The effect of ovine trophoblast protein-one on endometrial protein secretion and cyclic nucleotides

The effect of ovine trophoblast protein-one (oTP-1) on endometrial protein secretion was examined by using a dual radioisotope technique in which 3H- and 35S-methionine were employed to measure relative rates of protein release into the medium by endometrial explant cultures (Exp. I). Endometrium (200 mg) from Day (D) 12 of the cycle was cultured with either 5 micrograms/ml oTP-1, 5 micrograms/ml bovine serum albumin (BSA) or 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (DbcAMP). Culture media from control BSA and treated explant cultures were mixed. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and detected by fluorography. Individual protein spots were punched from gels, extracted, and their radioactive content measured. Ratios of 3H:35S were used to determine treatment effects. In Experiment II, 3H- and 14C-leucine were used for the dual radiolabel, and the DbcAMP treatment was omitted. In both experiments, a protein having a molecular weight (Mr) of about 70,000 and a pI approximately equal to 4 was increased (p less than 0.01) 200-400% by oTP-1. Secretion of several other endometrial proteins was also amplified in the presence of oTP-1. The polypeptides that increased in response to oTP-1 were inhibited by DbcAMP, and vica versa. In Experiment III, endometrial explants from D12 cyclic ewes were cultured for 4 h with either 5 micrograms/ml oTP-1 or 5 micrograms/ml BSA to determine whether oTP-1 influenced concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Concentrations of cAMP in oTP-1-treated endometrium were lower (p less than 0.1) than in BSA-treated endometrium (0.29 vs. 0.41 pmoles/mg tissue, respectively). Levels of cGMP were unaffected by oTP-1. In Experiment IV, endometrium from D14 of the cycle was incubated in medium alone or in medium containing either 2 micrograms/ml oTP-1, 1 microgram/ml oxytocin (OXY), or oTP-1-plus-OXY. None of the treatments significantly affected cAMP levels. In Experiment V, D16 endometrium was collected from pregnant and nonpregnant ewes that had received either 0 or 10 IU OXY i.v. cAMP was higher (p less than 0.01) in endometrium from pregnant ewes compared to nonpregnant ewes (27.9 vs. 13.0 pmoles/mg tissue, respectively), but OXY had no detectable effect on endometrial content of cAMP in either nonpregnant or pregnant ewes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium secretion in canine tracheal mucosa

Calcium (Ca) affects many cellular functions of the respiratory tract mucosa and might alter the viscoelastic properties of mucus. To evaluate Ca homeostasis in a respiratory epithelium we investigated transport of Ca by the canine tracheal mucosa. Mucosal tissues were mounted in Ussing-type chambers and bathed with Krebs-Henseleit solution at 37 degrees C. Unidirectional fluxes of 45Ca were determined in tissues that were matched by conductance and short-circuit current (SCC). Under short-circuit conditions there was a significant net Ca secretion of 1.82 +/- 0.36 neq . cm-2 . h-1 (mean +/- SE). Under open-circuit conditions, where the spontaneous transepithelial potential difference could attract Ca toward the lumen, net Ca secretion increased significantly to 4.40 +/- 1.14 compared with 1.54 +/- 1.17 neq . cm-2 . h-1 when the preparation was short-circuited. Addition of a metabolic inhibitor, 2,4-dinitrophenol (2 mM in the mucosal bath), decreased tissue conductance and SCC and slightly decreased the unidirectional movement of Ca from submucosa to lumen. Submucosal epinephrine (10 microM) significantly enhanced Ca secretion by 2.0 +/- 0.63 neq . cm-2 . h-1. Submucosal ouabain (0.1 mM) failed to inhibit Ca secretion. The data suggest that canine tracheal mucosa secretes Ca; this secretory process is augmented by epinephrine or by the presence of a transepithelial potential difference as found under in vivo conditions.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

The effect of oxytocin on cortisol and corticosterone secretion in cyclic gilts--in vivo and in vitro studies

Oxytocin (OT) may be implicated in the modulation of hypothalamo-pituitary-adrenal axis (HPA) at each level. In mature females the influence of OT on the HPA axis appeared to be dependent on ovarian steroid milieu and stress. In cyclic sows, the role of OT in the regulation of corticoid secretion is unknown. In the present study changes in plasma cortisol and corticosterone concentrations in response to exogenous OT (in vivo experiment) and a direct influence of OT on adrenocortical steroidogenesis (in vitro experiment) were determined in luteal- and follicular-phase gilts. In the luteal-phase gilts (n=5), OT injections increased both cortisol (p<0.01) and corticosterone (p<0.05) plasma concentrations, but in the follicular-phase gilts (n=5) only the concentration of cortisol (p<0.05) was elevated in response to the treatment. Areas under the cortisol and corticosterone curves calculated for 30 min period after the OT injection were statistically higher (p<0.05) during the luteal than the follicular phase. In the in vitro experiment, two doses of OT (10(-7) and 10(-6) M) increased (p<0.05) secretion of cortisol by porcine adrenocortical cells representing the luteal phase, but not the follicular phase. However, OT did not affect the release of corticosterone by the cells. Incubation of the cells with the OT-antagonist (10(-5) M) abolished the effects of OT on cortisol secretion. Thus, in the present study, stimulatory effects of OT on the hypothalamo-pituitary-adrenal axis were demonstrated in cyclic gilts. The changes in plasma corticoid concentrations in response to exogenous OT were more prominent during the luteal than the follicular phase of the estrous cycle. Moreover, the in vitro experiment revealed a possibility of direct action of OT on adrenocortical cells isolated from luteal phase gilts. These results suggest that OT may participate in the modulation of HPA axis activity in pigs.     

The effect of secretin on pentagastrin-stimulated secretion of gastric pepsin and acid in rats.

Rats with chronic gastric fistulas were stimulated for 12 or 24 h with constant intravenous infusion of pentagastrin. When secretin was also infused for the last half period of the experiment, respectively, 6 or 12 h, the volume of gastric secretion and HCl output were significantly reduced but the concentration of pepsin was significantly increased. The dissociated effect of secretin on gastric acid and pepsin secretion reported previously in man, dog and cat was also found in the rat.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

The effect of ethanol on prolactin secretion in vitro

In order to investigate the action of ethanol on the anterior pituitary gland, the effect of ethanol on prolactin secretion in vitro was studied. Ethanol significantly increased the in vitro incorporation of ³H-leucine into both prolactin contained within the pituitary gland and that released into the medium. The enhancement of ³H labelled-prolactin synthesis induced by ethanol was suppressed by cycloheximide. These results support the hypothesis that ethanol stimulates the in vitro synthesis and release of prolactin by the pituitary gland.     

Peptide YY interacts with secretin and duodenal acidification to inhibit gastric acid secretion

Previous studies have indicated that plasma levels of peptide YY (PYY) increase significantly after a meal. The purpose of this study was to characterize the interaction of PYY and secretin in the inhibition of gastric acid secretion, and to determine whether PYY can influence acid-induced inhibition of gastric acid secretion in conscious dogs. I.v. administration of PYY at 200 pmol/kg/h inhibited pentagastrin (1 microgram/kg/h)-stimulated gastric acid output (P less than 0.05). PYY further augmented i.v. secretin-induced inhibition of pentagastrin-stimulated gastric acid output by 32 +/- 7%, and intraduodenal hydrochloric acid-induced inhibition of pentagastrin-stimulated gastric acid output by 40 +/- 12%. The mean integrated release of secretin response to duodenal acidification (3.9 +/- 1.0 ng-[0-60] min/ml) was not affected by PYY (3.3 +/- 0.9 ng-[0-60] min/ml). The present study demonstrates that PYY can interact with secretin and duodenal acidification in an additive fashion to inhibit pentagastrin-stimulated gastric acid secretion. Our results suggest that several hormones that are released postprandially can interact with each other to inhibit gastric acid secretion.     

The effect of secretin and cholecystokinin-pancreozymin on the secretion of bile in the anaesthetized rabbit.

Effects of secretin and Cholecystokinin-Pancreozymin (CCK-PZ) on the secretion of bile in anaesthetized rabbits have been studied. Single injections of secretin (5.0 u.kg(-1) significantly increased the flow of bile irrespectively of whether the cystic duct was free or had been tied. A sustained increase in bile flow could be obtained by the continuous infusion of secretin. Cholecystokinin-Pancreozymin was effective in increasing the bile flow in doses of 1.0 u.kg(-1). Much of the effect could be attributed to contraction of the gallbladder but a significant increase in flow could still be elicited after ligation of the cystic duct. Our findings strongly suggest that the biliary secretion in rabbits is not as different from the general pattern as has previously been suggested.     

Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species.     

The effect of neurotensin and secretin on gastric acid secretion and mucosal blood flow in man

Neurotensin stimulates pancreatic secretion directly and by potentiating the effect of secretin. Neurotensin also inhibits gastric secretion. Secretin inhibits gastric secretion as well, but whether it also interacts with neurotensin is not known. Secretin is known to inhibit gastric mucosal blood flow (GMBF). The effect of neurotensin on GMBF is not known. Acid secretion (triple lumen perfused orogastric tube) and GMBF ([14C]aminopyrine clearance) were therefore measured in 6 subjects during neurotensin, secretin and neurotensin plus secretin infusions. Neurotensin plus secretin reduced acid secretion by a median 130 (range 34-394) mumol/min which was significantly greater than either neurotensin at 36 (7-67) mumol/min or secretin 54 (20-347) mumol/min alone (P less than 0.05). This effect appeared independent of GMBF. Neurotensin plus secretin reduced GMBF by 14 (12-27) ml/min but not significantly more than neurotensin at 11 (3-20) ml/min or secretin 18 (2-27) ml/min alone. Further, there was no correlation between changes in acid output and GMBF during infusion of the peptides. We conclude that the inhibitory effects of neurotensin and secretin on gastric secretion are at least additive and together they may function as an 'enterogastrone'.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid

The effect of luminal application of arachidonic acid on the alkaline secretion, prostaglandin generation, and mucus glycoprotein output and composition was studied in proximal and distal duodenum of conscious dogs. Surgically prepared duodenal loops were instilled in vivo for up to 2 h with saline (control) followed by various concentrations (12.5–100 μg/ml) of arachidonic acid. The experiments were conducted with and without intravenous pretreatment with indomethacin. The recovered instillates were assayed for the content of prostaglandin and HCO₃⁻, and used for the isolation of mucus glycoprotein. Exposure of duodenal mucosa to arachidonic acid led to concentration-dependent increase in the output of HCO₃⁻ and prostaglandin generation. In both cases this response was greater in the proximal duodenum. Pretreatment with indomethacin caused reduction in the basal HCO₃⁻ and prostaglandin output, and prevented the increments evoked by arachidonic acid. The proximal and distal duodenum displayed similar basal output and composition of mucus glycoprotein. Comparable increases in these glycoproteins were also obtained with arachidonic acid, the effect of which was abolished by indomethacin. Compared to basal conditions, mucus glycoproteins elaborated in response to arachidonic acid exhibited higher contents of associated lipids and covalently bound fatty acids, and contained less protein. The associated lipids of mucus glycoproteins elaborated in the presence of arachidonic acid showed enrichment in phospholipids and decrease in neutral lipids. The carbohydrate components in these glycoproteins also exhibited higher proportions of sialic acid and sulfate. The changes brought about by arachidonic acid were prevented by indomethacin pretreatment, and in both cases the glycoprotein composition returned to that obtained under basal conditions. The enrichment of mucus glycoprotein in lipids, sialic acid and sulfate in response to endogenous prostaglandin may be of significance to the function of this glycoprotein in the hostile environment of the duodenum.     

Bile secretion in the chicken: effects of secretin, cholecystokinin- pancreozymin and duodenal mucosal extract

The effects of i.v. administration of secretin, CCK-PZ, acid extracts from the duodenal mucosa and the duodenal acidification of the intestine on bile secretion were studied in anaesthetized chickens. Secretin and acid extracts from the duodenal mucosa, which increase bile flow, caused comparable modifications in bile composition; infusion of HCl to the duodenum only induced slight modifications. CCK-PZ caused a pronounced cholecystokinetic effect and, to a lesser degree, it also showed choleretic effects. The results suggest that in the hormonal regulation of bile secretion in the chicken CCK-PZ is more important than secretin and furthermore that the choleretic activity of the latter must be carried out by other secretin-like peptides.