Effect of acetic acid or trypsin application on rat colonic motility in vitro and modulation by two synthetic fragments of chromogranin A

The hypothesis that Chromogranin A (CgA)-derived peptides are involved in mechanisms modulating altered colonic motility was tested. Rat distal colonic strips were studied using an organ bath technique. Acetic acid (AA)-induced effects were characterized on spontaneous mechanical activities (SMA) in the presence of CgA4-16 or CgA47-66. In preparations with mucosa, AA induced a transient hyperactivity followed by a decrease in tone. The first phase is sensitive to tetrodotoxin (TTX) and capsaicin. The second phase was sensitive to BAYK8644 but insensitive to L-nitro-arginine-methyl-ester (L-Name)/apamin together. CgA4-16 or CgA47-66 alone produced no change on SMA. The administration of CgA4-16 prior to AA increased the duration of the excitatory component and reduced tone inhibition. CgA47-66 prior to AA only decreased duration of the excitatory phase. In preparations without mucosa, AA decreased tone. This effect was sensitive to BAYK8644 and CgA4-16. Trypsin decreased basal tone. This effect was suppressed by TTX, BAYK8644 or L-Name/apamin and were reduced by CgA4-16. AA-induced effects on rat colonic motility in vitro may be mediated through activation of primary afferents and an action at L-Type calcium channels. CgA-derived peptides are shown to decrease AA-induced effects on motility.     

Effects of GABA on circular smooth muscle spontaneous activities of rat distal colon

GABAergic regulation of intestinal motility through the modulation of non-adrenergic non-cholinergic (NANC) neurons remains poorly understood especially in rat colon where very few studies have been undertaken. Therefore, the effects of GABA on circular preparations of rat distal colon were investigated using classical organ bath chambers to record spontaneous mechanical activities (SMA). SMA was characterized by the occurrence of rhythmic phasic contractions (type-I) or by spontaneously occurring large contractions superimposed on small rhythmic contractions (type-II). In the presence of atropine and guanethidine (NANC conditions), these large contractions were inhibited by bicuculline, a GABA(A)-receptor antagonist as well as by TTX, L-NAME and apamin together, or L 732-138, a NK1-receptor antagonist. In NANC conditions, GABA induced a transient monophasic relaxation or a biphasic effect characterized by a relaxation followed by a tonic contraction in both type-I and -II preparations. Both the inhibitory and excitatory effects of GABA were blocked by TTX and L-NAME + apamin; the GABA-induced contraction was also sensitive to L 732-138. The responses to GABA were mimicked by the GABA(A)-receptor agonist, muscimol, whereas baclofen and CACA, respectively GABA(B) and GABA(C)-receptors agonists showed no effect. These results demonstrated that only GABA(A)-receptors seem to be involved in the regulation of SMA in rat distal colon in NANC conditions. Release of NANC inhibitory transmitter (NO and probably ATP) and NANC excitatory transmitter (maybe substance P) might be involved.     

The effect of a chromogranin A-derived peptide (CgA4-16) in the writhing nociceptive response induced by acetic acid in rats

The nociceptive effects of i.p administration of a synthetic peptide (CgA4-16) derived from chromogranin A (CgA) were studied on a model of inflammatory (somato-visceral) pain. Inflammatory mediators participate in controlling the activity of enterochromaffin cells that store and release chromogranins. Adult male Wistar rats were injected i.p with diluted acetic acid (AA) to induce abdominal writhes. Pharmacological agents were injected prior to CgA4-16 and/or AA together. While i.p CgA4-16 alone did not produce any effect, the peptide increased the number of abdominal constrictions induced by i.p AA administration in a dose-related manner. To determine the possible mechanisms involved in CgA4-16 produced pronociceptive effect, i.p diltiazem or indomethacin were tested. The pronociceptive effect induced by CgA4-16 was blocked by pretreatment of either substance. I.p administration of CGRP, substance P (SP) or capsaicin evoked dose-related abdominal writhing. CgA4-16, 20 min prior to CGRP or capsaicin, potentiated the nociceptive effects induced by CGRP or capsaicin, but not those induced by SP. Taken together, these data suggest for the first time that a CgA-derived peptide may modulate inflammatory pain.     

Time course of neural and contractile disturbances in a rat model of colitis induced by Trichinella spiralis

Colitis induced by Trichinella spiralis in rat induces alterations in the spontaneous motor pattern displayed by circular colonic muscle [Auli, M., Fernandez, E., 2005. Characterization of functional and morphological changes in a rat model of colitis induced by T. spiralis. Digestive Diseases and Sciences 50(8), 1432-1443]. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 microM), N-nitro-l-arginine (L-NOARG, 1 mM) and apamin (1 microM) were determined in vitro in the organ bath with circular muscle strips from sham-infected and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission.     

Inhibition of small conductance K+ -channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.     

Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), the neuronal blocker tetrodotoxin (TTX, 10(-6) M), or both on spontaneous contractions and on the production of basal NO. Both L-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by L-NAME and was inhibited by 38% by TTX. When tested together, L-NAME in the presence of TTX or TTX in the presence of L-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.     

Effects of a chromogranin-derived peptide (CgA 47-66) in the writhing nociceptive response induced by acetic acid in rats

Chromogranin A (CgA) is an acidic protein identified within a large variety of endocrine cells. Colocalized with catecholamines in chromaffin cells, CgA is a prohormone precursor of small biologically active peptides. Vasostatin (CgA 1-76) is the most conserved fragment of CgA and chromogranin A 47-66 peptide (CgA 47-66) possesses potent antimicrobial activities. The aim of this study was to test the hypothesis that CgA 47-66 may be involved in mechanisms modulating nociception. Thus, we used acetic acid (AA) which produces a delayed inflammatory response and episodes of abdominal writhing, a marker of pain, when injected intraperitoneally (i.p.) to rats. Administration (i.p.) of CgA 47-66 induced specific opposite dose-dependent effects depending on concentration. That is, CgA 47-66 below 0.5 mg/kg produced antinociceptive effects, whereas at 2 mg/kg it produced a marked pronociceptive effect. The latter effect was blocked by diltiazem and indomethacin. CgA 47-66-induced antinociceptive effects on AA-induced responses were reversed when the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF 9-41 was i.p. injected to animals prior to AA and CgA 47-66 administration. The administration of i.p. calcitonin gene-related peptide (CGRP) or substance P (SP) evoked dose-dependent abdominal writhing; this effect was abolished when CgA 47-66 was injected. The present data suggest, for the first time, that a fragment of CgA, CgA 47-66, possesses potent antinociceptive effects at low doses. Although the mechanism triggered by this peptide is unknown, CRF receptors are likely to be involved.     

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.     

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.     

Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.     

Nitrergic and purinergic regulation of the rat pylorus

The role of nitric oxide (NO) and ATP in the regulation of nonadrenergic, noncholinergic (NANC) inhibitory transmission in the pylorus remains unclear. In the presence of atropine and guanethidine, electric field stimulation induced NANC relaxations in a frequency-dependent manner (1-20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M). P(2X) purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 3 x 10(-5) M) and P(2Y) purinoceptor antagonist reactive blue 2 (2 x 10(-5) M) had no effect on NANC relaxations. However, the combined administration of L-NAME and PPADS, but not reactive blue 2, evoked greater inhibitory effects on NANC relaxation than that evoked by L-NAME alone. alpha-Chymotrypsin and vasoactive intestinal polypeptide antagonist did not affect NANC relaxations. ATP (10(-5)-10(-3) M) and P(2X) purinoceptor agonist alpha, beta-methyleneadenosine 5'-triphosphate (10(-7)-10(-5) M), but not P(2Y) purinoceptor agonist 2-methylthioadenosine 5'-triphosphate (10(-7)-10(-5) M), induced muscle relaxations in a dose-dependent manner, and relaxations were significantly reduced by PPADS and unaffected by TTX. These studies suggest that NO and ATP act in concert to mediate NANC relaxation of the rat pylorus. ATP-induced relaxation appears to be mediated by P(2X) purinoceptors located on smooth muscle cells.     

Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.     

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Functional evidence for a role of GABA receptors in modulating nerve activities of circular smooth muscle from rat colon in vitro

In the enteric nervous system, activation of neuronal GABA(A)- and GABA(B)-receptors has been shown to modulate neuronal activity. The consequences of this modulation depend on the location in the gastrointestinal tract or the animal species studied. These data illustrate the complexity of GABA-induced effects. Furthermore, the GABA(C)-receptor has been identified in a neuroendocrine cell line suggesting a modulating role of this third type of GABA receptor in intestinal functions. Therefore, the modulating role of GABA-receptor agonists was determined in circular preparations of rat distal colon during electrical nerve stimulation (NS) in vitro. Mechanical response to NS was characterized by a relaxation followed at the end of the stimulation by an off-contraction. In normal Krebs solution (basal conditions), muscimol and baclofen, respectively GABA(A)- and GABA(B)-agonists, induced a significant increase of the electrically induced off-contraction. The GABA(C) agonist, CACA, showed no significant effect on the response to NS. Excitatory effects of muscimol on the off-contraction were abolished in the presence of atropine. Furthermore, in the presence of atropine, muscimol increased the amplitude of the electrically induced relaxation; similarly the baclofen-induced increase of off-contraction amplitude was significantly lower than that observed in control conditions. Baclofen and muscimol effects on the off-contraction were abolished in the presence of hexamethonium or guanethidine. Furthermore, muscimol and baclofen did not induce any significant change on the response to NS in the presence of L-NAME and apamin together. Thus, it seems that in rat distal colon, GABA regulates significantly both excitatory (through GABA(A)- and GABA(B)-receptors) and inhibitory (through GABA(A)-receptors) neuronal activities. We also gave evidence for a possible interplay between GABAergic intrinsic neurons and adrenergic nerve terminals. Finally, it is shown for the first time the presence of the GABA vesicular transporter (VIAAT) around myenteric ganglia of rat colon.     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.     

P2Y1 receptors mediate inhibitory purinergic neuromuscular transmission in the human colon

Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2Y(1) receptors. We found that 1) ATP (1 mM) and adenosine 5'-beta-2-thiodiphosphate (ADPbetaS) (10 microM), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 microM) and apamin (1 microM) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited (IC(50) approximately 1 microM) by MRS 2179; 4) ADPbetaS reduced the IJP probably by a desensitization mechanism; 5) apamin (1 microM) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) P2Y(1) receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a P2Y(1) receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. P2Y(1) receptors can be an important pharmacological target to modulate smooth muscle excitability.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Sympathetic innervation of the rectum and bladder of the skate and parallel effects of ATP and adrenalin

1. Longitudinal muscles of the rectum of the skate are first briefly excited and then inhibited by stimulation of the sympathetic nerve fibres. 2. ATP, adrenalin and noradrenalin also produce inhibition. 3. 5HT is strongly excitatory but acetylcholine is only excitatory above 1 microM. 4. The rectum contracts strongly to mechanical stimulation; the response is not blocked by TTX. 5. The inhibitory actions of sympathetic stimulation or ATP were not blocked by guanethidine, propranalol, antazoline, theophylline or bee venom (apamin). 6. ATP continued to produce inhibition after the nerve response was blocked by TTX. 7. The urinary bladder gives slow rhythmic contractions, which are inhibited by nerve stimulation and by adrenalin but ATP has no action. 8. 5HT is strongly excitatory but acetylcholine has little action.     

Mechanisms of non-adrenergic non-cholinergic synaptic transmission in smooth muscle cells of the gastrointestinal tract

Non-adrenergic non-cholinergic (NANCh) inhibitory synaptic potentials in smooth muscle cells (SMC) of the gastrointestinal tract are of a complex transmitter and ion nature. A blocker of ATP receptors, suramin, blocks the fast component, while a blocker of NO synthase, L-NOARG, blocks the slow component of NANCh inhibitory synaptic potentials. In the presence of both suramin and L-NOARG, SMC respond to stimulation of the intramural plexus by generating a low-amplitude hyperpolarization, and VIP is likely to be the transmitter for this effect. Low-conductance Ca²⁺-dependent potassium channels are involved in generation of the fast component of NANCh inhibitory synaptic potentials, and these channels are effectively blocked by apamin. The slow component of this potential is generated by high-conductance Ca²⁺-dependent potassium channels. In the presene of both apamin and L-NOARG (or charibdotoxin), SMC respond to intramural stimulations with non-cholinergic excitatory synaptic potentials, and ATP application evokes depolarization. Both effects are blocked by suramin. In the presence of apamin, noradrenaline also evokes depolarization in SMC, and this effect, similarly to hyperpolarization under normal conditions, is blocked by phentolamine. Our studies allow us to suggest that in smooth muscles of the gastrointestinal tract there are two types of synaptic transmission: the excitatory cholinergic, adrenergic, and ATP-ergic transmission and the inhibitory adrenergic, ATP-ergic, NO-ergic, and VIP-ergic transmission.