Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO₃⁻ secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO₃⁻ secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO₃⁻ secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO₃⁻ secretion after intracisternal administration (1 nmol/rat). PGE₂ (300 μg/kg, iv) significantly increased HCO₃⁻ secretion in both the stomach and the duodenum. The potency of duodenal HCO₃⁻ secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE₂. The duodenal HCO₃⁻ secretory action of PACAP-27 as well as PGE₂ was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO₃⁻ secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO₃⁻ secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO₃⁻ secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO₃⁻ secretion.     

Involvement of prostaglandin E receptor EP3 subtype in duodenal bicarbonate secretion in rats

We investigated the involvement of prostaglandin E (PGE) receptor subtype EP3 in the regulatory mechanism of duodenal HCO₃⁻ secretion in rats. A proximal duodenal loop or a chambered stomach was perfused with saline, and HCO₃⁻ secretion was measured using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved through 10 min of exposure to 10 mM HCl in the duodenum or 100 mM HCl in the stomach. Various EP agonists or the EP4 antagonist were given i.v., while the EP1 or EP3 antagonist was given s.c. or i.d., respectively. Sulprostone (EP1/EP3 agonists) stimulated duodenal HCO₃⁻ secretion in a dose-dependent manner, and this response was inhibited by AE5-599 (EP3 antagonist) but not AE3-208 (EP4 antagonist). AE1-329 (EP4 agonist) also increased duodenal HCO₃⁻ secretion, and this action was inhibited by AE3-208 but not AE5-599. The response to PGE₂ or acidification in the duodenum was partially attenuated by AE5-599 or AE3-208 alone but completely abolished by the combined administration. Duodenal damage caused by mucosal perfusion with 150 mM HCl for 4 h was worsened by pretreatment with AE5-599 and AE3-208 as well as indomethacin and further aggravated by co-administration of these antagonists. Neither the EP3 nor EP4 antagonist had any effect on the gastric response induced by PGE₂ or acidification. These results clearly demonstrate the involvement of EP3 receptors, in addition to EP4 receptors, in the regulation of duodenal HCO₃⁻ secretion as well as the maintenance of the mucosal integrity of the duodenum against acid injury.     

Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.     

Prostaglandin E prevents indomethacin-induced gastric and intestinal damage through different EP receptor subtypes

Gastrointestinal ulcerogenic effect of indomethacin is causally related with an endogenous prostaglandin (PG) deficiency, yet the detailed mechanism remains unknown. We examined the effect of various PGE analogues specific to EP receptor subtypes on these lesions in rats and mice, and investigated which EP receptor subtype is involved in the protective action of PGE(2). Fasted or non-fasted animals were given indomethacin s.c. at 35 mg/kg for induction of gastric lesions or 10-30 mg/kg for intestinal lesions, and they were killed 4 or 24 h later, respectively. Various EP agonists were given i.v. 10 min before indomethacin. Indomethacin caused hemorrhagic lesions in both the stomach and intestine. Prior administration of 16,16-dimethyl PGE(2) (dmPGE(2)) prevented the development of damage in both tissues, and the effect in the stomach was mimicked by 17-phenyl PGE2 (EP1), while that in the small intestine was reproduced by ONO-NT-012 (EP3) and ONO-AE-329 (EP4). Butaprost (EP2) did not have any effect on either gastric or intestinal lesions induced by indomethacin. Similar to the findings in rats, indomethacin caused gastric and intestinal lesions in both wild-type and knockout mice lacking EP1 or EP3 receptors. However, the protective action of dmPGE(2) in the stomach was observed in wild-type and EP3 receptor knockout mice but not in mice lacking EP1 receptors, while that in the intestine was observed in EP1 knockout as well as wild-type mice but not in the animals lacking EP3 receptors. These results suggest that indomethacin produced damage in the stomach and intestine in a PGE(2)-sensitive manner, and exogenous PGE(2) prevents gastric and intestinal ulcerogenic response to indomethacin through different EP receptor subtypes; the protection in the stomach is mediated by EP1 receptors, while that in the intestine mediated by EP3/EP4 receptors.     

Phosphatidylinositol 3-kinase is involved in prostaglandin E2-mediated murine duodenal bicarbonate secretion

Prostaglandin E(2) (PGE(2)) plays an important role in the regulation of duodenal bicarbonate (HCO(3)(-)) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE(2)-mediated duodenal HCO(3)(-) secretion. Murine duodenal mucosal HCO(3)(-) secretion was examined in vitro in Ussing chambers by pH-stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol 3-kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. PGE(2)-stimulated duodenal HCO(3)(-) secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT-5720 by 23% and 20%, respectively; the Ca(2+)-influx inhibitor verapamil by 26%; and the calmodulin antagonist W-13 by 24%; whereas the PI3K inhibitors wortmannin and LY-294002 reduced PGE(2)-stimulated HCO(3)(-) secretion by 51% and 47%, respectively. Neither the MAPK inhibitor PD-98059 nor the tyrosine kinase inhibitor genistein altered PGE(2)-stimulated HCO(3)(-) secretion. PGE(2) application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and Akt phosphorylation. These results demonstrated that PGE(2) activates PI3K in duodenal mucosa and stimulates duodenal HCO(3)(-) secretion via cAMP-, Ca(2+)-, and PI3K-dependent signaling pathways.     

Involvement of dopamine receptor subtypes in dopaminergic modulation of aldosterone secretion in rats

The postulation that dopamine (DA) may tonically inhibit aldosterone (ALDO) secretion has arisen from the finding that metoclopramide, a non-selective DA receptor antagonist with prominent non-dopaminergic actions, stimulates ALDO secretion. Experiments were performed to determine: (a.) the ability of several non-specific and subtype-specific DA receptor antagonists to stimulate ALDO secretion, (b.) the subtype DA receptor involved in regulating ALDO secretion, and (c.) if ALDO responses were associated with changes in plasma Na+(pNa), K+(pK), or osmolality (pOsm). Blood samples were withdrawn from carotid arterial catheters in conscious, fasted male Sprague-Dawley rats before and following intra-arterial administration of lactated Ringer's placebo, furosemide (10 mg/kg), or one of several DA receptor antagonists. Furosemide stimulated ALDO, decreased pK, and left pNa and pOsm unchanged. The non-selective DA receptor antagonists metoclopramide (0.2, 0.6 mg/kg), rs-sulpiride (0.2 mg/kg), and haloperidol (0.1 mg/kg), and the DA-2 receptor antagonists domperidone (0.1 mg/kg) and s-sulpiride (0.1 mg/kg) each stimulated ALDO, and left pNa, pK, and pOsm unchanged. Conversely, the DA-1 receptor antagonists SCH 23390 (0.03, 0.1 mg/kg) and r-sulpiride (0.1 mg/kg) failed to stimulate ALDO, and left pNa, pK, and pOsm unaltered. These studies suggest that ALDO secretion in rats is modulated by a mechanism involving DA-2, but not DA-1 subtype receptors, and that the ALDO responses to DA receptor antagonism are independent of changes in pNa, pK, and pOsm.     

Expression and distribution of somatostatin receptor subtypes in the pancreatic islets of mice and rats.

Somatostatin acts on specific membrane receptors (sst(1-5)) to inhibit exocrine and endocrine functions. The aim was to investigate the distribution of sst(1-5) in pancreatic islet cells in normal mice and rats. Pancreatic samples from five adult C57BL/6 mice and Sprague-Dawley rats were stained with antibodies against sst(1-5) and insulin, glucagon, somatostatin, or pancreatic polypeptide (PP). A quantitative analysis of the co-localization was performed. All ssts were expressed in the pancreatic islets and co-localized on islet cells to various extents. A majority of the beta-cells expressed sst(1-2) and sst(5) in mouse islets, while < or =50% in the rat expressed sst(1-5). The expression of sst(1-5) on alpha-cells did not differ much among species, with sst(2) and sst(5) being highly expressed. About 70% of the delta-cells expressed sst(1-4) in the rat pancreas, whereas 50% of the islet cells expressed sst(1-5) in the mouse. Furthermore, 60% of the PP-cells expressed sst(1-5) in the mouse, while the rat islets had lower values. Co-expression with the four major islet hormones varies among species and sst subtypes. These similarities and differences are interesting and need further evaluation to elucidate their physiological role in islets.     

Prostaglandin E2 secretion by oviductal transport-stage equine embryos.

This study was conducted to identify embryonic products whose secretion was temporally associated with the oviductal transport period of the mare. Chemicals secreted by oviductal-transport-stage equine embryos were identified by incubating Day 6 or Day 7 early uterine embryos with 35S-methionine/cysteine, 3H-progesterone, or 3H-arachidonic acid for 24 h, and subsequently identifying radioactively labeled proteins (SDS-PAGE; n = 3 embryos), steroids (HPLC; n = 3 embryos), or prostaglandins (HPLC; n = 3 embryos) in the culture medium. Early uterine embryos secreted 116.1 +/- 45.5 pg of prostaglandin (PG) E2/embryo, 1.0 +/- 0.2 pg of 17 alpha-hydroxy progesterone/embryo, 4.8 +/- 0.6 pg of androstenedione/embryo, and 11.5 +/- 4.5 pg of PGF2 alpha/embryo. They did not secrete detectable quantities of protein, testosterone, or estradiol-17 beta. A second experiment was conducted to measure temporal changes in embryonic PGE2 secretion during the oviductal and early uterine period. Day 3, Day 4, Day 5, and Day 6 embryos (n = 8 embryos/day) were incubated with 3H-arachidonic acid for 24 h, and the concentration of 3H-PGE2 in the culture medium was subsequently measured by HPLC. Embryos did not secrete detectable amounts of PGE2 prior to the expected time of oviductal transport (Day 3 and Day 4). They secreted 5.7 +/- 1.0 pg of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly (p less than 0.01) higher amounts (42.0 +/- 11.5 pg) of PGE2/embryo immediately after uterine entry (Day 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic receptor subtypes involved in hippocampal circuits

Muscarinic receptors modulate hippocampal activity in two main ways: inhibition of synaptic activity and enhancement of excitability of hippocampal cells. Due to the lack of pharmacological tools, it has not been possible to identify the individual receptor subtypes that mediate the specific physiological actions that underlie these forms of modulation. Light and electron microscopic immunocytochemistry using subtype-specific antibodies was combined with lesioning techniques to examine the pre- and postsynaptic location of m1-m4 mAChR at identified hippocampus synapses. The results revealed striking differences among the subtypes, and suggested different ways that the receptors modulate excitatory and inhibitory transmission in distinct circuits. Complementary physiological studies using m1-toxin investigated the modulatory effects of this subtype on excitatory transmission in more detail. The implications of these data for understanding the functional roles of these subtypes are discussed.     

Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP > AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.     

Prostaglandin E and mitogenic stimulation of human lymphocytes in serum-free medium

Sera used in cell cultures contain significant amount of prostaglandins (PGs). In order to vaoid any effects of contaminating PGs, the present study employed a serum-free culture medium and confirmed the inhibitory effect of prostaglandin E (PGE) on the human lymphocyte activation which had been observed previously employing a serum-containing medium. PGE₁ displayed a significantly stronger inhibitory effect on the cells than previously shown. Furthermore, reported enhancement of PGE synthesis by mitogen-activated lymphocytes could not be reproduced.     

Prostaglandin stimulation of gastrointestinal transit in post-operative ileus rats

The prostaglandins PGF_2α, PGE₂ and 16,16-dimethyl PGE₂, when administered intravenously, orally, subcutaneously or intraduodenally to laparotomized rats, decreased gastric emptying, small intestinal transit and colonic transit as compared to unoperated controls. All three prostaglandins increased colonic transit above that found with unoperated controls. This activity was independent of small intestinal fluid accumulation (i.e., enteropooling) since ligating the ileal-cecal junction had no effect on colonic transit. Small intestinal transit was increased, but not normalized, by PGE₂ and 16,16-dimethyl PGE₂. 16,16-Dimethyl PGE₂ completely restored gastric emptying when given intravenously to laparotomized rats at doses greater than 5.0 μg/kg. This effect on gastric emptying lasted approximately 4 hrs. Thus, 16,16-dimethyl PGE₂, when given intravenously, normalized gastric emptying, significantly increased small intestinal transit, and made the colon hypermotile. Prostaglandins may be beneficial in the treatment of post-operative ileus and other conditions of sluggish gastrointestinal propulsion.     

Role of endothelin receptor subtypes in volume-stimulated ANF secretion

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.     

Prostaglandin E inhibition of mitogen stimulation in patients with multiple sclerosis

Kirbey et al have reported that leukocyte function from patients with multiple sclerosis is not suppressed by PGE2, as are normal leukocytes. We examined the ability of PGE2 (0.01-0.5 microgram/ml) to suppress Phytohemagglutinin induced 3H-thymidine incorporation in peripheral blood lymphocytes from multiple sclerosis patients and normals. There was no difference in sensitivity between the two groups. There was also no difference in activity of the prostaglandin producing suppressor cell between the multiple sclerosis patients and controls.     

Prostaglandin E2 secretion by day-6 to day-9 equine embryos.

Prostaglandin E2 (PGE2) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE2 secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27 +/- 0.39), tended to increase (P less than 0.1) on Day 7 (0.57 +/- 0.88), and increased significantly (P less than 0.05) on Day 8 (2.23 +/- 0.86) and Day 9 (4.13 +/- 0.71). Embryo diameter at the start of the incubation period was linearly correlated (P less than 0.01) to embryonic PGE2 secretion.     

Prostaglandin E inhibition of mitogen stimulation in patients with multiple sclerosis

Kirbey et al have reported that leukocyte function from patients with multiple sclerosis is not suppressed by PGE₂, as are normal leukocytes. We examined the ability of PGE₂ (0.01–0.5 μg/ml) to suppress Phytohemagglutinin induced ³H-thymidine incorporation in peripheral blood lymphocytes from multiple sclerosis patients and normals. There was no difference in sensitivity between the two groups. There was also no difference in activity of the prostaglandin producing suppressor cell between the multiple sclerosis patients and controls.     

Prostaglandin F2alpha (PGF2alpha) and prolactin secretion in rats.

Plasma prolactin and F-prostaglandins (PGF) were measured anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF2alpha (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpormazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF2alpha administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpormazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. Theese results indicate that PGF2alpha can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpormazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.