Electrolyte Secretion Research Articles

Rotavirus infection stimulates the Cl- reabsorption process across the intestinal brush-border membrane of young rabbits.

Rotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl(-) into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl(-) concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl(-) absorption. At 7 dpi, luminal Cl(-) concentrations were slightly increased, indicating a moderate net Cl(-) secretion. By using a rapid filtration technique, (36)Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% +/- 24% increase in all Cl(-) uptake activities (Cl(-)/H(+) symport, Cl(-) conductance, and Cl(-)/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl(-) reabsorption process across the intestinal BBM. We propose that the massive Cl(-) reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion.

The V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti: localization and activity.

The V-type H(+)-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H(+)-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H(+)-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H(+)-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO(3)(-)-sensitive ATPase activity accounted for 50-60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na(+)/K(+)-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H(+)-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na(+) and K(+) secretion and estimates of the H(+) flux mediated by the V-type H(+)-ATPase suggest a 1:1 stoichiometry for Na(+)/H(+) and K(+)/H(+) exchange transport across the apical membrane.

Immunobiology of epithelial chemokines in the intestinal mucosa

Surgery 2003;133:601-7.

Regulation of Cl- secretion by alpha2-adrenergic receptors in mouse colonic epithelium

Previous studies have shown that α2 adrenoceptor (α2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of α2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that α2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that α2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 μg ml−1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+–K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 μm). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of α2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that α2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by α2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

Regulation of Cl- secretion by alpha2-adrenergic receptors in mouse colonic epithelium.

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation, and effects of ouabain and bumetamide

Isolated salivary glands of Periplaneta americana were used to measure secretion rates and, by quantitative capillary electrophoresis, Na +, K +, and Cl − concentrations in saliva collected during dopamine (1 μM) and serotonin (1 μM) stimulation in the absence and presence of ouabain (100 μM) or bumetanide (10 μM). Dopamine stimulated secretion of a NaCl-rich hyposmotic saliva containing (mM): Na + 95 ± 2; K + 38 ± 1; Cl - 145 ± 3. Saliva collected during serotonin stimulation had a similar composition. Bumetanide decreased secretion rates induced by dopamine and serotonin; secreted saliva had lower Na +, K + and Cl − concentrations and osmolarity. Ouabain caused increased secretion rates on a serotonin background. Saliva secreted during dopamine but not serotonin stimulation in the presence of ouabain had lower K + and higher Na + and Cl − concentrations, and was isosmotic. We concluded: The Na +-K +-2Cl − cotransporter is of cardinal importance for electrolyte and fluid secretion. The Na +/K +-ATPase contributes to apical Na + outward transport and Na + and K + cycling across the basolateral membrane in acinar P-cells. The salivary ducts modify the primary saliva by Na + reabsorption and K + secretion, whereby Na + reabsorption is energized by the basolateral Na +/K +-ATPase which imports also some of the K + needed for apical K + extrusion.

Co-expression of adrenomedullin and adrenomedullin receptors in rat epididymis: distinct physiological actions on anion transport.

Adrenomedullin (AM) has been found in the brain as well as in various peripheral tissues, including reproductive organs such as the testis and the prostate. Here, we report the expression of AM in the rat epididymis and its role in anion secretion. Whole-epididymal extracts had 35.3 +/- 1.4 fmol of immunoreactive AM per mg of protein, and immunocytochemical studies showed positive AM immunostaining in the epithelial cells. By solution-hybridization-RNase protection assay, preproAM mRNA was detected at high levels in the epididymis. Gel filtration chromatography of AM showed two peaks, with the predominant one eluting at the position of authentic rat AM (1-50). Specific binding of AM to the epididymis, which could be displaced by calcitonin gene-related peptide, was observed. The epididymis also bound to calcitonin gene-related peptide, and this was displaceable by AM. Furthermore, the epididymis was shown to co-express mRNA encoding the calcitonin receptor-like receptor and receptor activity-modifying proteins, RAMP1/RAMP2. The corpus region had the highest AM level and gene expression and the lowest active peptide:precursor ratio. However, mRNA levels of the receptor and the receptor activity-modifying proteins were similar in all regions. In monolayer cultures derived from the rat epididymal cells, AM stimulated short-circuit current on the luminal side in a dose-dependent manner. Our results demonstrate the presence of AM, preproAM mRNA, AM receptors, and specific-binding sites in the rat epididymis as well as the possible role of AM in the regulation of electrolyte and fluid secretion in the epididymis.

Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance.

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an (125)I(-) efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a (86)Rb(+) efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca(2+) concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger (125)I(-) efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I(sc) increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl(-) gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I(sc) increase from apically permeabilized PDEC subject to a luminal-to-serosal K(+) gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca(2+)-activated apical Cl(-) channels and basolateral K(+) channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.

Regulation of mucin and fluid secretion by conjunctival epithelial cells.

Tears play a vital role in the health and protection of the cornea and conjunctiva. The tear film consists of multiple layers and different glands secrete each layer. Because of many and varied requirements of the ocular surface cells, the volume, composition and structure of the tear film must be exquisitely controlled. If any layer of the tear film is disrupted or altered, the entire tear film is affected, often with deleterious effects. This chapter reviews the current knowledge of the neural and growth factor regulation of electrolyte, water and protein secretion from the goblet and stratified squamous cells of the conjunctiva as well as the mechanisms used for fluid secretion. The evidence presented in this review suggests that parasympathetic nerves stimulate goblet, but not stratified squamous, cell secretion. Sympathetic nerves stimulate stratified squamous, but not goblet, cell secretion, while P2Y(2) agonists stimulate secretion from both cell types. Growth factors regulate goblet cell secretion, but their effects on stratified squamous cell secretion are unknown.

Guanylin and Functional Coupling Proteins in the Human Salivary Glands and Gland Tumors: Expression, Cellular Localization, and Target Membrane Domains

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated secretion of an electrolyte-rich fluid is a major but incompletely understood function of the salivary glands. We provide molecular evidence that guanylin, a bioactive intestinal peptide involved in the CFTR-regulated secretion of electrolyte/water in the gut epithelium, is highly expressed in the human parotid and submandibular glands and in respective clinically most relevant tumors. Moreover, in the same organs we identified expression of the major components of the guanylin signaling pathway, ie, guanylin-receptor guanylate cyclase-C, cGKII, and CFTR, as well as of the epithelial Cl(-)/HCO(3)(-) anion exchanger type 2 (AE2). At the cellular level, guanylin is localized to epithelial cells of the ductal system that, based on its presence in the saliva, is obviously released into the salivary gland ducts. The guanylin-receptor guanylate cyclase-C, cGKII, CFTR, and AE2 are all confined exclusively to the apical membrane of the same duct cells. These findings implicate guanylin as intrinsic regulator of electrolyte secretion in the salivary glands. We assume that duct epithelial cells synthesize and release guanylin into the saliva to regulate electrolyte secretion in the ductal system by an intraductal luminocrine signaling pathway. Moreover, the high expression of guanylin in pleomorphic adenoma and Warthin tumors (cystadenolymphoma), the most common neoplasms of salivary glands, predicts guanylin as a significant marker in tumor pathology.

The electrolyte/fluid secretion stimulatory peptides guanylin and uroguanylin and their common functional coupling proteins in the rat pancreas: a correlative study of expression and cell-specific localization.

Guanylin and uroguanylin are intestinal or urine peptides that stimulate epithelial electrolyte/fluid secretion by activating the cystic fibrosis gene product (CFTR). Because CFTR is essentially involved in the electrolyte secretion of the pancreatic duct cells, the rat pancreas was investigated for expression and cell-specific localization of guanylin and uroguanylin; expression of major components of the guanylin signaling pathway, i.e., the guanylin/uroguanylin receptor guanylate cyclase C (GC-C), cGMP-dependent protein kinase II, and CFTR; and expression of the epithelial Cl-/HCO3- exchanger AE2. Reverse transcriptase polymerase chain reaction analyses revealed high expression of guanylin, uroguanylin, and the functional coupling proteins in the rat pancreas. At the cellular level, guanylin and uroguanylin were localized by immunohistochemistry to the centroacinar cells and proximal duct cells of the exocrine pancreas. The guanylin/uroguanylin receptor GC-C, cGKII, CFTR, and AE2 were all found in the same segments of the ductal system, where they were confined to the apical membrane of centroacinar cells and proximal duct epithelial cells, a circumstance suggesting that both peptides may act through the ductal lumen. In view of the well-known functional significance of guanylin and uroguanylin, the presence and cell-specific expression not only of the both peptides but also of their common functional coupling proteins implicates a regulatory function of these peptides in the electrolyte/fluid secretion within the rat exocrine pancreas.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells.

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCD(i)) and terminal (hIMCD(t)) inner medullary regions of human kidneys. AVP, PGE(2), and forskolin increased cAMP in both hIMCD(i) and hIMCD(t) cells. The effects of AVP and PGE2 were greatest in hIMCD(i); however, forskolin increased cAMP to the same extent in hIMCD(i) and hIMCD(t). Basal short-circuit current (I(SC)) of hIMCD(i) monolayers was 1.4 +/- 0.5 microA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE(2), and forskolin increased I(SC); the current was reduced by blocking PKA, apical Cl- channels, basolateral NKCC1 (a Na+ - K+ - 2Cl- cotransporter), and basolateral Cl-/HCO(3)(-) exchangers. In fluid transport studies, hIMCD(i) monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCD(t) monolayers, basal current was not different from zero and cAMP had no effect on I(SC). We conclude that AVP and PGE2 stimulate cAMP-dependent Cl- secretion by hIMCD(i) cells, but not hIMCD(t) cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

Reactive Nitrogen Species Modulate the Effects of Rhein, an Active Component of Senna Laxatives, on Human Epithelium In Vitro

ABSTRACTBackgroundSenna laxatives are used worldwide. However, their misuse can lead to chronic mucosal inflammation with the accumulation of pigment‐laden leukocytes and may cause colon cells to undergo apoptosis. This study explores the mechanisms by which rhein, an active component of senna, acts on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes.MethodsHuman colonic adenocarcinoma (CaCo‐2) monolayer cells, in the presence or in the absence of rhein, were used to monitor the production of reactive nitrogen species using the Griess reaction. Modified Ussing chambers were used to study electrolyte secretion. The capacity to recruit human polymorphonuclear leukocytes was evaluated using masked well chemotaxis chambers. Rhein‐induced apoptosis was investigated by counting apoptotic nuclei stained with Hoechst 33258 dye.ResultsRhein caused a dose‐dependent increase in short‐circuit current that was abolished in chloride‐free bathing buffer or by preincubating with 100 μmol/L NG‐nitro‐L‐arginine (L‐NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 μmol/L rhein, induced nitrate production. Supernatants obtained from CaCo‐2 cultures after incubation with 50 μmol/L rhein stimulated a time‐dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 μmol/L L‐NAME, whereas rhein per se was not active. Neutralizing antibodies anti–interleukin‐8 (IL‐8) and anti‐ENA78 also inhibited chemotaxis. Overnight rhein incubation produced an increased number of apoptotic cells in the culture supernatant that was significantly decreased by preincubation with 100 μmol/L L‐NAME. Light‐degraded rhein had no effects on CaCo‐2 monolayers.ConclusionsThe integrity of rhein is crucial to generating nitric oxide, which mediates, with different time courses, ion secretion, chemotaxis, and apoptosis of human‐derived cells.

Reactive nitrogen species modulate the effects of rhein, an active component of senna laxatives, on human epithelium in vitro.

Senna laxatives are used worldwide. However, their misuse can lead to chronic mucosal inflammation with the accumulation of pigment-laden leukocytes and may cause colon cells to undergo apoptosis. This study explores the mechanisms by which rhein, an active component of senna, acts on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. Human colonic adenocarcinoma (CaCo-2) monolayer cells, in the presence or in the absence of rhein, were used to monitor the production of reactive nitrogen species using the Griess reaction. Modified Ussing chambers were used to study electrolyte secretion. The capacity to recruit human polymorphonuclear leukocytes was evaluated using masked well chemotaxis chambers. Rhein-induced apoptosis was investigated by counting apoptotic nuclei stained with Hoechst 33258 dye. Rhein caused a dose-dependent increase in short-circuit current that was abolished in chloride-free bathing buffer or by preincubating with 100 micromol/L NG-nitro-L-arginine (L-NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 micromol/L rhein, induced nitrate production. Supernatants obtained from CaCo-2 cultures after incubation with 50 micromol/L rhein stimulated a time-dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 micromol/L L-NAME, whereas rhein per se was not active. Neutralizing antibodies anti-interleukin-8 (IL-8) and anti-ENA78 also inhibited chemotaxis. Overnight rhein incubation produced an increased number of apoptotic cells in the culture supernatant that was significantly decreased by preincubation with 100 micromol/L L-NAME. Light-degraded rhein had no effects on CaCo-2 monolayers. The integrity of rhein is crucial to generating nitric oxide, which mediates, with different time courses, ion secretion, chemotaxis, and apoptosis of human-derived cells.

Syndrome aigu d'irradiation : les atteintes du système gastro-intestinal

Acute syndrome of radiation: injuries to the gastrointestinal tract. Exposure to ionising radiation at medium to high doses results in the manifestation of mixed pathologies. Following the analysis of several radiation accidents it is clear that intestinal injury influences patient survival. However the appearance of the classically defined gastrointestinal syndrome is not always evident. Nevertheless injury to the gastrointestinal tract, in particular loss of barrier function, seems to play an important role in the development of Multiple Organ Failure such as reported in the recent accident at Tokai Mura. lonising radiation overexposure results in changes in intestinal motility and nutrient, fluid and electrolyte absorption and secretion all which may contribute to the genesis of diarrhoea. In addition to modified cellular transport properties for nutrients or electrolytes, important loss of epithelial cells is also a major contributing factor. Intestinal functions are controlled by many factors such as neurotransmitters, locally released mediators from endocrine cells or immunocompetent cells in addition to luminal agents. To date, treatment of radiation-induced gastrointestinal injury is mainly symptomatic. However treatments such as growth factors, anti-inflammatory cytokines as well as cellular transplantation remain to he validated in the radiation accident situation.

Effect of octreotide on fluid absorption and secretion by the normal human jejunum and ileum in vivo.

We hypothesized that part of the non-specific antidiarrhoeal effect of octreotide is mediated by a proabsorptive or antisecretory effect on small intestinal active electrolyte transport. To measure the effect of octreotide on net absorption, the jejunum and ileum of normal human subjects were perfused with a balanced electrolyte solution; to measure the effect of octreotide on normal active chloride secretion, the jejunum was perfused with a bicarbonate-free solution. During perfusion of a balanced electrolyte solution, octreotide increased basal net fluid absorption in the jejunum and ileum by about 40 mL/h per 30 cm. In the jejunum, octreotide markedly inhibited basal and sham feeding-stimulated active chloride secretion and inhibited water secretion by 28 and 51 mL/h per 30 cm, respectively. Octreotide causes an increase in the net epithelial cell absorption rate of a balanced electrolyte solution in the normal jejunum and ileum. In the jejunum, this proabsorptive effect is mediated mainly by the reduction of normal active electrolyte secretion, rather than by stimulation of normal active electrolyte absorption. These results support the hypothesis that part of the antidiarrhoeal action of octreotide is due to its effects on active electrolyte transport mechanisms by normal epithelial cells of the small intestine.

A comparative molecular field analysis (COMFA) of the structural determinants of heat-stable enterotoxins mediating activation of guanylyl cyclase C.

The heat-stable enterotoxin binds to and activates guanylyl cyclase C (GC-C), regulating fluid and electrolyte secretion in intestinal epithelial cells. A COMFA model was developed to predict the primary interactions between GC-C agonists and their receptor. This model predicts that the amide backbone of Cys(5)-Cys(6)-Glu(7)-Leu(8), the beta carbon atoms of Cys(5)-Cys(6), and the side chains of Pro(12), Ala(13), and Ala(15) comprise the primary interactions of GC-C agonists with the receptor surface.

Enteric nerves and diarrhoea.

This review article discusses the importance of the enteric nervous system for the fluid and electrolyte secretion evoked by luminal secretagogues in the small intestine. The first part of the review summarizes observations on augmented secretion caused by cholera toxin, which has been the subject of extensive studies in the past. The latter part reviews studies of the participation of the enteric nervous system in other secretory states of the gut. The involvement of the enteric nervous system in the pathophysiology of intestinal secretory states opens up potential new sites of actions for drugs in the treatment of diarrhoea. This is discussed in the final part of this review.

Cyclooxygenase-2 Regulates Apoptosis in Rat Epididymis Through Prostaglandin D21

In previous studies, cyclooxygenase (COX)-1 and COX-2 isozymes have been detected in the rat epididymis. COX-1 mediates electrolyte and fluid secretion induced by a number of peptide hormones, including bradykinin, angiotensin, and endothelin, via local formation of prostaglandin (PG) E2; however, the physiological role of COX-2 remains largely unknown. Marked apoptotic cell death in the rat epididymis following androgen depletion has been reported. Because expression of both COX isozymes is dependent on androgen, we investigated whether these isozymes control apoptosis in the epididymis. Apoptosis was detected in rat epididymal epithelial cells by in situ staining using the TUNEL method and by the presence of internucleosomal DNA fragmentation using capillary electrophoresis with laser-induced fluorescence detection. Specific COX inhibitors were used to delineate the roles of the 2 isozymes. There was no significant apoptotic cell death in normal and specific COX-1 inhibitor (SC-560)-treated epididymal cells. However, application of a specific COX-2 inhibitor (NS-398) induced apoptosis in a dose- and time-dependent manner. A similar apoptotic effect of COX-2 inhibitor was seen in the in vivo study. The drastic DNA fragmentation induced by COX-2 inhibitor could be reversed completely by PGD2 and partially by PGE2. In addition, the protective effect of PGD2 against COX-2 inhibition was significantly blocked by a PGDP-receptor antagonist, BWA868C. These results indicate that the COX-2 products PGD2 and, to a lesser extent, PGE2 control apoptosis in cultured rat epididymal cells in vitro.

Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl(-) secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl(-) secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl(-), by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca(2+)-ATPase inhibitor cyclopiazonic acid and in low-Ca(2+) buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.