Rat Proximal Colon Research Articles

Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels.

1. The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. 2. Apamin (1-100 nM) produced a concentration-dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) and the joint application of apamin plus L-NAME had no additive effects. Apamin (0.1 microM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM - 10 microM) or 8-Br-cyclic GMP (1-100 microM). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (0.1-5 microM), inhibitor of NO-stimulated guanylate cyclase, produced a concentration-dependent increase of the spontaneous contractions. ODQ (1 microM) in the presence of apamin (0.1 microM) did not produce any further increase in the contraction amplitude, whereas after L-NAME (100 microM) it decreased the spontaneous contractions. ODQ (1 microM) reduced the SNP inhibitory effects. 4. Zaprinast (1-50 microM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration-dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 microM) or L-NAME (100 microM). 5. These results suggest that small conductance Ca2+-dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin-sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal.

The protein tyrosine kinase pathway is not involved in the regulation of K+ transport across the rat colon.

The protein tyrosine kinase inhibitor, genistein, is known to activate the cystic fibrosis transmembrane regulator (CFTR) Cl- channel and to inhibit K+ currents across the rat colonic epithelium. The aim of the present study is to answer the question whether these effects are involved in the regulation of transepithelial K+ transport. Therefore, the action of genistein on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake and efflux of Rb+ in mucosa-submucosa preparations. All effects of genistein (5 x 10(-5) mol L(-1)) were tested in the presence of a low concentration of forskolin (2 x 10(-7) mol L(-1)), because prestimulation of the cAMP pathway has been shown to be a prerequisite for a secretory action of genistein. Forskolin caused an increase in the serosa-to-mucosa flux of Rb+ (J(Rb)sm) thereby stimulating net K+ secretion in the proximal and distal colon. None of these effects was further enhanced after administration of genistein. Neither mucosal uptake of Rb+, representing mainly the activity of the H+-K+-ATPase in the distal colon, nor serosal Rb+ uptake, representing, e.g. the activity of the Na+-K+-2Cl- cotransporter, were affected by genistein. Also the efflux of Rb+ across the apical or the basolateral membrane, an indicator for the apical and basolateral K+ conductance, was unchanged in the presence of genistein. These results demonstrate that the K+ channels inhibited by genistein are not involved in transepithelial K+ transport.

Inhibition of spontaneous smooth muscle contractions in rat and rabbit intestine by blockers of the thromboxane A2 pathway.

The effect of inhibitors of the thromboxane A2 pathway on spontaneous contractions of intestinal smooth muscle preparations was studied. The thromboxane A2 antagonists Bay u3405, SK and F 88046 and KW-3635 concentration-dependently inhibited both the amplitude and the frequency of spontaneous contractions of the longitudinal muscle from the rat proximal colon. A concentration-dependent inhibition of the myogenic contractions was also observed with the thromboxane A2 synthase inhibitor U-51605, and with the combined cyclooxygenase/lipoxygenase inhibitor nordihydroguaiaretic acid, whereas indomethacin, a pure cyclooxygenase inhibitor, was ineffective. None of these inhibitors affected the contractile response evoked by the cholinergic agonist carbachol, excluding non-specific actions on intestinal motility. A similar response was observed for the rabbit jejunum, which, in contrast to the rat colon, exhibits more regular, high-frequency spontaneous contractions, which were inhibited by Bay u3405, SK and F 88046 and KW-3635 in a concentration-dependent manner, whereas the response to carbachol remained unaffected. These results suggest a role for thromboxane A2 in the generation and/or facilitation of spontaneous smooth muscle contractions in the gut.

Modulation by nitric oxide of spontaneous mechanical activity in rat proximal colon.

1. In order to examine the role of nitric oxide (NO) in the tonic neural inhibition in rat proximal colon, the effects of N(omega)-nitro-L-arginine methyl ester (L-NAME) were studied on the spontaneous contractions of circular muscle (monitored as intraluminal pressure changes) and of longitudinal muscle (detected as isometric tension changes). 2. L-NAME (3 x 10(-6)-3 x 10(-4) M) caused a concentration-dependent increase in the amplitude of circular contractions, without affecting those of longitudinal muscle. This effect was prevented by L-arginine (1-5 x 10(-3) M), but not D-arginine. 3. In the presence of tetrodotoxin (10(-6) M), which per se induced increase of the pressure waves, L-NAME (10(-4) M) caused no further effects on the amplitude of the spontaneous contractions. 4. The response to L-NAME (10(-4) M) was unaffected by atropine (10(-6) M), guanethidine (10(-6) M), hexamethonium (up to 3 x 10(-4) M) or alpha-chymotrypsin (up to 5 U ml(-1)). 5. NK2 receptor antagonists, SR 48968 (3 x 10(-6) M) or MEN 10627 (10(-6) M), produced a reduction of the amplitude of the pressure waves but failed to affect the contractile response to L-NAME (10(-4) M). 6. These findings suggest that tonic production of NO from inhibitory neurones influences the degree of contractions of circular muscle. An involvement of an inhibitory peptide as well as disinhibition of cholinergic or NK2-tachykinergic excitatory neurotransmission in the mechanism of NO action can be ruled out.

Colonic H-K-ATPase beta-subunit: identification in apical membranes and regulation by dietary K depletion.

P-type ATPases require both alpha- and beta-subunits for functional activity. Although an alpha-subunit for colonic apical membrane H-K-ATPase (HKcalpha) has been identified and studied, its beta-subunit has not been identified. We cloned putative beta-subunit rat colonic H-K-ATPase (HKcbeta) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat beta-subunits. Northern blot analysis demonstrates that this HKcbeta is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcbeta mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase beta1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcbeta mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcbeta fusion protein established expression of a 45-kDa HKcbeta protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcbeta protein expression only in apical membranes. Physical association between HKcbeta and HKcalpha proteins was demonstrated by Western blot analysis performed with HKcbeta antibody on immunoprecipitate of apical membranes of rat distal colon and HKcalpha antibody. Tissue-specific upregulation of this beta-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcalpha protein provide compelling evidence that HKcbeta is the putative beta-subunit of colonic H-K-ATPase.

Connexin43 Expression in Network-Forming Cells at the Submucosal-Muscular Border of Guinea Pig and Dog Colon

The tissue-specific expression of connexin subtypes in gap junctions between the interstitial cells and smooth muscle cells in the submuscular plexus of the colon has a functional importance in relation to intestinal pacemaker activity. Immunocytochemical observations of two types of connexin molecules, connexin43 and connexin45, were made with a confocal laser scanning microscope on cryosections of freshly frozen dog, guinea pig, mouse and rat proximal colon. Connexin43 immunoreactivity appeared as a series of dots along the submuscular plexus of guinea pig and dog. In contrast, connexin43 immunoreactivity was not found in mouse and rat colon. Connexin43 immunoreactivity was not observed in the colon muscular layer in the four animal species examined. In double-stained materials with a marker for either vimentin or smooth muscle actin, connexin43 immunoreactivity was colocalized with vimentin immunoreactivity, whereas it was not with either smooth muscle actin immunoreactivity or phalloidin reactivity. This indicated that the connexin43-expressing cells possess a vimentin-positive fibroblast-like nature rather than a smooth muscle-like one. In addition, in guinea pig colon, connexin43 immunoreactivity colocalized with c-Kit immunoreactivity. In conclusion, network-forming cells are connected by connexin43 gap junctions in the submuscular plexus of guinea pig and dog colon, most likely indicating that interstitial cells act as an intestinal pacemaker and conductive system.

Mechanisms of carbachol-induced alterations in K + transport across the rat colon

The effect of carbachol, an agonist of the Ca 2+ pathway, on K + transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake, and efflux of Rb +, a marker for K +, in mucosa–submucosa preparations. Unidirectional ion flux measurements revealed that carbachol stimulated K + secretion in the proximal colon by a marked increase in the serosa-to-mucosa flux ( J sm Rb) and a more moderate rise in the mucosa-to-serosa flux ( J ms Rb). In the distal colon carbachol had no effect on J ms Rb, but J sm Rb was reduced after a transient increase finally resulting in an inhibition of K + secretion. Carbachol caused a stimulation of mucosal Rb + uptake in the distal colon, which was diminished in the presence of inhibitors of the apical H +–K +-ATPase, vanadate and ouabain. In contrast, in the proximal colon the serosal Rb + uptake was enhanced by carbachol, an effect, which could be prevented by bumetanide, an inhibitor of the basolateral Na +–K +–2Cl −-cotransporter. Efflux experiments revealed that carbachol caused a transient increase of apical and basolateral Rb + permeability in both colonic segments. In the distal colon, stimulated K + efflux to the serosal side was reduced by quinine, efflux to the mucosal side was blocked by tetraethylammonium. In the proximal colon, carbachol-activated apical and basolateral K + efflux were inhibited by Ba 2+. In conclusion, these data suggest that in the distal colon carbachol stimulates the H +–K +-ATPase and the basolateral K + efflux through quinine-sensitive K + channels, whereas in the proximal colon carbachol induces K + secretion due to a stimulation of the basolateral Na +–K +–2Cl −-cotransporter and an increased efflux to the luminal side via Ba 2+-sensitive apical K + channels.

NPY receptor subtypes involved in the contraction of the proximal colon of the rat

Experiments were designed to determine the receptor subtype(s) involved in the contraction of the rat proximal colon to NPY. In this tissue, mRNA of Y 2 and Y 4 NPY receptor subtypes were highly expressed, whereas Y 5 mRNA levels were very low and Y 1 mRNA levels were intermediate. NPY analogues induced contractions with the following order of potency: rPP>hPP=PYY=NPY=[Leu 31, Pro 34]NPY>NPY (2–36)=[ d-Trp 32]NPY>NPY (13–36). Responses to NPY, PYY and NPY (13–36) were not or partially affected by tetrodotoxin, in contrast to the responses to [Leu 31,Pro 34]NPY, rPP, hPP and [ d-Trp 32]NPY which were fully blocked. Atropine did not inhibit the contractions to NPY, PYY and [Leu 31,Pro 34]NPY but significantly affected those to NPY (13–36), [ d-Trp 32]NPY, rPP and hPP. The specific Y 1 receptor antagonist BIBP 3226 was ineffective but JCF 104 and JCF 105 (two compounds with preferential affinity toward the hY 5 receptor versus the hY 1 or hY 2 receptor) abolished the contractions provoked by the NPY analogues. These results suggest that NPY activates three receptor subtypes, a Y 2 subtype possibly by a direct action on the smooth muscle cells, as well as a Y 4 and a Y 5 (or `Y 5-like') subtype which, respectively, release acetylcholine and an unknown neurotransmitter.

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 µM), guanethidine (1 µM), indomethacin (10 µM), and Nomega -nitro-L-arginine methyl ester (L-NAME) (100 µM). The NK1 receptor antagonist, SR 140,333 (up to 3 µM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 µM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [ betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.Key words: NK1 receptor, NK2 receptor, nonadrenergic, noncholinergic (NANC) excitatory junction potential, intestine.

Characterization of 5-HT Receptors in Rat Proximal Colon

1. In view of its multiple sites of action, we investigated the activity of 5-hydroxytryptamine (5-HT, serotonin) on various potential receptors in the isolated proximal colon of rats. 2. 5-HT induced concentration-dependent contractions of colonic strips (pEC 50=7.54±0.12). 3. The 5-HT 1 receptor agonist, 5-carboxamidotryptamine, induced concentration-dependent contractions (pEC 50=5.93±0.27); however, neither the 5-HT 3 receptor-agonist, phenylbiguanide, nor the 5-HT 4 receptor-agonist, renzapride, caused contractions at concentrations as high as 10 −4 M. 4. The 5-HT 1/2 receptor antagonist, methiothepin, caused concentraction-dependent nonsurmountable antagonism. The 5-HT 3 receptor antagonist, tropisetron, inhibited the contractions to a concentration of 5-HT⩾10 −6 M. Ketanserin had no effect on responses to 5-HT. 5. Tetrodotoxin and atropine had no effect on responses to 5-HT. 6. We conclude that contractions to 5-HT are mediated by 5-HT 1-like and probably 5-HT 3 receptors that activate tetrodotoxin insensitive mechanisms.

Dietary Maltitol Decreases the Incidence of 1,2-Dimethylhydrazine-Induced Cecum and Proximal Colon Tumors in Rats

Maltitol is fermented in the colon due to only partial hydrolysis in the small intestine. In the present study, we examined effects of dietary maltitol on dimethylhydrazine-induced intestinal tumor in rats. In experiment 1, rats were fed a fiber-free diet or diets supplemented with 1 or 5 g/100 g maltitol for 27 wk. Each group of rats was injected with dimethylhydrazine or vehicle alone for the first 14 wk of the experimental period. Maltitol supplementation at 1 g/100 g of the diet significantly reduced tumor incidence in the cecum and the 5% supplement reduced tumor incidence in both the cecum and proximal colon in dimethylhydrazine-treated rats. In experiment 2, we investigated the effect of the 1 g/100 g maltitol diet on the short chain fatty acid concentrations in cecal contents of placebo and dimethylhydrazine-treated rats. Intake of the 1 g/100 g maltitol diet doubled (P < 0.05) the concentration of butyrate but did not affect acetate or propionate in the cecal contents. These results suggest that dietary maltitol has a protective effect against dimethylhydrazine-induced tumors in rat cecum and proximal colon and that butyrate produced by bacterial fermentation of maltitol in the cecum may be involved in the protection.

Nonionic diffusion of short-chain fatty acids across rat colon.

Short-chain fatty acid (SCFA) transport across the colon may occur by nonionic diffusion and/or via apical membrane SCFA-/HCO3- exchange. To examine the relative importance of these processes, stripped segments of rat (Ratus ratus) proximal and distal colon were studied in Ussing chambers, and the unidirectional fluxes of radiolabeled SCFA butyrate, propionate, or weakly metabolized isobutyrate were measured. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or 1 or 5 mM HCO3- Ringer, decreases in mucosal pH stimulated mucosal-to-serosal flux (Jm-->s) of all SCFA, decreases in serosal pH stimulated serosal-to-mucosal flux (Js-->m), and bilateral pH decreases stimulated both fluxes equally. These effects were observed whether the SCFA was present on one or both sides of the tissue, in both proximal and distal colon, in the absence of luminal Na+, and in the presence of either luminal or serosal ouabain. Changes in intracellular pH or intracellular [HCO3-] did not account for the effects of extracellular pH. Luminal Cl- removal, to evaluate the role of apical membrane Cl-/SCFA- exchange, had no effect on Jm-->s but decreased Js-->m 32% at pH 6.5 and 22% at 7.2. Increasing SCFA concentration from 1 to 100 mM, at pH 6.4 or 7.4, caused a linear increase in Jm-->s. We conclude that SCFA are mainly transported across the rat colon by nonionic diffusion.

5-(N,N-Dimethyl)-Amiloride to Discriminate the Unidirectional Electrolyte Transports in Rat Small Intestine and Proximal Colon In Vivo

The effect of dimethyl-amiloride (DMA), a selective Na +/H + exchange blocker, was studied on electrolyte net fluxes and unidirectional fluxes of Na and Cl at four levels of rat intestine in vivo in basal conditions. DMA was applied intraluminally at concentrations of 10 −4 and 10 −3 M in the model of ligated loops prepared from duodenum, proximal jejunum, distal ileum and ascending colon in fasted Sprague Dawley rats. Two iso-osmotic test solutions were used: (1) hypo-ionic: Na + 80 mM and (2) iso-ionic: Na + 148 mM, pH 8.2. 22Na was placed in the loop and 36Cl was given by intravenous route at the beginning of the experiment. Na +/H + was calculated by two different means, one was based on pH variation following amiloride inhibition of Na influx, the other on the calculation of the passive Na transport. The quantitative evaluation shows that Na/H exchange largely contributes to the electroneutral absorption and luminal pH regulation. The exchanger activity decreases from duodenum, jejunum, ileum and colon where it is completed by K/H exchange to assure low colon luminal pH.

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon.

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 microM), guanethidine (1 microM), indomethacin (10 microM), and N(omega)-nitro-L-arginine methyl ester (L-NAME) (100 microM). The NK1 receptor antagonist, SR 140,333 (up to 3 microM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 microM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.

Endogenous CCK inhibits colonic contractions in unrestrained conscious rats

As cholecystokinin octapeptide (CCK-OP) and feeding have been reported to relax the circular muscle contractions of the proximal colon in unrestrained conscious rats under fasting conditions, the, action of cholecystokinin, released after duodenal infusion of a low residual diet of clinimeal, was studied on the motor activity of the proximal colon in unrestrained conscious rats. We used an implantable telemetry system with a miniature strain gauge force transducer introduced into the rat proximal colon. By using a specific radioimmunoassay system for CCK, plasma levels of CCK before and after duodenal infusion of clinimeal (0.5, 1.0, 2.0 ml) were determined at 0, 5, 10, 20, 30, 60 min in the portal blood. The clinimeal infusion caused a significant increase in CCK levels of the portal plasma during 5 and 30 min. This increase was in a dose-dependent manner. In accordance with this increase in plasma CCK, the motor activity of the proximal circular muscle was suppressed significantly. A bolus injection of the CCK A receptor antagonist, loxiglumide, CR 1505 (0.1, 0.5 and 1.0 mg/kg ip), prior to clinimeal blocked the inhibitory action of CCK on the motor activity in a concentration-dependent manner. These data suggest that endogenous CCK released by a residual diet is involved in the mechanism of inhibition of motor activity in the proximal colon.

Regulation of apical and basolateral K+ conductances in rat colon.

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K+ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K+ in the rat proximal and distal colon. 2. Administration of nystatin (100 micrograms ml-1 at the mucosal side), in the presence of Na+ and in the presence of a serosally directed K+ gradient, stimulate a large increase in short-circuit current (ISC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na(+)-K(+)-ATPase and of a current cross a quinine-sensitive basolateral K+ conductance. 3. The pump current, measured as Na(+)-dependent or scilliroside-sensitive current in the absence of a K+ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5 x 10(-6) mol 1(-1)). 4. The current across the basolateral K+ conductance, measured as current in the presence of a serosally directed K+ gradient either in the absence of Na+ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5 x 10(-5) mol 1(-1)), but inhibited by forskolin (5 x 10(-6) mol 1(-1)). 5. Basolateral K+ depolarization induced a negative ISC in both colonic segments, which was inhibited by the K+ channel blocker quinine (10(-3) mol 1(-1)) at the mucosal side), but was resistant to tetraethylammonium (5 x 10(-3) mol 1(-1) at the mucosal side). This K+ current across an apical K+ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl- conductance under these conditions. 6. These results demonstrate that an increase in intracellular Ca2+ concentration induced by carbachol causes an increase in the basolateral and the apical K+ conductance, thereby inducing K+ secretion in parallel with an indirect support of Cl- secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K+ conductance; a mechanism which might contribute to the inhibition of K+ absorption.

Inhibition of mechanical activity by neurotensin in rat proximal colon: involvement of nitric oxide.

The aim of the present study was to define the nature of inhibitory action of neurotensin in rat proximal colon. Mechanical activity was detected as changes of intraluminal pressure. Neurotensin (10(-10) to 10(-7) M), in the presence of atropine (10(-6) M), guanethidine (10(-6) M), and nifedipine (10(-8) M), induced a tetrodotoxin-insensitive inhibitory effect characterized by the complete disappearance of the spontaneous phasic contractions. The inhibitory effect of neurotensin (10(-7) M) was abolished by scorpion venom (Leiurus quinquestriatus hebraeus) (10(-6) g/ml) or high K+ (40 mM KCl), whereas it persisted in the presence of omega-conotoxin GVIA, (10(-7) M). N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M to 3 x 10(-4) M) antagonized the inhibitory response to neurotensin but did not affect the response to norepinephrine. L-Arginine (5 x 10(-3) M) prevented the effect of L-NAME. The contractile action of neurotensin, observed in the absence of atropine and nifedipine, was potentiated by L-NAME (10(-4) M); L-arginine (5 x 10(-3) M) prevented the L-NAME effect. The present results suggest that in rat proximal colon the inhibitory action of neurotensin is largely due to production of nitric oxide (NO), likely released from neural prejunctional sites. Ca2+ influx through N-type channels is not a required step in the mechanism of neurotensin inhibitory action.

Nitric oxide is involved in the inhibitory action of cholecystokinin octapeptide (CCK-OP) on proximal colonic motility

To determine whether nitric oxide (NO) is a possible mediator in the inhibitory action of CCK-octapeptide (CCK-OP) on circular muscle contractions of the rat proximal colon, contractile activities of the circular muscle were recorded in the proximal colon of unrestrained conscious rats in the fasting state using a miniature strain gauge force transducer and an implantable telemetry system. Regular and rhythmic phasic contractions were observed during the fasted condition, similar to the myoelectric migrating complex seen in intestinal contractions of the fasting dog. These phasic contractions were almost completely inhibited after intraperitoneal (i.p.) administration of CCK-OP at a dose of 15 μg/kg body weight. Nω-nitro-arginine, methyl ester ( l-NAME), at doses of 20 and 200 mg/kg i.p. administered prior to i.p. injection of CCK-OP, prevented the inhibitory action on the fasting phasic contractions. The degree of prevention was dose-dependent. 100 mg/kg body weight i.p. injection of l-arginine inhibited the circular muscle contractions. The same dose of d-arginine had no action on contractions of the circular muscle of the proximal colon in the fasted rat. From these data, we conclude that NO is one possible mediator in the inhibitory mechanism of CCK-OP on smooth muscle motor activity of the rat proximal colon in vivo.

Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine

BACKGROUND & AIMS: Escherichia coli heat-stable enterotoxins (STa) provoke electrogenic Cl- secretion in the intestine through a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent signal transduction pathway. The cGMP receptor involved in the activation of the Cl- channel is not known with certainty but may comprise either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (cAK) or cGMP-dependent protein kinase (cGK) type II. The aim of this study was to discriminate between these possibilities using specific kinase inhibitors.METHODS: Intestinal electrogenic Cl- secretion was determined by measuring short-circuit current (Isc) in a Ussing chamber.RESULTS: The general protein kinase inhibitors staurosporine and H-8 inhibited rat cGK II activity in vitro with 50% inhibitory concentration values of 4 nmol/L and 3 mumol/L, respectively, which are lower than those reported for cAK. Both staurosporine and H-8, when added to rat proximal colon at concentrations that did not affect the Isc response to 8-bromo-cAMPS, inhibited the STa- and 8-bromo-cGMP- provoked Isc response for more than 80%. Furthermore, the relative specific cGK inhibitor Rp isomer of 8-(chlorophenylthio)-cGMP, but not the cAK inhibitor RP isomer of (Rp) 8-bromo-cAMPS, inhibited the Isc response to submaximal levels of STa in rat proximal colon.CONCLUSIONS: These data provide further evidence for an important role of cGK II in STa-mediated Cl- secretion in native rat intestinal epithelium.(Gastroenterology 1997 Feb;112(2):437-43)

Mode and mechanism of neurotensin action in rat proximal colon

This study examined the mechanism of action of neurotensin on intraluminal pressure in rat proximal colon. The direct and indirect contractile response to neurotensin (100 nM) was abolished in Ca 2+-free solution, and was antagonized by nifedipine (1–5–10 nM) and potentiated by Bay K 8644 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate) (10–100–1000 nM). Neurotensin, in the presence of nifedipine (10 nM) and atropine (1 μM), induced a tetrodotoxin-insensitive inhibitory effect, which was antagonized by SR 48692 (2[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy-phenyl)pyrazol-3-yl) carbonyl amino]tricyclo (3.3.1.1. 3.7) decan-2-carboxylic acid) (300 nM) or apamin (0.1 μM). The results demonstrate that the neurotensin response is dependent on the influx of Ca 2+ via L-type channels and results from summation of excitatory and inhibitory effects.