Mechanism of a nitric oxide donor NOR 1-induced relaxation in longitudinal muscle of rat proximal colon

We previously suggested that nitric oxide (NO)-mediated relaxation of the rat proximal colon is not associated with change in cyclic GMP content. We further studied the intracellular mechanism of NO-induced relaxation by measuring changes in tension and intracellular Ca2+ concentration ([Ca2+]i), simultaneously. NOR 1, NO donor, relaxed the longitudinal muscle of the rat proximal colon, which was precontracted by carbachol, with a concomitant decrease in [Ca2+]. ODQ, an inhibitor of soluble guanylate cyclase, partially inhibited the relaxant effect of only higher concentrations of NOR 1, but Rp-8-Br-cGMPS, an inhibitor of cyclic GMP-dependent protein kinase (PKG), did not have any effects on the relaxant effect of NOR 1. When the preparations were transferred to normal solution after the treatment with thapsigargin, an inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, in the absence of Ca2+, contraction with a concomitant increase in [Ca2+]i occurred. NOR 1 did not show significant effects on the tension and [Ca2+]i in thapsigargin-treated preparations. In high K+-precontracted preparations, NOR 1 relaxed the preparations with a slight change in [Ca2+]i. The relaxant effect was significantly inhibited by ODQ and Rp-8-Br-cGMPS. These results suggest that NO induces the relaxation preferentially by acting thapsigargin-sensitive function of SR and in turn decreasing [Ca2+]i, although a cyclic GMP-PKG pathway is suggested under the experimental conditions of a high K+ concentration.     

Characterization of muscarinic receptor-mediated cationic currents in longitudinal smooth muscle cells of mouse small intestine

In mouse intestinal smooth muscle cells held at -50 mV, carbachol evoked an atropine-sensitive inward current in the intracellular presence of Cs(+). The current response consisted of an initial peak followed by a smaller plateau component on which oscillatory currents frequently arose. Results from various experimental procedures indicated that the inward current is a muscarinic receptor-operated cationic current (mI(cat)) sensitive to cytosolic Ca(2+) concentration ([Ca(2+)](i)) and that the initial peak and oscillatory components are contaminated by Ca(2+)-activated Cl(-) currents. Under conditions of [Ca(2+)](i) buffered to 100 nM, the mI(cat) response to cumulative carbachol applications was inhibited competitively by an M(2)-selective antagonist but non-competitively by an M(3)-selective one. Also it was severely reduced by pertussis toxin (PTX) treatment or a phospholipase C (PLC) inhibitor. Comparative analysis of mI(cat) in mouse and guinea-pig intestinal myocytes indicated that the underlying channels resemble between those myocytes in agonist sensitivity, current-voltage relationship, and unitary conductance. The results suggest that in mouse intestinal myocytes, mI(cat) arises mainly via an M(2)/M(3) synergistic mechanism involving PTX-sensitive G-proteins and PLC activity in the absence of current modulation by [Ca(2+)](i) changes, as described for guinea-pig ileal mI(cat). The channels underlying mI(cat) are also indistinguishable in gating properties between both types of myocytes.     

A method for measurement of muscarinic receptor-mediated responses in dissociated single colon longitudinal smooth muscle cells.

We previously reported a simple method of acutely preparing dissociated smooth muscle cells from urinary bladder tissue, but the feasibility of this method has not been well ascertained. In the present study, we assessed whether this method is applicable for measuring muscarinic receptor function in intestinal smooth muscle cells. Single smooth muscle cells were prepared from the longitudinal muscle tissue of guinea pig colon by the enzymatic dissociation with papain and hyaluronidase, followed by collagenase digestion. Muscarinic responses in the isolated smooth muscle cells were measured by intracellular Ca(2+) mobilization and extracellular acidification through Fura-2 fluorometry and Cytosensor microphysiometry, respectively. A single, viable population of colon longitudinal smooth muscle cells (approximately 6 x 10(6) cells/animal) was obtained. In these cells, carbachol (muscarinic agonist) induced Ca(2+) mobilization and extracellular acidification over the concentration range similar to that previously reported to produce contraction of the intact colon muscle strips. Atropine (nonselective muscarinic antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, M(3)-selective antagonist) inhibited the Ca(2+) mobilization with potencies approximately 3 log units greater than that for methoctramine (M(2)-selective antagonist). For extracellular acidification, the potency differences between these antagonists was approximately 2 log units. In addition, the carbachol-induced extracellular acidification was inhibited by 5-[N-ethyl-N-isopropyl]-amiloride, a selective inhibitor of the Na(+)/H(+) exchanger. These findings indicate that in isolated colonic smooth muscle cells, M(3) receptors are predominantly involved in Ca(2+) mobilization, while a mixed population of M(2) and M(3) receptors seems to contribute to extracellular acidification. Our results further suggest the role of the Na(+)/H(+) exchanger in muscarinic-mediated extracellular acidification. Consequently, our method produces viable isolated colonic smooth muscle cells that display physiologically appropriate responses to muscarinic receptor activation, and the method may be applicable for several types of nonvascular smooth muscle tissues.     

Origin of Ca2+ necessary for carbachol-induced contraction in longitudinal muscle of the proximal colon of rats

The origin of Ca2+ necessary for carbachol (CCh)-induced contraction of longitudinal muscle of the proximal colon of rats was studied. CCh induced contraction of the muscle consisting of two phases, phasic and tonic phases, with a concomitant biphasic increase in [Ca2+]i. After removal of Ca2+ from the bathing solution of the colonic segments, CCh-induced contraction was rapidly inhibited; there was almost complete inhibition 1 min after the removal. Nicardipine, a blocker of voltage-dependent calcium channel, also significantly inhibited CCh-induced contraction. On the other hand, treatment of the colonic segments with thapsigargin, an inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, did not significantly affect the contraction except causing a slight decrease in the rate of contraction. These results suggest that Ca> entering through voltage-dependent calcium channels, but not released from SR, is essential for CCh-induced contraction of longitudinal muscle of the proximal colon of rats. This strict dependency of the CCh-induced contraction on extracellular Ca2+ was discussed in relation to the results obtained in the fundus of rats.     

Desensitization of beta-adrenoceptor- and prostaglandin E1 receptor-mediated human vascular smooth muscle relaxation.

Regulation of beta-adrenoceptors in animal tissues and human cell cultures has been extensively described; on the other hand, relatively little is known about regulation of beta-adrenoceptors in human tissues in vivo. Both beta-adrenoceptors and the prostaglandin E1 (PGE1) receptors stimulate vasodilation. We wondered if prolonged infusion of isoproterenol or PGE1 would cause desensitization of smooth muscle relaxation and used the dorsal hand-vein compliance technique to investigate this question. After constructing a dose-response curve to either the beta-agonist isoproterenol or to PGE1 in a phenylephrine preconstricted vein, isoproterenol (271 ng/min), PGE1 (956 pg/min), or saline was infused for 4 h in separate experiments. There was no change in the ED50 or Emax for either isoproterenol or PGE1 after saline infusion. After a 4-h infusion of isoproterenol, the maximal vasodilator response to isoproterenol was significantly (p less than 0.01) attenuated from 61 +/- 33% to 19 +/- 10%, while the ED50 significantly increased (p less than 0.01) from a geometric mean of 37 to 197 ng/min. After infusion of isoproterenol, the mean maximum PGE1-induced venorelaxation of 129 +/- 29% was modestly but significantly (p less than 0.05) blunted to 96 +/- 35%, while the ED50 of PGE1 increased significantly (p less than 0.01) from a geometric mean of 81 to 398 pg/min. A 4-h infusion of PGE1 significantly (p less than 0.01) attenuated the maximum response to PGE1 from 73 +/- 35 to 28 +/- 16%. The maximal vasodilatory response to isoproterenol was also significantly blunted (p less than 0.05) from 62 +/- 35 to 42 +/- 41%, with no change in ED50.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of 5-HT receptors mediating contraction and relaxation of the longitudinal muscle of guinea-pig distal colon in vitro

A range of agonists and antagonists were used to characterize the receptors through which 5-hydroxytryptamine (5-HT) contracts and relaxes the longitudinal muscle of segments of guinea-pig distal colon, in vitro. 5-HT contracted the longitudinal muscle over the concentration range 10^–9 to 10^–4 mol/l. The 5-HT3 receptor agonist, 2-methyl-5-HT, produced concentration dependent contractions over the range 10^–6 to 10^–4 mol/l. 5-methoxytryptamine, an agonist at 5-HT4 receptors, caused contractions over a concentration range of 10^–8 to 10^–4 mol/l. The 5-HT4 antagonist, SDZ 205-557 (5 × 10^–7 mol/l) substantially suppressed the responses to low concentrations of 5-HT and to 5-methoxytryptamine, but had no effect on the responses to higher concentrations of 5-HT. In contrast, the 5-HT3 antagonist, granisetron (10^–6 mol/l), blocked the effect of 2-methyl-5-HT and substantially depressed responses to high concentrations of 5-HT, but had no effect on lower concentrations of 5-HT. Granisetron produced a small reduction in the response to 5-methoxytryptamine. Tetrodotoxin (TTX) (3 × 10^–7 mol/l) almost abolished the response to 5-methoxytryptamine and markedly suppressed the response to 2-methyl-5-HT, but the responses to 5-HT were only partially reduced. The 5-HT, antagonist, methiothepin 10^–6 mol/l. depressed the response to 5-HT 10^–7 to 10^–4 mol/l. and blocked its TTX insensitive component. The 5-HT₂ antagonist, ketanserin, in concentrations up to 10^–5 mol/l, had no effect on the contractions evoked by 5-HT.The response to 5-HT was substantially depressed by hyoscine (3 × 10^–6 mol/l. The tachykinin antagonist, spantide 10^–5 mol/l. depressed the response to 5-HT but to a lesser extent than hyoscine. Spantide and hyoscine combined completely blocked the contractile responses to 5-HT Responses to 2-methyl-5-HT were partially suppressed by hyoscine (3 x 10^–6 mol/l. and spantide (10^–5 mol/l) and completely blocked when both byoscine and spantide were present. Contractions evoked by 5-methoxytryptamine were partially blocked by hyoscine (3 × 10^–6 mol/l) and were unaffected by spantide (10^–5 mol/l), but a combination of hyoscine and spantide completely blocked such responses.When the excitatory transmission was blocked with hyoscine (3 × 10^–6 mol/l) and spantide 10^–5 mol/l) and the tone of the muscle raised, an inhibitory response to 5-HT was revealed that had a threshold concentration between 10^–7 mol/l) and 3 × 10^–7 mol/l, and a maximum effect at 10^–4 mol/l. It was blocked by TTX (3 × 10^–7 mol/l) and granisetron 10^–6 mol/l. while N-nitro-l-arginine (NOLA) (10^–4 mol/l) and SDZ 205-557 (5 × 10^–7 mol/l) had no effect. Apamin A 10^–6 mol/l. partially suppressed this response.It is concluded that 5-HT3, 5-HT4 and 5-HT₁-like receptors mediate contraction of the longitudinal muscle of the distal colon. The 5-HT3 and 5-HT4 receptors are located on the excitatory motor neurons innervating the longitudinal muscle and the 5-HT₁-like receptor is located on the muscle. 5-HT3 receptors are also found on inhibitory neurons to the muscle. Correspondence to: D. J. Woollard at the above address     

ATP通过P2受体调节大鼠近端结肠纵行肌的舒张与收缩反应(英文)

目的腺苷三磷酸(ATP)对大鼠离体远端结肠纵行肌运动的影响已明确,对近端结肠纵行肌的影响可能不同,但未有报告,为此对此进行观察并探讨其受体机制。方法观察静息张力时或预收缩时0.1μmol·L-1～1mmol·L-1ATP和1～100μmol·L-1腺苷对大鼠近端结肠纵行肌的抑制和兴奋作用。结果在静息张力下,1μmol·L-1～1mmol·L-1ATP对大鼠近端结肠纵行肌产生3种效应,即抑制自发性收缩反应,一过性轻度降低基础张力(0.05～0.08g),随后产生浓度依赖性收缩反应(0.04～0.44g)。0.1μmol·L-1河豚毒素不影响ATP的上述作用。在静息张力下,1～100μmol·L-1腺苷对近端结肠纵行肌未产生明显的收缩反应。应用5羟色胺(5HT)或乙酰胆碱(ACh)预收缩标本时,1μmol·L-1～1mmol·L-1ATP产生明显的浓度依赖性舒张反应(23.2%～94.6%,5HT预收缩;24.8%～92.4%,ACh预收缩),而腺苷引起的舒张反应明显小于ATP。结论在大鼠离体近端结肠纵行肌,ATP主要通过嘌呤嘧啶(P)2受体介导收缩反应,部分通过P1受体介导舒张反应。     

Essential role of the interstitial cells of Cajal in nitric oxide-mediated relaxation of longitudinal muscle of the mouse ileum

The role of interstitial cells of Cajal (ICC) in electrical field stimulation (EFS)-induced neurogenic responses in ileum was studied by using the ICC-deficient mutant (SLC-W/W(V)) mouse and its wild type. In the immunohistochemical study with anti-c-Kit antibody, ICC was observed in the myenteric plexus (MY) and deep muscular plexus (DMP) region in the wild type. In the mutant, ICC-MY were lost, only ICC-DMP were present. EFS induced a rapid contraction of the ileal segments from the wild type mouse in the direction of longitudinal muscle. In the mutant mouse, onset of contraction was delayed and its rate was slowed. EFS induced nonadrenergic, noncholinergic (NANC) relaxation in the presence of atropine and guanethidine in the wild type. A nitric oxide synthase inhibitor inhibited the relaxation and L-arginine reversed it. In the mutant, EFS did not induce NANC relaxation. There was no difference between the responsiveness of the segments from wild type and mutant mice to exogenously added acetylcholine or Nor-1. Taking into account the selective loss of ICC-MY in the mutant mice, it seems likely that ICC-MY have an essential role in inducing nitric oxide-mediated relaxation of longitudinal muscle of the mouse ileum and that ICC-MY partly participate in EFS-induced contraction.     

Investigation of the 5-HT receptor mediating relaxation in guinea-pig proximal colon.

5-Hydroxytryptamine (5-HT) induced concentration-related relaxations (EC50 = 9.1 microM) of guinea-pig proximal colon pretreated with ketanserin (1 microM) and ondansetron (10 microM). This 5-HT-induced effect was neuronally mediated since it was blocked by tetrodotoxin (0.3 microM). 5-Carboxamidotryptamine was a full agonist and ten times more potent than 5-HT. alpha-Methyl-5-HT was a partial agonist. 2-Methyl-5-HT was without effect. Methysergide and metergoline were antagonists of 5-HT producing parallel shifts at 0.1 microM but unsurmountable antagonism at higher concentrations. pKB values of 8.0 and 7.3 were calculated for methysergide and metergoline, respectively. This study has identified a 5-HT-induced relaxation of guinea-pig proximal colon which is mediated via a neuronal 5-HT1-like receptor. However, the subtype has yet to be established.     

Sodium nitroprusside regulates the relaxation of the longitudinal muscle in the gut

Nitric oxide (NO) has been shown to mediate nonadrenergic-noncholinergic relaxation in gastrointestinal (GI) smooth muscle cells. As GI smooth muscles relaxations are partly dependent on NO, we decided to investigate the effect of sodium nitroprusside (SNP) on the longitudinal muscle contraction of the isolated guinea pig ileum. Increasing concentrations of SNP (10(-10)M, 10(-9)M, 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) reduced ileum contractions stimulated by electrical stimulation (ES) (8-76%; p < 0.05) and by acetylcholine (Ach) (23-62%; p < 0.05) significantly and in a concentration-dependent manner. Furthermore, treatment with an inhibitor of the soluble guanylate cyclase, methylene blue (10 mM), antagonized significantly the relaxing effect of SNP (0-39%; p < 0.05, p < 0.01, p < 0.001 for ES- and 4-27%; p < 0.05 for Ach-induced contractions). The results show that treatment with 1 microM manganese-containing superoxide dismutase (MnSOD) and 10 microM L-arginine (L-arg) caused a significant decrease in SNP induced relaxations (6-55%; p < 0.05, p < 0.001 and 2-46%; p < 0.05, p < 0.01 for ES- and 15-28%; p < 0.05, p < 0.01, p < 0.001 and 12-32%; p < 0.05, p < 0.01 for Ach-induced contractions, respectively). In conclusion, our data suggest that SNP, which releases NO, is able to depress longitudinal muscle contraction of the isolated guinea pig ileum, suggesting that exogenous application of NO inhibits intestinal contractions of smooth muscle cells and that cGMP mediates the response to NO. In addition, MnSOD and L-arg decreased the relaxing effect of SNP on the isolated ileum of the guinea pig.     

Inhibition of the NANC relaxation of the guinea-pig proximal colon longitudinal muscle by the purinoceptor antagonist PPADS, inhibition of nitric oxide synthase, but not by a PACAP/VIP antagonist.

The effects of the P(2)-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl- 2('),4(')- disulphonic acid (PPADS), the nitric oxide (NO) synthase inhibitor N(G)-nitro- l -arginine (l -NOARG), the K(+)-channel blocker apamin, the pituitary adenylate cyclase activating peptide (PACAP) antagonist PACAP(6-38) and the sensory neuron-blocking drug capsaicin were examined on the non-adrenergic, non-cholinergic (NANC) relaxation evoked by electrical field stimulation in the longitudinal muscle of the guinea-pig proximal colon. Both PPADS (50 microm) and l -NOARG (100 microm) significantly inhibited the NANC relaxation. In the presence of l -NOARG, PPADS inhibited and apamin (100 nm) practically abolished the response. Capsaicin slightly but significantly enhanced the NANC relaxation at 10, but not at 1 Hz stimulation frequency. PACAP(6-38) (3 microm) had no effect on the NANC relaxation, although it abolished the relaxant effect of exogenous PACAP(1-27) (10 nm) and reduced that of exogenous vasoactive intestinal polypeptide (VIP, 30-100 nm) by about 60 %. PPADS (50 microm) inhibited the relaxant action of exogenous adenosine 5(')-triphosphate (ATP; 1 and 10 microm), the inhibition being stronger at 1 microm ATP. These data indicate that an exogenous P(2)-purinoceptor stimulant (possibly ATP) and NO are involved in the NANC relaxation of the guinea-pig colon. The 'non-nitrergic' apamin-sensitive component of the response might also include an unidentified transmitter. No evidence has been found for a mediating role of PACAP/VIP-like peptides.     

Beta 1-adrenoceptors mediate smooth muscle relaxation in mouse isolated trachea.

1. The relaxant effects to the beta-adrenoceptor agonists isoprenaline, adrenaline, noradrenaline, RO363, procaterol and fenoterol were investigated in carbachol-contracted mouse isolated tracheal preparations. 2. The order of potencies for those beta-adrenoceptor agonists that induced full relaxation of carbachol-contracted mouse tracheal preparations was isoprenaline greater than RO363 greater than noradrenaline = adrenaline greater than fenoterol. The EC50 value of isoprenaline for relaxation was 46 nM. The beta 1-adrenoceptor-selective agonist, RO363 was ten times more potent than the beta 2-adrenoceptor-selective agonist, fenoterol. The highly beta 2-adrenoceptor-selective agonist procaterol was a partial relaxant and induced only 28 +/- 4% relaxation. 3. Relaxations induced by noradrenaline and isoprenaline were not significantly affected by the neuronal uptake inhibitor, cocaine (10 microM) or by the extraneuronal uptake inhibitor, deoxycorticosterone acetate (25 microM) respectively. The alpha-adrenoceptor agonist methoxamine induced no observable elevation of mouse tracheal smooth muscle tone. 4. Schild plots for the beta-adrenoceptor antagonists, atenolol and betaxolol (beta 1-adrenoceptor-selective) and ICI 118,551 (beta 2-adrenoceptor-selective) were linear, with slope values approaching unity. Mean pA2 values derived for atenolol, betaxolol and ICI 118,551 for antagonism of beta-adrenoceptor-mediated relaxation were 7.1, 8.4 and 7.2, respectively. These data were independent of the use of isoprenaline or noradrenaline as the agonist. 5. These findings indicate that beta-adrenoceptor-mediated relaxations of mouse isolated trachea occur predominantly through activation of beta 1-adrenoceptors.     

Effects of purines on the longitudinal muscle of the rat colon.

1. Adenosine and adenosine 5'-triphosphate (ATP) have been reported to cause relaxation of the rat colon longitudinal muscle preparation; the purinoceptors mediating this effect were investigated by use of a series of agonists and antagonists. 2. The tissue was precontracted with carbachol (1 microM), and the purines induced reversible relaxations with a potency order of 5'-N-ethylcarboxamidoadenosine (NECA) greater than N6-cyclopentyladenosine (CPA) = adenosine 5'-(alpha, beta-methylene) triphosphonate (AMPCPP) greater than adenosine = adenylyl 5'-(beta, gamma-methylene) disphosphonate (AMPPCP) = ATP. The P1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (3 microM) shifted to the right the log concentration-response curves of all these agonists except for AMPCPP, indicating that they all act via P1-purinoceptors. The order of potency of the adenosine analogues and the relatively high concentrations of the antagonist required indicated that these receptors are of the A2 subtype. The P2-selective antagonist suramin (300 microM) inhibited responses to AMPCPP, but not to the other agonists. 3. The dephosphorylation of the nucleotides was studied by high performance liquid chromatography following incubation with the longitudinal muscle preparation for up to 30 min. ATP was rapidly degraded, largely to adenosine, and AMPPCP and AMPCPP were also degraded, although more slowly, to adenosine and adenosine 5'-(alpha, beta-methylene) diphosphonate (AMPCP) respectively. AMPCP, like AMPCPP, caused relaxations by acting on P2-purinoceptors, as it was also inhibited by suramin (300 microM). Incubation of the tissue with adenosine deaminase abolished responses to adenosine, reduced those to ATP and AMPPCP, but had no effect on those to AMPCPP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that ATP or a related purine is an excitatory neurotransmitter in the longitudinal muscle of mouse distal colon

Background and purpose:     

Involvement of cyclic AMP - PKA pathway in VIP-induced, charybdotoxin-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats

The intracellular mechanism of vasoactive intestinal peptide (VIP)-induced, charybdotoxin (ChTx)-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats was studied. A single pulse or 100 pulses at 10 Hz of electrical field stimulation (EFS) induced rapid transient relaxation or that with a subsequent contraction of the longitudinal muscle in the presence of atropine and guanethidine, respectively. Rp-8 bromo cAMPS, an inhibitor of cyclic AMP dependent protein kinase (PKA), at 30 microM inhibited the relaxations induced by EFS with a single or 100 pulses maximally by about 80 or 60%, respectively. It also inhibited VIP (300 nM)-induced relaxation by 82%. VIP (100 nM - 1 microM) increased the cyclic AMP content of longitudinal muscle myenteric plexus preparations obtained from the distal colon. ChTx at 100 nM almost completely inhibited 8 bromo cyclic AMP-induced relaxation of the distal segments. EFS with two or three pulses at 10 Hz induced inhibitory junction potentials consisting of two phases, rapid and subsequent slow hyperpolarization in the membrane potential of longitudinal smooth muscle cells. Rp-cAMPS, another inhibitor of PKA, inhibited the delayed slow hyperpolarization. It also inhibited the exogenously added VIP-induced hyperpolarization of the cell membrane. Thus, the present study suggests that activation of PKA via activation of VIP receptors is associated with activation of ChTx-sensitive K(+) channels in relaxation of longitudinal muscle of the distal colon of Wistar-ST rats. British Journal of Pharmacology (2000) 129, 140 - 146     

Evidence against VIP as the inhibitory transmitter in non-adrenergic, non-cholinergic nerves supplying the longitudinal muscle of the mouse colon.

Vasoactive intestinal peptide (VIP) had two types of effects on the longitudinal muscle of the mouse distal colon. At low concentrations (10(-8) M) VIP induced a contraction which seemed to be related to the production of prostaglandins as it was abolished after preincubation with indomethacin (10(-6) M). At higher concentrations (3 X 10(-8) and 10(-7) M) VIP induced relaxations which developed slowly and were related to stimulation of the adenylate cyclase activity of the smooth muscle cells. There is no evidence that VIP is the non-adrenergic, non-cholinergic transmitter released by electrical stimulation in this preparation and responsible for rapid relaxation of the smooth muscle.     

Nociceptin inhibits tonic nitric oxide release in the mouse isolated proximal colon

Nociceptin/orphanin FQ, the endogenous ligand of the opioid receptor-like (ORL1) receptor, caused contractions in the isolated colon of the mouse. Tetrodotoxin and the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine also produced contractions which were quantitatively similar to those seen in response to nociceptin. In the presence of either tetrodotoxin or Nomega-nitro-L-arginine, nociceptin was unable to cause a further contraction, whereas the muscarinic receptor agonist carbachol elicited a contractile response. Nociceptin had no contractile activity in colonic preparations contracted by Nomega-nitro-L-arginine then relaxed by addition of the NO donor sodium nitroprusside. These data suggest that nociceptin causes contractions of the mouse proximal colon by inhibiting the tonic, neuronal release of NO.     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.