Activin exhibited minimal suppression of thyrocyte DNA synthesis and did not exhibit suppressive effects on TSH receptor mRNA expression. Phosphorylated Smad1/5/8 was detected in the lysates of porcine thyrocytes treated with BMP-2, -4, -6, and -7. However, in the presence of TSH, BMP-6 and -7 failed to activate Smad1/5/8 phosphorylation and 3TP-reporter activity, whereas BMP-2 and -4 maintained clear activation of the BMP signaling regardless of the presence of TSH. This diverged regulation of thyroid BMP system by TSH is most likely due to the reduction of ALK-6 expression caused by TSH. Thus, the thyroid BMP system is functionally linked to TSH actions through modulating TSH receptor expression and TSH, in turn, selectively inhibits BMP signaling. Given that BMP system is present in human thyroid and the expression pattern of ALK-2 and BMPRII is different between follicular adenomas and normal thyroid tissues, the endogenous BMP system may be involved in regulating thyrocyte growth and TSH sensitivity of human thyroid adenomas.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.