A comparison of thyrotropin- and dibutyryl cyclic AMP- induced thyroglobulin iodination in cultured human thyroid cells

The relative degree of 125-I labelling of thyroglobulin-- bound mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in isolated, cultured human thyroid cells has been compared following exposure of 125-I supplemented cells to 100 mU/ml of bovine thyrotropin (TSH) or 1.0 mM dibutyryl cyclic AMP (dBcAMP) for 96 hours. Pronase digestion of the lysed cells and Sephadex G-10 fractionation of the digested lysates revealed a predominance of [125-I]MIT over [125-I]DIT in both sets of experimental cells as well as in controls. Levels of [125-I]DIT, however, were only enhanced above control values in cells incubated with TSH. These findings suggest that an increase in availability of intracellular iodide, following cellular exposure to TSH, may facilitate a preferential synthesis of DIT relative to that of MIT. This theory offers an explanation for the differential effects of TSH and dibutyryl cyclic AMP on the levels of newly--synthesised T4 recovered from the cells used in this study, and from the culture medium in a previous investigation.     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Dibutyryl cyclic AMP affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyaluronic acid to form large aggregates. These results suggest that the increase in hyaluronate synthesis caused by dibutyryl cyclic AMP mayt have destabilized the interaction of proteoglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those which are observed in cartilage of osteoarthritic joints.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

A calcium ionophore (A-23187, 20 mug/ml) stimulted 14C-1-glucose oxidation in dog thyroid slices to an extent equivalent to that obtained by the optimal concentration of dibutyryl cyclic AMP (1mM). Furthermore, the ionophore augmented the stimulation by dibutyryl cyclic AMP much more than the simple additive effect. The ionophore also enhanced the effect of TSH, but to a lesser extent. Under conditions where organic binding was blocked, T/M ratio of radioiodine concentration was lowered in slices by the ionophore; the findings similar to those obtained with TSH and dibutyryl cyclic AMP. The ionophore exhibited a slightly depressive effect on the basal cyclic AMP level. The elevation by TSH of cyclic AMP levels was also slightly depressed by the ionophore, but statistically insignificant in most cases. These results indicate that calcium ion may play an important role in the TSH regulation of iodide transport and glucose metabolism in the thyroid, in some cases by augmenting the effects of cyclic AMP.     

Chronic and acute effects of thyrotropin on phosphatidylinositol turnover in cultured porcine thyroid cells

The 32P incorporation into phospholipids of isolated porcine thyroid cells, cultured for 1-4 days, has been studied in subsequent 2-h incubations. Along with culture ageing, decreased 32P incorporation into total phospholipid of control cells was observed. The presence of 40 munits/ml TSH during the 2 h incubation yielded a relative increase in labelling of phosphatidylinositol, named 'acute phospholipid effect'. A chronic treatment of the cells with TSH concentration ranging from 0.1 to 10 munits/ml ensured the maintenance of a high turnover rate of total phospholipids. The analysis of individual phospholipids revealed that 1-day culture cells in the presence of 0.1 munits/ml TSH presented a strong increase of phosphatidylinositol labelling. This 'chronic phospholipid effect' of TSH can be reproduced by a chronic treatment with dibutyryl cyclic AMP (10(-3)M) or prostaglandin E2 (10(-6)M), which did not evoke a classical phospholipid effect in a 2 h incubation. If TSH (40 munits/ml) is added to the cells in a 2 h incubation, control cells show the classical phospholipid effect whereas cells chronically treated with TSH, dibutyryl cyclic AMP or prostaglandin E2 presented a 'reverse phospholipid effect' i.e. a relative decrease in phosphatidylinositol labelling. 10(-4)M cycloheximide presence during the last 12 h of culture prevented the establishment of the 'chronic phospholipid effect' and of its consequence, 'the reverse phospholipid effect'. On the basis of these results a scheme is proposed in keeping with current hypotheses concerning phosphatidylinositol metabolism.     

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone.

While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10^?6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.     

Biphasic inhibitory and stimulatory effects of thyrotropin on the mitotic activity of thyroid explants cultured in vitro.

An influence of thyrotropin (TSH), dibutyryl cyclic AMP (dbcAMP), triiodothyronine (T3) and thyroxine (T4) on the mitotic incidence in the organ-cultured rat thyroid was studied. The study has revealed biphasic inhibitory and stimulatory effects of TSH on the thyroid mitotic activity. The inhibitory effect occurred 14 h after an exposure of the thyroid explants to TSH and was enhanced by T3. A similar inhibitory effect was observed after an exposure of the explants to dbcAMP. 24 h after the exposure of the thyroid explants to TSH a stimulatory effect on the mitotic incidence was observed. The latter effect was not reproduced by dbcAMP.     

Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

Plasminogen activator production by parietal endoderm cells: Stimulation by a protein from pituitary

It has been shown previously that dibutyryl cyclic AMP increases the production of plasminogen activator in mouse parietal endoderm cells. This fact suggested that the production of plasminogen activator by parietal endoderm cells may be under the control of a hormone acting via adenylate cyclase. We have cultured rat parietal endoderm cells in the absence of serum and show that they respond to dibutyryl cyclic AMP with an increase in plasminogen activator production and a change in morphology. We describe the existence of a compound from pituitary which is capable of stimulating plasminogen activator secretion in these cells. Relatively impure preparations of ovine and bovine TSH contain significant amounts of activity, whereas more highly purified preparations of TSH, and all other pituitary hormones tested, are inactive, indicating that the factor is not a known pituitary hormone. The active compound was characterized using ovine and bovine TSH as a source, and it is macromolecular and proteinaceous, and depends on protein synthesis for its effect. The stimulation is enhanced by methylisobutylxanthine, a phosphodiesterase inhibitor, suggesting that the event is mediated by cyclic AMP. This observation leads to the prediction that the coaddition of dibutyryl cAMP and the active compound at nonsaturating concentrations should be additive. Instead, the stimulation is synergistic, and depends on the addition of dibutyryl cyclic AMP first when the compounds are added sequentially. Finally, we show that mouse teratocarcinoma cells chemically induced to differentiate to a cell type indistinguishable from parietal endoderm respond to a source of the compound by increasing plasminogen activator production.     

Cyclic AMP release from perfused dog thyroid lobes as a sensitive indicator of TSH activation of the secretion process. Evidence for two types of thyroid secretion latency

It has been demonstrated in various types of thyroid tissue preparations that cyclic AMP (cAMP) released into the medium reflects the amount of cAMP in the cells. In the present study employing perfused dog thyroid lobes the dynamics of cAMP release were compared to those of thyroxine (T4) and triiodothyronine (T3) release. The experiments gave evidence that even the lowest concentrations of TSH which stimulate hormone release (in this study 1 microU/ml) also activate the cAMP system; the very high levels of cAMP obtained by stimulation with high concentrations of TSH (in this study 10,000 microU/ml) are not accompanied by corresponding high increases in hormone release. On the contrary the T4 and T3 release is lower than during stimulation with more moderate concentrations of TSH (100 microU/ml). Hence studies employing high concentrations of TSH and measurements of cAMP as indicator of activity of secretory processes should be interpreted very cautiously; the prolonged lag in thyroid hormone secretion observed after stimulation with low concentrations of TSH is accompanied by a corresponding lag in activation of the cAMP system. This pattern suggest that the duration of late secretory processes such as thyroglobulin pinocytosis and hydrolysis is independent of the degree of stimulation and not involved in the variations in secretion latency.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Induction of alkaline phosphatase in choriocarcinoma cells by 1-beta-D-arabinofuranosyl-cytosine, mitomycin C, phleomycin, and cyclic nucleotides.

Alkaline phosphatase is induced in cultured human choriocarcinoma cells by three inhibitors of DNA synthesis which alter DNA structure: 1-β-D-arabinofuranosyl-cytosine, mitomycin C, and phleomycin. No induction is observed with the inhibitors, hydroxyurea and thymidine, which do not alter DNA structure. Cyclic AMP, analogs of cyclic nucleotides, and sodium butyrate also induce alkaline phosphatase in these cells. Among the cyclic nucleotides tested, dibutyryl cyclic AMP is the best inducer, whereas dibutyryl cyclic GMP is a poor inducer. Induction of alkaline phosphatase by inhibitors of DNA synthesis or by exposure to dibutyryl cyclic AMP appears to utilize different mechanisms. Maximum induction is observed after simultaneous addition of both types of inducers at the concentrations found to be optimal for each inducer alone. Under these conditions, the induced activity is equal to or greater than the sum of the activities induced by each inducer. RNA synthesis and protein synthesis are required for induction. Dibutyryl cyclic AMP added to cultures of choriocarcinoma cells is not degraded in the culture medium, but is extensively degraded in the cells. Nevertheless, significant amounts of dibutyryl and monobutyryl cyclic AMP are found intracellularly throughout the experiment. Since the cellular uptake of dibutyryl cyclic AMP is extremely slow, the amount of butyrate released by intracellular degradation cannot account for the observed induction. Neither the rate of uptake nor the stability of dibutyryl cyclic AMP are changed by the addition of 1-β-D-arabinofuranosyl-cytosine to the culture medium. Furthermore, 1-β-D-arabinofuranosyl-cytosine inhibits the induction by sodium butyrate. The results indicate that butyrate is not the major mediator of induction by dibutyryl cyclic AMP.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Stimulatory effect of dibutyryl cyclic GMP on acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells.

We previously reported that endogenous nitric oxide (NO) is involved in the peripheral control of gastric acid secretion induced by some secretagogues, and that endogenous NO is involved in the acid secretion process via histamine release from histamine-containing cells. However, the stimulus-secretion coupling in the cells remains to be clarified. In the present study, we investigated the effect of dibutyryl cyclic GMP on gastric acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells, in comparison with those of dibutyryl cyclic AMP. Dibutyryl cyclic GMP (300 microM) produced a slight but significant increase of gastric acid secretion, which was completely inhibited by the histamine-H2 receptor antagonist famotidine. In contrast, dibutyryl cyclic GMP (1 mM) markedly inhibited histamine-induced acid secretion. Dibutyryl cyclic AMP (100 microM) produced a sustained increase of gastric acid secretion. The pretreatment with famotidine partially inhibited dibutyryl cyclic AMP-induced gastric acid secretion. Dibutyryl cyclic GMP and dibutyryl cyclic AMP significantly increased the histamine release from gastric mucosal cells. These results suggest that both intracellular cyclic GMP and cyclic AMP act as second messengers for histamine release in the histamine-containing cells, probably ECL cells. On the other hand, in gastric parietal cells, cyclic AMP has a stimulatory effect on gastric acid secretion, whereas cyclic GMP has an inhibitory effect.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.