Control of ion transport in mouse proximal and distal colon by prolactin.

The lactogenic hormone prolactin (PRL) has been known to affect Ca(2+) and electrolyte transport in the intestinal epithelium. In the present study we analyzed ion transport in mouse proximal and distal colon, and acute changes induced by PRL. In the proximal colon, carbachol activated a Ca(2+) dependent Cl(-) secretion that was sensitive to DIDS and NFA. In the distal colon, both ATP and carbachol activated K(+) secretion. Ca(2+) -activated KCl transport in proximal and distal colon was inhibited by PRL (200 ng/ml), while amiloride sensitive Na(+) absorption and cAMP induced Cl(-) secretion remained unaffected. Luminal large conductance Ca(2+) -activated K(+) (BK) channels were largely responsible for Ca(2+) -activated K(+) secretion in the distal colon, and basolateral BK channels supported Ca(2+) -activated Cl(-) secretion in the proximal colon. Ca(2+) chelating by BAPTA-AM attenuated effects of carbachol and abolished effects of PRL. Both inhibition of PI3 kinase with wortmannin and blockage of MAP kinases with SB 203580 or U 0126, interfered with the acute inhibitory effect of PRL on ion transport, while blocking of Jak/Stat kinases with AG 490 was without effects. PRL attenuated the increase in intracellular Ca(2+) that was caused by stimulation of isolated colonic crypts with carbachol. Thus PRL inhibits Ca(2+) dependent Cl(-) and K(+) secretion by interfering with intracellular Ca(2+) signaling and probably by activating PI3 kinase and MAP kinase pathways.     

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.     

Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl(-) secretion and an inverse response of the short-circuit current (I(sc)) toward stimulation with carbachol (CCh). Alternative Cl(-) channels are found in airway epithelia and have been attributed to residual Cl(-) secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I(sc) upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca(2+)-dependent Cl(-) conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I(sc) were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I(sc) in CF rectal biopsies but caused a negative I(sc) in non-CF subjects. The CCh-induced negative I(sc) in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I(sc) was significantly enhanced in CF and was caused by activation of a luminal K(+) conductance, as shown by the use of the K(+) channel blockers Ba(2+) and tetraethylammonium. Moreover, a cAMP-dependent luminal K(+) conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl(-) channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl(-) conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca(2+)- and cAMP-dependent K(+) secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.     

Diminished nitroprusside-induced relaxation of inflamed colonic smooth muscle in mice.

The dextran sodium sulphate (DSS) induced colitis in mice was used as a experimental model to study the contractility of murine longitudinal colonic smooth muscle during inflammation. Smooth muscle segments of proximal, middle and distal colon were mounted in organ baths. Smooth muscle contraction was induced by carbachol showing an aboral increase in activity, whereas in the inflamed middle colonic segment a marked decrease in activity was observed. The dilatative effect of sodium-nitroprusside (SNP) as a nitric oxide donor was investigated after precontraction by carbachol. Both in normal and DSS segments administration of SNP to isolated mouse colonic smooth muscle preparations caused regional differences in relaxation, the highest relaxation seen in normal proximal colonic tissue. However, this relaxation was markedly reduced in inflamed proximal preparations, associated with a diminished cGMP contents.     

Stimulation of chloride secretion by baicalein in isolated rat distal colon

The effect of baicalein on mucosal ion transport in the rat distal colon was investigated in Ussing chambers. Mucosal addition of baicalein (1-100 microM) elicited a concentration-dependent short-circuit current (I(sc)) response. The increase in I(sc) was mainly due to Cl(-) secretion. The presence of mucosal indomethacin (10 microM) significantly reduced both the basal and subsequent baicalein-evoked I(sc) responses. The baicalein-induced I(sc) were inhibited by mucosal application of diphenylamine-2-carboxylic acid (100 microM) and glibenclamide (500 microM) and basolateral application of chromanol 293B (30 microM), a blocker of K(v)LQT1 channels and Ba(2+) ions (5 mM). Treatment of the colonic mucosa with baicalein elicited a threefold increase in cAMP production. Pretreating the colonic mucosa with carbachol (100 microM, serosal) but not thapsigargin (1 microM, both sides) abolished the baicalein-induced I(sc). Addition of baicalein subsequent to forskolin induced a further increase in I(sc). These results indicate that the baicalein evoked Cl(-) secretion across rat colonic mucosa, possibly via a cAMP-dependent pathway. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. Baicalein may stimulate Cl(-) secretion via a cAMP-independent pathway or have a direct effect on cystic fibrosis transmembrane conductance regulator.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Changes in number of serotonin-containing cells and serotonin levels in the intestinal mucosa of rats with colitis induced by dextran sodium sulfate

The role of serotonin in the pathogenesis of inflammatory bowel disease has not been fully studied. We examined the changes in the serotonin level and the density of serotonin-containing enterochromaffin (EC) and mast cells in the intestinal mucosa of dextran sodium sulfate (DSS)-induced colitis in rats. Rats were treated with 1.5% DSS for 1 month. Serotonin levels were biochemically measured and the density of epithelial EC cells and mucosal mast cells was quantified by serotonin immunohistochemistry. DSS caused malnutrition due to chronic diarrhea. Infiltrated inflammatory cells were microscopically observed in the colonic wall with intact epithelium. The serotonin content in the mucosa/submucosa tissue was increased in the proximal and distal colon in DSS-treated rats, compared to that in control rats. The density of EC cells in the epithelium also increased in the proximal and distal colon in DSS-treated rats. In contrast, the density of mast cells in the lamina propria dramatically increased in the distal, but not in the proximal colon in DSS-treated rats. This discrepancy implies the serotonin released from EC cells and from mast cells may play different roles in the pathogenesis of DSS-induced colitis. Accepted: 30 July 1999     

Electrogenic ion secretion in proximal,mid and distal colon from fed and starved mice

1. Electrogenic ion transport was monitored in vitro as the short-circuit current (Isc in μA/cm²) across proximal, mid and distal colon removed from fed and 48 hr-starved Swiss albino mice (Mus muscaris).2. Electrogenic secretion was induced either with serosal bethanechol (muscarinic agonist), DMPP (nicotinic agonist) or dibutyryl-cyclic AMP (DbcAMP). Proximal and distal colon from starved mice showed greater electrogenic secretion in response to bethanechol than those from the fed controls while DMPP and DbcAMP did not activate the hypersecretion.3. In the distal colon, starvation induced a large increase in the basal Isc that was unaffected by mucosal amiloride but was inhibited by tetrodotoxin (TTX) and by diphenylamine-2-carboxylic acid (DPC) unlike the fed basal Isc. Bethanechol activated a biphasic response consisting of a transient decrease in the Isc followed by a sustained increase both of which were significantly greater in the starved than the fed tissue and were inhibited by TTX, DPC and atropine but not hexamethonium.4. Starvation enhances the secretory response to muscarinic activation in proximal and distal colon and induces an increased basal electrogenic (Cl ⁻) secretion in the distal colon stimulated by an augmented neural tone.     

Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis

Background

Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohn''s disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.

Methods

C57BL/6 mice were administered with 3.5% of DSS in drinking water for various times. Clinical and histological features were determined using standard criteria. Myeloperoxidase (MPO) activity, transepithelial permeability and proinflammatory mediators were determined in whole colon or proximal and distal parts of colon.

Results

As expected after administration of DSS, mice manifest loss of body weight, shortening of colon length and bloody feces. Histological manifestations included shortening and loss of crypts, infiltration of lymphocytes and neutrophil, symptoms attenuated after DSS withdrawal. The MPO value, as inflammation indicator, also increases significantly at all periods of DSS treatment, and even after DSS withdrawal, it still held at very high levels. Trans-mucosal permeability increased during DSS treatment, but recovered to almost control level after DSS withdrawal. The production of proinflammatory mediators by colonic mucosa were enhanced during DSS treatment, and then recovered to pre-treated level after DSS withdrawal. Finally, enhanced expression of proinflammatory mediators also revealed a different profile feature in proximal and distal parts of the colon.

Conclusion

Experimental colitis induced by DSS is a good animal model to study the mechanisms underlying the pathogenesis and intervention against IBD, especially UC.     

PACAP provides colonic protection against dextran sodium sulfate induced colitis

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a crucial role in immunity and inflammation. Our aim was to obtain insight in the role of PACAP in experimental colitis in mice and thus its possible role in inflammatory bowel disease. PACAP-deficient (PACAP-/-) mice and wild-type control mice were challenged by colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and difference of cytokine production in the proximal and distal colon. After DSS administration, mortality was more severe in PACAP-/- mice versus wild-type control mice. The histological score and the disease activity index of PACAP-/- mice were significantly higher than those of wild-type control mice. In proximal colon, production of IL-1beta and IL-6 in PACAP-/- mice were significantly upregulated on day 8 after DSS administration, compared to wild-type control mice. In distal colon, furthermore, production of IFNgamma, IL-1beta, IL-6, IL-12, and KC were significantly higher in PACAP-/- mice than in wild-type control mice on day 4. Our findings indicate that PACAP regulates the production of pro-inflammatory cytokine in the experimental colitis.     

Effects of inhibitors and specific ion-free salines on segmental fluid secretion by the Malpighian tubules of the black field cricket Teleogryllus oceanicus

The effects of inhibitors and specific ion-free salines on fluid secretion rates in the distal and main segments showed that there were major differences in secretory mechanisms in the two segments. Both main and distal segments of the Malpighian tubules were sensitive to DIDs, SITS and acetazolamide but in different ways. The evidence suggests that the main segment does not contain a Cl(-)/HCO(3)(-) exchanger in the basal membrane, whereas the distal segment may do so. Secretion in both segments was K(+) dependent. Ba(2+) markedly reduced fluid secretion by the main segment and K(+) entry into the cells of the main segment is suggested to be predominantly via K(+) channels. Entry of K(+) may be primarily by other routes, such as Na K ATPase, in the distal segment. In the distal segment secretion was highly Mg(2+) dependent. Both segments were sensitive to amiloride analogs suggesting the presence of apical cation/H(+) exchangers.     

Characterisation of chloride currents across the proximal colon in CftrTgH(neoim)1Hgu congenic mice

It was the aim of the present study to investigate chloride secretion across the proximal colon of Cftr ^{TgH(neoim)1Hgu} congenic mice. Stripped epithelia were incubated in Ussing chambers and the electrophysiological data were compared between cystic fibrosis (CF) animals and wild type (WT) animals. In comparison with the control animals, all Cftr ^{TgH(neoim)1Hgu} congenic mice had a distinctly reduced basal chloride secretion and a reduced chloride secretion after stimulation with carbachol and forskolin. When comparing chloride secretion across the proximal colon between WT animals, all mice showed a comparable pattern of response to carbachol and forskolin but quantitative differences, BALB/c exhibiting the highest and HsdOla:MF1 exhibiting the lowest increase in Cl current. Likewise, all CF animals showed the same reaction pattern to carbachol and forskolin, but there was no distinct difference that lasted for the whole measurement. To investigate interferences between Ca- and cyclic adenosine monophosphate-activated pathways of Cl secretion in CF animals, we studied epithelia from CF/3CF/1F1 animals with a mixed background. In these animals, the levels of the carbachol or forskolin-induced chloride currents did not depend on the prestimulation with the respective other secretagogue. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, which blocks calcium-activated chloride channels, reduced the current response to carbachol by about 23%. This result, obtained in BALB/c-Cftr ^{TgH(neoim)1Hgu} mice, indicates that alternative chloride channels might be present in the proximal colon of these mice. In contrast, there was no evidence for alternative chloride conductances in BALB/c WT animals, but we cannot exclude that in WT mice a higher chloride secretion via Cftr-channels may have masked an alternative chloride secretion.     

Dextran sodium sulfate-induced colitis reveals nicotinic modulation of ion transport via iNOS-derived NO

In normal colon, ACh elicits a luminally directed Cl- efflux from enterocytes via activation of muscarinic receptors. In contrast, in the murine model of dextran sodium sulfate (DSS)-induced colitis, an inhibitory cholinergic ion transport event due to nicotinic receptor activation has been identified. The absence of nicotinic receptors on enteric epithelia and the ability of nitric oxide (NO) to modulate ion transport led us to hypothesize that NO mediated the cholinergic nicotinic receptor-induced changes in ion transport. Midportions of colon from control and DSS-treated mice were examined for inducible NO synthase (iNOS) expression by RT-PCR and immunofluorescence or mounted in Ussing chambers for assessment of cholinergic-evoked changes in ion transport (i.e., short-circuit current) with or without pretreatment with pharmacological inhibitors of NO production. iNOS mRNA and protein levels were increased throughout the tissue from DSS-treated mice and, notably, in the myenteric plexus, where the majority of iNOS immunoreactivity colocalized with the enteric glial cell marker glial fibrillary acidic protein. The drop in short-circuit current evoked by the cholinomimetic carbachol in tissue from DSS-treated mice was prevented by selective inhibitors of iNOS activity [N6-(1-iminoethyl)-lysine HCl and N-[3-(aminomethyl)benzyl]acetamidine] or an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or by removal of the myenteric plexus. Thus, in this model of colitis, a "switch" occurs from muscarinic to nicotinic receptor-dominated control of cholinergic ion transport. The data indicate a novel pathway involving activation of nicotinic receptors on myenteric neurons, resulting in release of NO from neurons or enteric glia and, ultimately, a dampening of stimulated epithelial Cl- secretion that would reduce secretory diarrhea.     

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.     

Effect of somatostatin on electrogenic ion transport in the duodenum and colon of the mouse, Mus domesticus

In this study, we have used the mouse intestine and the Ussing short circuit technique to compare the effects and mechanism of action of somatostatin (SST, 0.1 microM) on cAMP- and Ca(2+)-mediated ion secretion in the duodenum and colon of the Swiss-Webster mouse. The cAMP-dependent secretagogues, prostaglandin E(2) (1 microM) and dibutyryl-cAMP (150 microM) increased short circuit current (I(sc)) in both regions, but only the colonic response was inhibited by SST. This inhibition was independent of enteric nerves, suggesting a direct action on the epithelial cells. The Ca(2+)-dependent secretagogue carbachol (10 microM) stimulated a transient increase in I(sc) in both intestinal segments. In the duodenum, SST partially inhibited this increase in I(sc) and both the responses to carbachol and SST were independent of enteric nerves. In the colon, while SST inhibited the carbachol induced increase in I(sc), pre-treatment with tetrodotoxin (750 nM) profoundly inhibited the carbachol induced increase in I(sc), thus markedly reducing the inhibitory effect of SST. This indicates an involvement of the enteric nervous system in the response to carbachol and the action of SST in the colon. These data indicate marked regional differences within the mouse intestine of the effects of SST on ion secretion and demonstrate different mechanisms of action of SST in the duodenum and colon.     

Cellular mechanism for potentiation of Ca2+-mediated Cl- secretion by the flavonoid baicalein in intestinal epithelia

Flavonoids belong to a large group of plant polyphenols that are consumed daily in large amounts. Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl(-) secretion across rat colonic mucosa. The current study examines the effect of baicalein on Cl(-) secretion in human colonic epithelial (T84) cells and its interaction with Ca(2+)- and cAMP-dependent secretagogues. We have employed a technique that allows concurrent monitoring of short-circuit current (I(SC)) and [Ca(2+)](i) in polarized epithelium. Basolateral application of baicalein induced a concentration-dependent increase in I(SC). The increase in I(SC) was because of Cl(-) secretion and was not accompanied by any discernible increase in [Ca(2+)](i). Baicalein acted synergistically with Ca(2+)- but not cAMP-dependent secretagogues. In the presence of baicalein, the carbachol and histamine induced increases in I(SC) that were markedly potentiated while increases in [Ca(2+)](i) were not significantly enhanced. Baicalein treatment uncoupled Cl(-) secretion from inhibitory effects normally generated by muscarinic activation. Baicalein treatment also resulted in increased cAMP content and activated PKA activity. Nystatin permeabilization studies revealed that baicalein stimulated an apical Cl(-) current but did not activate any basolateral K(+) current. These data suggest that baicalein potentiates Ca(2+)-mediated Cl(-) secretion through a signaling pathway involving cAMP and protein kinase A, most likely through the cystic fibrosis transmembrane conductance regulator in the apical membrane.     

Intestinal permeability and contractility in murine colitis.

We developed an in vitro organ bath method to measure permeability and contractility simultaneously in murine intestinal segments. To investigate whether permeability and contractility are correlated and influenced by mucosal damage owing to inflammation, BALB/c mice were exposed to a 10% dextran sulphate sodium (DSS) solution for 8 days to induce colitis. The effect of pharmacologically induced smooth muscle relaxation and contraction on permeability was tested in vitro. Regional permeability differences were observed in both control and 10% DSS-treated mice. Distal colon segments were less permeable to 3H-mannitol and 14C-PEG 400 molecules compared with proximal colon and ileum. Intestinal permeability in control vs. 10% DSS mice was not altered, although histologic inflammation score and IFN-gamma pro-inflammatory cytokine levels were significantly increased in proximal and distal colon. IL-1beta levels were enhanced in these proximal and distal segments, but not significantly different from controls. Any effect of pharmacologically induced contractility on intestinal permeability could not be observed. In conclusion, intestinal permeability and contractility are not correlated in this model of experimentally induced colitis in mice. Although simultaneous measurement in a physiological set-up is possible, this method has to be further validated.     

Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system

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). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 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 I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.     

Disturbances in epithelial ionic secretion in different experimental models of colitis

This paper studies the disturbances in ionic secretion in the colon of rats with different models of acute and chronic colitis measured as changes in short-circuit current. The aim was to verify whether the reported inhibition of basal and stimulated secretion in the trinitrobenzene sulfonic acid and mytomicin C models are applicable to experimental colitis as such. All models showed remarkable similarity in ion transport as determined in Ussing chambers, with downregulated basal as well as carbachol evoked secretion. The EC(50) of carbachol was unchanged in all cases. Iodoacetamide and oxazolone colitis models were notable exceptions in that the dose response curves for carbachol were unaltered compared to controls. The reason is unclear but seems to be unrelated to either interferon gamma or interleukin 4 levels or to the severity of the inflammatory response.     

Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion

Our goal was to examine the sidedness of effects of the purinergic agonist, uridine 5'-triphosphate (UTP), on Cl(-) secretion in intestinal epithelial cells. We hypothesized that UTP might exert both stimulatory and inhibitory effects. All studies were conducted with T84 intestinal epithelial cells. UTP induced Cl(-) secretion in a concentration-dependent fashion. Responses to serosally added UTP were smaller and more transient than those evoked by mucosal addition, but there was no evidence that mucosal responses involved cAMP-dependent mechanisms. Pretreatment with serosal UTP inhibited subsequent Ca(2+)-dependent Cl(-) secretion induced by carbachol or thapsigargin, or secretion induced by mucosal UTP, in a manner that was reversed by a tyrosine kinase inhibitor. The inhibitory effect of serosal UTP on Cl(-) secretion was not additive with that of carbachol, known to exert its inhibitory effects through the tyrosine kinase-dependent generation of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)]. Moreover, responses to both serosal and mucosal UTP were reduced by prior treatment of T84 cells with carbachol. Finally, serosal, but not mucosal, UTP evoked an increase in Ins(3,4,5,6)P(4). We conclude that different signaling mechanisms lie downstream of apical and basolateral UTP receptors in epithelial cells, at least in the intestine. These differences may be relevant to the use of UTP as a therapy in cystic fibrosis.