Regulation Of Anion Secretion Research Articles

199 Identification of a Uroguanylin-GUCY2C Brain Gut Axis That Regulates Feeding in Rat

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS) to release secretagogues. We have previously demonstrated that extracellular calcium-sensing receptor (CaSR) agonists can abrogate toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Based on our observation that CaSR is expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, we hypothesized that CaSR agonists may also function through neuronal pathways. To examine this possibility, rat colon segments containing intact ENS were isolated, mounted on Ussing chambers and perfused. Basal and cyclic nucleotide-stimulated anion secretions were monitored by measuring changes in short circuit current (Isc). CaSR was activated by R-568 either before or after cyclic nucleotide stimulation to mimic diarrhea prevention or treatment, respectively, and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of Isc in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. Activation of CaSR with R-568 significantly reduced basal and forskolin-induced Isc. A similar inhibitor effect of R-568 on Isc was achieved following the forskolin stimulation, suggesting that this agonist might be useful both for diarrhea prevention and treatment. Interestingly, when colon tissues were pretreated with TTX to inhibit the ENS activity, the inhibitory effects of R-568 were either significantly attenuated or abolished, indicating that R-568 exerts its antisecretory effect on the intestine primarily by inhibiting the ENS. The antisecretory effect of R-568 appears to be largely mediated by the submucosal Meissner's plexus because removal of the muscular layers and the myenteric Auerbach's plexus from the colon did not significantly reduce the inhibitory effect of R568. Our results suggest a new model in which the neuronal component of intestinal fluid transport can be modulated by the inhibitory effects of CaSR on the ENS. The ability of CaSR agonist to reduce secretagogue-stimulated anion secretion suggests that modulation of CaSR activity may provide a new therapy for secretory diarrheas and other ENS-mediated conditions.

198 Bitter Stimuli Induce ERK Activation in Mouse and Human Cell Lines

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS) to release secretagogues. We have previously demonstrated that extracellular calcium-sensing receptor (CaSR) agonists can abrogate toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Based on our observation that CaSR is expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, we hypothesized that CaSR agonists may also function through neuronal pathways. To examine this possibility, rat colon segments containing intact ENS were isolated, mounted on Ussing chambers and perfused. Basal and cyclic nucleotide-stimulated anion secretions were monitored by measuring changes in short circuit current (Isc). CaSR was activated by R-568 either before or after cyclic nucleotide stimulation to mimic diarrhea prevention or treatment, respectively, and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of Isc in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. Activation of CaSR with R-568 significantly reduced basal and forskolin-induced Isc. A similar inhibitor effect of R-568 on Isc was achieved following the forskolin stimulation, suggesting that this agonist might be useful both for diarrhea prevention and treatment. Interestingly, when colon tissues were pretreated with TTX to inhibit the ENS activity, the inhibitory effects of R-568 were either significantly attenuated or abolished, indicating that R-568 exerts its antisecretory effect on the intestine primarily by inhibiting the ENS. The antisecretory effect of R-568 appears to be largely mediated by the submucosal Meissner's plexus because removal of the muscular layers and the myenteric Auerbach's plexus from the colon did not significantly reduce the inhibitory effect of R568. Our results suggest a new model in which the neuronal component of intestinal fluid transport can be modulated by the inhibitory effects of CaSR on the ENS. The ability of CaSR agonist to reduce secretagogue-stimulated anion secretion suggests that modulation of CaSR activity may provide a new therapy for secretory diarrheas and other ENS-mediated conditions.

Regulation of anion secretion by nitric oxide in human airway epithelial cells.

Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester, reduced short- circuit current (I(sc)), whereas an NO donor, S-nitrosoglutathione (GSNO), increased I(sc), with an EC50 approximately 1.2 microM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, prevented activation of I(sc) by NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on I(sc). Interestingly, GSNO elevated intracellular Ca2+ concentration ([Ca2+]i) but had no effect on I(sc) activated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.

LOCAL REGULATION OF ANION SECRETION BY PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN HUMAN COLONIC T84 CELLS

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel hypothalamic peptide, which has been shown to exert various functions in a number of tissues, including exocrine and endocrine tissues. The present study investigated the role of local PACAP in the control of anion secretion by the human colonic T84 cell. Both bioactive forms of PACAP-27 and PACAP-38 gave rise to a dose-dependent increase in the short-circuit current (ISC). However, there was a reversal in the order of potency observed at different concentration ranges for the two bioactive forms. PACAP-27 was greater than PACAP-38 when the peptide concentrations were below 10n m; PACAP-38 was greater than PACAP-27 in the range of 10–80n m. The effects of both PACAP forms were restricted to the apical aspect of the T84 cell. The ISCresponses to both PACAP-27 and PACAP-38 were suppressed respectively by the non-selective Cl−channel blocker, diphenylamine-dicarboxylic acid (DPC), by the Ca2+dependent Cl−channel blocker, diisothiocyanatostilbene-disulfonic acid (DIDS) and by the Ca2+chelator, BAPTA-AM, indicating the involvement of Ca2+. The expression of PACAP was demonstrated and localized specifically to the perinuclear cytoplasm of the T84 cell using immunocytochemistry, indicating its epithelial origin. Thus, the present data suggest that, in addition to the well-known cAMP-dependent pathway, PACAP may play a role in regulating colonic Cl−secretion via a Ca2+-dependent pathway, perhaps through two distinct PACAP receptor subtypes. Moreover, the regulation of anion secretion by T84 cells may be mediated by locally formed PACAP in an autocrine or paracrine fashion.

Serotonin via 5‐HT1B and 5‐HT2B receptors stimulates anion secretion in the rat epididymal epithelium

1. The short-circuit current (Isc) technique was used to study the role of 5-hydroxytryptamine (5-HT) in the regulation of anion secretion in cultured rat cauda epididymal epithelia. 2. 5-HT, the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) and the 5-HT2B-selective agonist alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) added basolaterally stimulated Isc in a dose-dependent manner with EC50 values of 0.4, 20 and 0.3 microM, respectively. No other agonists for 5-HT receptors had any effect. 3. The pattern of responses to 5-HT was biphasic. Pretreating the tissues with the 5-HT1B-selective antagonist isamoltane (200 microM) and the 5-HT2B-selective antagonist rauwolscine (200 microM) inhibited the rapid transient phase by 55 and 45 %, whereas the sustained phase could only be blocked by rauwolscine. 4. Removal of chloride or bicarbonate or both from the normal Krebs-Henseleit solution reduced the responses to 5-HT, 5-NOT and alpha-methyl-5-HT to varying degrees. The results suggest that 5-HT1B- and 5-HT2B-mediated responses were mainly due to chloride and bicarbonate secretion, respectively. 5. Manipulation of the cAMP and Ca2+ signal transduction pathways with chemical agents provided evidence that the responses to 5-HT were mediated through cAMP. 6. Piroxicam pretreatment abolished the Isc response to alpha-methyl-5-HT but not to 5-NOT, indicating that the 5-HT2B-mediated response, but not the 5-HT1B-mediated response, is dependent on prostaglandin synthesis. 7. Immunohistochemical studies showed that 5-HT-like immunoreactivity was detected in nerve fibres and in small granular cells surrounding the epididymal tubules. 8. It is suggested that the 5-HT released from serotonergic nerve endings and/or from mast cells regulates electrolyte and fluid secretion in the epididymis.

Regulation of anion secretion by cyclo-oxygenase and prostanoids in cultured epididymal epithelia from the rat.

1. The role of cyclo-oxygenase (COX) in the regulation of anion secretion (measured as short- circuit current, Isc) in cultured epididymal epithelia from immature rats was investigated. 2. COX inhibitors attenuated the increase of anion secretion caused by bradykinin (LBK) but had no effect on that caused by PGE2, suggesting that prostaglandin synthesis mediates the secretory response of the tissues to LBK. 3. The apparent IC50 values for indomethacin, piroxicam and L-745,337 in inhibiting the LBK-induced Isc were 0.14, 1.34 and 15.7 microM, respectively. This order of potency: indomethacin > piroxicam > L-745,337 >> DFU suggests the involvement of the COX-1 isozyme in the mediation of the secretory response to LBK. 4. Among the COX products (prostaglandins, thromboxane and prostacyclins) tested, only PGE2 and, to a much lesser extent, PGF2alpha stimulated anion secretion by cultured rat epididymal epithelia. 5. The effect of PGE2 was mimicked by 11-deoxyl PGE1, a specific prostaglandin E (EP)2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist, indicating that cyclic AMP-coupled EP2/4 receptors are involved in the LBK-stimulated anion secretion. 6. A reverse transcriptase-polymerase chain reaction study detected the expression of COX-1 and COX-2 mRNA in intact rat epididymis and in cultured epididymal epithelia. The expression of COX-1 mRNA was reduced by LBK by 44 %. 7. Immunohistochemical studies demonstrated the presence of COX-1 immunoreactivity in the basal cells of the intact rat epididymis. By comparison, COX-2 immunoreactivity was detected in the apical pole of the principal cells. 8. The role of COX in the formation of the epididymal microenvironment and the implication of long term administration of non-steroidal anti-inflammatory drugs (NSAIDs) on male fertility are discussed.

Stimulation of anion secretion by beta-adrenoceptors in the mouse endometrial epithelium.

1. Regulation of anion secretion by adrenoceptors in primary culture of mouse endometrial epithelium was investigated using the short circuit current (ISC) technique. 2. Adrenaline stimulated a sustained increase in the ISC in a concentration-dependent manner. The adrenaline-induced ISC could be inhibited by pretreatment with diphenylamine 2,2'-dicarboxylic acid (DPC) or replacement of external Cl- and HCO3-, but not by amiloride or replacement of Na+ in apical solution. 3. The concentration-dependent responses of the adrenaline-induced ISC to the Cl- channel blockers glibenclamide and DPC were examined and exhibited IC50 values of 380 and 960 microM, respectively. 4. The effect of various adrenoceptor agonists on the ISC was examined. The order of potency appeared to be isoprenaline > adrenaline > noradrenaline, while no response was elicited by the alpha-adrenoceptor agonist methoxamine, indicating a predominant involvement of beta-adrenoceptors. 5. The beta-adrenoceptor antagonist propranolol was found to be much more effective than the alpha-adrenoceptor antagonist phentolamine in inhibiting the ISC responses induced by all adrenoceptor agonists examined. 6. The effect of adrenaline on the ISC was mimicked by an adenylate cyclase activator, forskolin, but suppressed by the adenylate cyclase inhibitor MDL 12,330A, indicating the involvement of cAMP. 7. Our results demonstrate that anion secretion by the mouse endometrial epithelium is regulated by beta-adrenoceptors and involves a cAMP-dependent mechanism.

Paracrine/autocrine regulation of anion secretion in the epididymis: role of angiotensin II.

Accumulated evidence has suggested that anion secretion in the epididymis may be subject to local paracrine/autocrine control in addition to neural and humoral regulation. The present paper reviews recent studies presenting evidence for the presence of major components of a tissue renin-angiotensin system in the rat epididymis and the regulatory effect of angiotensin II (Ang II) on epididymal anion secretion. The mechanisms for local generation of Ang II and its local action are discussed. The importance of paracrine/autocrine roles of Ang II in the maintenance of epididymal functions, as well as sperm functions is also addressed.

The role of calcitonin gene-related peptide in the regulation of anion secretion by the rat and human epididymis.

A study was carried out to investigate the role of the calcitonin gene-related peptide (CGRP) in the regulation of electrolyte transport in the rat and human epididymis. In monolayer cultures derived from the rat cauda epididymal cells, CGRP stimulated the short-circuit current (SCC) in a dose-dependent manner with the EC50 (concentration required to produce 50% of the response) at 15 nmol/l. This effect of CGRP was seen when the peptide was added to the basolateral aspect of the cells; apical addition having negligible effect. The CGRP-induced rise in the SCC was dependent on the presence of chloride in the bathing solution. Calcitonin had no effect on the SCC and did not affect the CGRP-induced rise in the SCC. The effect of CGRP on secretion was inhibited in a competitive fashion by the CGRP receptor antagonist CGRP(8-37). In contrast to bradykinin, angiotensin II and endothelin I, the effect of CGRP was independent of prostaglandin synthesis. Measurement of intracellular adenosine 3':5'-cyclic monophosphate showed a time- and dose-dependent increase upon stimulation with CGRP. CGRP also stimulated the SCC in monolayers grown from the human epididymis. The current could be inhibited by apical application of the chloride channel blocker, diphenylamine-2-carboxylate. Immunoreactive CGRP was found in the epithelia of rat and human cauda epididymidis. It is suggested that CGRP may regulate the electrolyte and fluid secretion in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.