Non-cholinergic Inhibitory Junction Potentials Research Articles

Nitric oxide inhibits smooth muscle responses evoked by cholinergic nerve stimulation in the guinea pig gastric fundus.

In circular smooth muscle tissues of the guinea pig gastric fundus, transmural nerve stimulation (TNS) evoked an atropine-sensitive cholinergic excitatory junction potential (e.j.p.) and, after inhibiting the e.j.p. with atropine, an apamin-sensitive nonadrenergic noncholinergic (NANC) inhibitory junction potential (i.j.p.). The amplitude of e.j.p.s was similar when the frequency of TNS was low (<0.5 Hz), but it decreased successively (depression phenomenon) when the frequency was high (>1 Hz). The depression phenomenon was attenuated after inhibiting the production of nitric oxide (NO) with N(omega)-nitro-L-arginine (NOLA), but was not altered by inhibiting the i.j.p. with apamin. The e.j.p.s were increased in amplitude by the inhibition of cholinesterase activity, but they were decreased by NO produced from SNP with no alteration of their depression phenomenon. Isometric twitch contractions were depressed during high-frequency TNS. NOLA caused an increase in the amplitude of twitch contractions and the attenuation of their depression that changed the transient contraction produced by high-frequency TNS (1 Hz) to a tetanic one. SNP reduced the amplitude of twitch contractions, with no alteration of the depression phenomena. Contractions produced by low concentrations of acetylcholine, but not by high concentrations, were attenuated by SNP, with no alteration of the membrane depolarization. The results suggest that NO produced during TNS has inhibitory actions on cholinergic transmission; the depression of e.j.p.s is mainly prejunctional events, and the depression of mechanical responses is mainly postjunctional events.

Effect of exogenous ATP on canine jejunal smooth muscle.

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 +/- 0.6 mV) and EFS evoked a NANC IJP (17 +/- 0.4 mV). Omega-conotoxin GVIA (100 nM) and a low-Ca(2+), high-Mg(2+) solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 microM) and N(G)-nitro-L-arginine (100 microM). Oxyhemoglobin (5 microM) partially (38.8 +/- 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-(N, O-bis-[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine , pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, alpha, beta-methylene ATP, 2-methylthioadenosine 5'-triphosphate tetrasodium salt, and adenosine 5'-O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.

Neuroeffector transmission to different layers of smooth muscle in the rat penile bulb

1. Using intracellular recording techniques, two distinct layers of smooth muscle were identified in the rat penile bulb. The inner muscle layer (parenchyma) exhibited spontaneous action potentials, while the outer sheet (sac) was electrically quiescent. 2. In the parenchyma, transmural stimulation initiated non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (IJPs) which were abolished by Nomeganitro-L-arginine (LNA) or 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The amplitude of IJPs was reduced by ouabain, dinitrophenol or decreasing the extracellular potassium concentration ([K+]o) but not by several K+ channel blockers. 3. The parenchyma also received an excitatory innervation mediated by alpha-adrenoceptors which caused a contraction that was not associated with a membrane potential change. 4. In the sac, transmural stimulation initiated two component excitatory junction potentials (EJPs) mediated by alpha-adrenoceptors and associated action potentials. The initial component was more dramatically suppressed than the secondary component by caffeine, ryanodine or cyclopiazonic acid (CPA). Lowering of the extracellular chloride concentration ([Cl-]o) selectively inhibited the rapid component of EJPs, while niflumic acid was less potent. 5. These results suggest that IJPs in the parenchyma result from the release of NO which stimulates sodium pump activity following the activation of guanylate cyclase. In the sac, the activation of alpha-adrenoceptors initiates EJPs by releasing Ca2+ from intracellular stores which activates Ca2+-activated channels.

Cyclic GMP-associated apamin-sensitive nitrergic slow inhibitory junction potential in the hamster ileum.

1. The mediators of non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (i.j.ps) in the circular smooth muscle cells of the hamster ileum were studied. 2. Electrical field stimulation (EFS; 0.5 ms duration, 15 V) of the intramural nerves with a train of five pulses at 20 Hz evoked a rapidly developing hyperpolarization (fast i.j.p.) followed by a sustained hyperpolarization (slow i.j.p.). 3. NG-nitro-L-arginine methyl ester (L-NAME; 50 - 200 microM) and NG-nitro-L-arginine (L-NNA; 50 - 200 microM), NO synthase inhibitors, inhibited or abolished the EFS-induced fast and slow NANC i.j.ps. The effects of these NO synthase inhibitors were reversed by L-arginine (5 mM) but not by D-arginine (5 mM). 4. Exogenously applied nitric oxide (NO; 1 - 100 microM) induced concentration-dependent hyperpolarizations. 5. Oxyhaemoglobin (5 - 50 microM), NO scavenger, inhibited only the slow i.j.p., and the NO-induced hyperpolarization. 6. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM) and cystamine (10 mM), guanylate cyclase inhibitors, inhibited only the slow i.j.p. Zaprinast (100 microM), a phosphodiesterase type V inhibitor, enhanced the amplitude and duration of the slow i.j.p. 7. Apamin (100 nM), a small conductance Ca2+-activated K+ channel blocker, inhibited only the slow i.j.p., and NO-induced hyperpolarization. A high concentration of 8-bromoguanosine 3':5'-cyclic monophosphate (8-bromo-cGMP; 1 mM)-induced membrane hyperpolarization which was blocked by apamin. 8. These results suggest that NO, or a related compound, may be the inhibitory transmitter underlying the apamin-sensitive NANC slow i.j.p. and cyclic GMP mediates the slow i. j.p. in the hamster ileum. It is also likely that NO, without involvement of guanylate cyclase is associated with the fast i.j.p.

Junctional Transmissions in Smooth Muscle of the Guinea Pig Duodenum and Their Modulation by the Prokinetic Agent DQ-2511

Junctional transmissions and their modulation by DQ-2511, a novel prokinetic agent, were investigated in smooth muscle of the guinea pig duodenum. Transmural nerve stimulation evoked a cholinergic excitatory junction potential, a nonadrenergic, noncholinergic inhibitory junction potential, and a substance P mediated slow depolarization. DQ-2511 showed dual effects on the slow depolarization: a low concentration (10^–9 mol/l) enhanced and high concentrations (>10^–6 mol/l) tended to inhibit. The depolarization produced by exogenously applied substance P was enhanced by low concentrations of DQ-2511. The results suggest that the prokinetic actions of DQ-2511 involve an enhancement of transmission from substance P neurons due to an increase in sensitivity of postjunctional receptors to substance P.

Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO.

1. The rat colonic circular muscle displays cyclic episodes of myenteric potential oscillations (MPOs), each of them associated with a spontaneous contraction. Nifedipine 1 microM abolished both MPOs and their associated contractions. TTX (1 microM) increased the amplitude and frequency of spontaneous contractions. 2. Electrical field stimulation (EFS) induced a non-adrenergic non-cholinergic (NANC) inhibitory junction potential (IJP), with two phases: an initial fast hyperpolarization (characterized by IJP amplitude) and a sustained hyperpolarization (characterized by IJP duration). 3. Sodium nitroprusside (10 microM) hyperpolarized and abolished spontaneous contractions even in presence of TTX or 1 microM apamin. ATP (100 microM) also hyperpolarized and abolished spontaneous contractions but its effects were decreased by TTX and abolished by apamin. 4. Suramin (100 microM) or apamin reduced the amplitude of the IJPs, but did not affect their duration. Incubation with L-NOARG (1 mM) reduced the duration but not the amplitude of the IJPs. In presence of L-NOARG plus suramin or L-NOARG plus apamin, both duration and amplitude of the IJPs were reduced but a residual IJP could still be recorded. 5. We conclude that the mechanical and electrical cyclic activity of the rat colonic circular muscle is modulated but not originated by the enteric nervous system and involves L-type calcium channel activity. EFS induces release of NANC inhibitory neurotransmitters which hyperpolarize and relax smooth muscle cells. Both ATP and NO are involved in IJP generation: ATP is responsible for the first phase of the IJPs involving activation of apamin-sensitive potassium channels, whereas NO initiates the second phase which is independent of the activation of such channels.

Effects of suramin on electrical and mechanical activities in antrum smooth muscle of the guinea-pig stomach.

The effects of suramin on electrical and mechanical responses produced by adenosine triphosphate (ATP), acetylcholine and transmural nerve stimulation were observed in antrum smooth muscle isolated from the guinea-pig stomach. Suramin (>10(-6) M) inhibited the non-adrenergic non-cholinergic inhibitory junction potential, with no alteration of the resting membrane potential, slow wave and the ATP-induced responses (hyperpolarization and inhibition of slow waves). The amplitude, but not the frequency, of spontaneous rhythmic contraction was inhibited by suramin (>10(-5) M), with no alteration of electrical responses of the membrane. Transmural nerve stimulation elicited cholinergic excitatory and non-adrenergic non-cholinergic inhibitory responses on the spontaneous contraction, and suramin inhibited only the latter. Suramin did not alter the ATP-induced inhibition of spontaneous contraction. The contractions produced by low concentrations (<10(-7) M), but not high concentrations (10(-6) - 10(-5) M), of acetylcholine were inhibited by suramin. It is concluded that in smooth muscle of the guinea-pig antrum, suramin inhibits contractions produced spontaneously and by low concentrations of acetylcholine, with no relation to the electrical responses of the membrane. Parallel inhibition by suramin of the electrical and mechanical responses elicited by excitation of non-adrenergic non-cholinergic inhibitory nerves may not be causally related to the inhibition of ATP-receptors.

Apamin- and nitric oxide-sensitive biphasic non-adrenergic non-cholinergic inhibitory junction potentials in the rat anococcygeus muscle.

1. Changes in membrane potential following electrical field stimulation (EFS; 1, 2 and 5 pulses at 5 Hz, 0.5 ms duration, 60-80 V) of non-adrenergic non-cholinergic (NANC) inhibitory nerves in the rat isolated anococcygeus muscle were measured using standard intracellular recording techniques. Resting membrane potential ranged between -60 and -70 mV. 2. In the presence of guanethidine (30 microM), atropine (1 microM), propranolol (1 microM) and phentolamine (0.05 microM) to establish NANC conditions, the membrane potential depolarized to between -40 and -50 mV. Under these conditions, EFS caused pulse-dependent, tetrodotoxin (1 microM)-sensitive biphasic inhibitory junction potentials (IJPs) comprising a fast onset and time-to-peak phase followed by a second, slower phase that delayed repolarization. The duration of NANC IJPs ranged between 10 and 20 s. 3. Inhibition of small-conductance Ca2+-activated K+ channels with apamin (0.1 microM) selectively blocked the first fast phase of the NANC IJP, whereas inhibitors of large-conductance Ca2+-activated K+ channels (charybdotoxin and iberiotoxin) and ATP-sensitive K+ channels (glibenclamide) all had no effect on NANC IJPs. 4. Both the nitric oxide synthase inhibitor N G-nitro-L-arginine (L-NOARG; 100 microM) and the inhibitor of soluble guanylate cyclase 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (ODQ; 10 microM) had no effect on the first fast phase of the NANC IJP. Each treatment, however, markedly inhibited the slow phase with the duration of the IJP reduced to between 1 and 3 s. The L-NOARG-resistant fast phase of the NANC IJP was almost abolished by the subsequent addition of apamin (0.1 microM). 5. In conclusion, the present study demonstrates unequivocal NANC nerve-mediated biphasic IJPs in the rat isolated anococcygeus. We propose that nitric oxide (NO), via activation of cGMP-dependent K+ channels, and a non-NO inhibitory factor which activates apamin-sensitive K+ channels contribute to NANC nerve-evoked IJPs in the rat anococcygeus.

Electrical properties of colonic smooth muscle in spontaneously non-insulin-dependent diabetic rats.

Electrical properties of colonic smooth muscle were investigated in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model animal for spontaneous non-insulin-dependent diabetes mellitus (NIDDM), and the results were compared with those obtained from the Long-Evans Tokushima Otsuka (LETO) rat, a control of OLETF rat. At experiments (aged 60-80 weeks), blood glucose level was about 171 mg/dl in LETO rats and 370 mg/dl in OLETF rats. Feces in the colon were restricted to the proximal region in LETO rats and distributed widely in the whole colon in OLETF rats. In both LETO and OLETF rats, the circular smooth muscle strips of the isolated distal colon revealed two types of spontaneous electrical response, slow wave and transient hyperpolarization. The resting membrane potential was smaller in OLETF rats than in LETO rats by about 3 mV, but it was not positively related with the blood glucose level. The amplitude of hyperpolarization produced by noradrenaline (NA) was smaller in OLETF rats than in LETO rats. Transmural nerve stimulation evoked a non-adrenergic, non-cholinergic (NANC) inhibitory junction potential (i.j.p.) in both LETO and OLETF rats; the amplitude of the i.j.p. was smaller in OLETF rats than in LETO rats, while the latency of the i.j.p. was longer in OLETF rats than in LETO rats. Thus, in the distal colon, NIDDM may cause a depolarization of the membrane, an attenuation of NANC inhibitory transmission and a reduction in reactivity of adrenoceptors to NA. These results suggest that the constipation appearing with diabetes mellitus involves dysfunction of both the enteric autonomic nerves and the smooth muscles in the colon.

Tachykinin NK 1 and NK 2 receptors mediate non-adrenergic non-cholinergic excitatory neuromuscular transmission in the guinea-pig stomach

By using selective tachykinin NK 1 and NK 2 receptor antagonists and agonists, we studied the excitatory non-adrenergic non-cholinergic transmission to the circular muscle of the corpus of guinea-pig stomach by the sucrose-gap method. After elimination of inhibitory junction potentials by apamin (0.1 μM), l-nitroarginine (30 μM) and tetraethylammonium (10 mM), electrical field stimulation (10 Hz) in the presence of atropine (1 μM) and nifedipine (1 μM) evoked a pure excitatory junction potential and contraction. The selective tachykinin NK 2 receptor antagonist, MEN 11420, concentration-dependently inhibited the non-adrenergic non-cholinergic excitatory junction potential (EC 50=0.09 μM) and contraction (EC 50=0.04 μM) evoked by electrical field stimulation. On the other hand, the selective NK 1 receptor antagonist GR 82334 (3 μM) only slightly (by about 30%) inhibited the excitatory junction potential while leaving the contraction unaffected. The combined administration of GR 82334 (1 μM) and MEN 11420 (0.3 μM) produced an additive inhibition of the excitatory junction potential, significantly larger than that produced by each antagonist alone. In the presence of both GR 82334 (1 μM) and MEN 11420 (0.3 μM), the P 2 purinoreceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (30 μM) remarkably inhibited the fast component of the excitatory junction potential. In the presence of atropine (1 μM), indomethacin (3 μM) and guanethidine (3 μM) either the selective NK 2 receptor agonist, [ βAla 8]neurokinin A (4–10) (0.01 μM), or the selective NK 1 receptor agonist, [Sar 9]substance P sulfone (0.3 μM), produced tetrodotoxin-(1 μM) and nifedipine-(1 μM) resistant depolarization and contraction. The [Sar 9]substance P sulfone (0.3 μM)-induced contraction, but not that induced by [ βAla 8]neurokinin A (4–10) (0.01 μM), was potentiated by apamin (0.1 μM) plus l-nitroarginine (30 μM). In the presence of atropine (1 μM), indomethacin (3 μM), guanethidine (3 μM), apamin (0.1 μM) and l-nitroarginine (30 μM), the selective tachykinin NK 2 and NK 1 receptor agonists, [ βAla 8]neurokinin A (4–10) and [Sar 9]substance P sulfone, both produced a concentration-dependent depolarization and contraction of the circular muscle. MEN 11420 inhibited the responses to [ βAla 8]neurokinin A (4–10) without affecting the responses to [Sar 9]substance P sulfone, while GR 82334 inhibited the responses to [Sar 9]substance P sulfone but not that to [ βAla 8]neurokinin A (4–10). These data provide evidence that tachykinin NK 2 receptors predominantly mediate the non-adrenergic non-cholinergic excitatory transmission to the circular muscle of the corpus of guinea-pig stomach. In addition, after blocking the non-adrenergic non-cholinergic inhibitory junction potential by apamin, l-nitroarginine and tetraethylammonium, the fast component of the non-adrenergic non-cholinergic excitatory junction potential could be mediated by adenosine triphosphate.

The possible role of ATP and PACAP as mediators of apaminsensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 microM each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2. The P2 purinoceptor agonist, alpha, beta methylene ATP (alpha, beta mATP, 100 microM) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 microM) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/- 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3. Apamin (0.1 microM) nearly abolished (about 90% inhibition) the NANC i.j.p. and the alpha, beta mATP-induced hyperpolarization, markedly reduced the alpha, beta mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation was unaffected. 4. Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e.j.p.). In the presence of TEA, alpha, beta mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5. The combined application of apamin (0.1 microM) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, alpha, beta mATP and PACAP elicited a biphasic response : depolarization and small contraction followed by hyperpolarization and relaxation. 6. The P2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the alpha, beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P2 purinoceptor antagonist, suramin (100 microM) inhibited to a similar extent (60-80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by alpha, beta mATP or PACAP. 7. PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 microM of PPADS and 300 microM of suramin inhibited the e.j.p. by about 40%). 8. We conclude that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating excitatory P2 purinoceptors. The subtypes of P2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P2 purinoceptors may be located extrajunctionally.

Characterization of the membrane conductance changes underlying the apamin-resistant NANC inhibitory junction potential in the guinea-pig proximal and distal colon

The nature of the electrically- or stretch-evoked nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (UPs) in circular smooth muscle cells of the guinea-pig proximal and distal colon were investigated using standard intracellular microelectrode recording techniques. We have confirmed that the NANC UP, recorded in the presence of hyoscine (1 μM) and nifedipine (1 μM), can be divided into two components with apamin (250 nM), a blocker of the small conductance Ca 2+-activated K + channels. Both the apamin-sensitive and the apamin-resistant components of the UP were blocked by tetrodotoxin (1.6 μM) or by lowering the external Ca 2+ concentration (to 0.25 mM). The apamin-sensitive UP was also blocked by ω-conotoxin GVIA (100 nM), a blocker of ‘N-type’ Ca 2+ channels. The apamin-resistant UP and rebound post-stimulus depolarization (PSD) were reduced upon exposure to either N G- l-arginine (NOLA), an inhibitor of nitric oxide synthase (NOS), or the nitric oxide (NO) scavenger, haemoglobin. The effects of NOLA were partially reversed in the presence of excess l-arginine, a substrate for NOS, suggesting that NO, or a related NO-donor compound, is likely to be the apamin-resistant inhibitory transmitter. Blockade of either the apamin-sensitive or apamin-resistant UP was associated with membrane depolarization and a decrease in the membrane conductance in the absence of nerve stimulation. In the proximal colon, the apamin-resistant UP and PSD could both be demonstrated to arise from an increase in the membrane conductance after subtraction of a non-linear background conductance. The hyperpolarization upon repetitive NANC nerve stimulation was mimicked by the NO donor, S-nitroso- l-cysteine (2.5–25 RM), which evoked a transient apamin-sensitive, but w-conotoxin GVIA resistant, component followed by a slower apamin-resistant component. These results suggest that neurally-released NO has a number of actions in the guinea-pig colon, causing apamin-resistant hyperpolarization and depolarization, as well as directly opening apamin-sensitive K + channels.

Non-adrenergic, non-cholinergic inhibitory junction potential in rat colonic circular muscle is partly sensitive to ω-conotoxin GVIA and resistant to L-, P- or Q-type calcium channel blockers

The effects of several Ca 2+ channel blockers were evaluated on inhibitory junction potential (IJP) evoked in rat colonic circular muscle by electrical field stimulation (EFS). Glass microelecrodes were used to record membrane potential of smooth muscle cells. IJPs were tetrodotoxin-sensitive (1 μM) and disappeared in Ca 2+-free solution. L-type calcium channels blockers, such as nifedipine (1 μM) or verapamil (1 μM), did not affect IJPs. IJPs were significantly reduced by ω-conotoxin GVIA (300 nM), an N-type Ca 2+ channel blocker. IJPs were resistant to ω-agatoxin IVA (50 nM), a P-type Ca 2+ channel blocker, and ω-conotoxin MVIIC (1 μM), which blocks both N- and Q-type Ca 2+ channels at micromolar concentrations. We conclude that the release of NANC neurotransmitter-mediating IJPs in the rat colon evoked by EFS involves N-type Ca 2+ channels. The fact that ω-conotoxin GVIA does not abolish the IJPs suggests a putative role for L-, P- or Q-type Ca 2+ channels.

Dual effects of trimebutine on electrical responses of gastric smooth muscles in the rat

The effects of trimebutine on the electrical properties of smooth muscle membranes were studied in the isolated rat stomach, the objective being to elucidate the dual actions of this drug on gastric motility. Transmural nerve stimulation elicited a cholinergic excitatory junction potential (e.j.p.) and a nonadrenergic noncholinergic inhibitory junction potential (i.j.p.), and trimebutine inhibited the e.j.p. more than the i.j.p., with no significant change in the acetylcholine-induced depolarization. Trimebutine reduced the interval and, at high concentrations, the amplitude of slow waves. In enzymatically dispersed single cells, the Ca 2+ current elicited by depolarization of the membrane was also inhibited by trimebutine. Thus, trimebutine increases slow wave frequency and inhibits cholinergic transmission and Ca 2+ influx. The former would enhance while the latter two would depress gastric motility.

Neuronal tachykinin NK2 receptors mediate release of non-adrenergic non-cholinergic inhibitory transmitters in the circular muscle of guinea-pig colon.

The aims of this study were: (i) verify the usefulness of the recently described non-peptide antagonist, SR 142801, for blocking tachykinin NK3 receptors in the circular muscle of the guinea-pig colon and (ii) after occlusion of NK3 receptors by SR 142801, test the hypothesis that tachykinins may activate non-adrenergic non-cholinergic inhibitory neurons via non-NK3 receptors. In sucrose gap, we found that SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced atropine (1 microM)-sensitive depolarization, action potentials and contractions of circular muscle of guinea-pig colon without affecting the cholinergic excitatory junction potential and contraction produced by single pulse electrical field stimulation. Likewise, SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced non-adrenergic non-cholinergic hyperpolarization and relaxation of the circular muscle, without affecting the non-adrenergic non-cholinergic inhibitory junction potentials and relaxation produced by single pulse electrical field stimulation. Therefore, SR 142801 is a suitable tool to occlude neuronal NK3 receptors in guinea-pig colon. In the presence of SR 142801 (0.1 microM), atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM) and nifedipine (1 microM) superfusion with neurokinin A (0.3 microM) produced depolarization on which a series of inhibitory junction potentials were superimposed. The incidence, number and amplitude of the inhibitory junction potentials evoked by neurokinin A was partly reduced by pretreatment with either apamin (0.1 microM) or L-nitroarginine (30 microM) and was totally blocked by pretreatment with apamin plus L-nitroarginine or by tetrodotoxin (1 microM). None of these treatments affected the depolarization and contraction produced by neurokinin A. The NK1 receptor selective antagonist, GR 82,334 (3 microM), did not affect the responses to neurokinin A, which were abolished by the NK2 receptor-selective antagonist GR 94,800 (0.1 microM). Substance P (0.3 microM) produced a large depolarization of the membrane but was poorly effective in producing superimposed inhibitory junction potentials. The NK1 receptor-selective agonist [Sar9]substance P sulfone (0.3 microM) produced large depolarization without inducing superimposed inhibitory junction potentials, while the NK2 receptor-selective synthetic agonist [beta-Ala8]neurokinin A(4-10) (0.3 microM) produced depolarization and superimposed inhibitory junction potentials. We conclude that neurokinin A, in addition to direct excitation and contraction of circular muscle activates, via neuronal NK2 receptors, inhibitory non-adrenergic non-cholinergic motorneurons. Thus, neuronal NK2 receptors should be considered as targets for endogenous tachykinins in enteric circuitries leading to descending relaxation in guinea-pig colon.

Excitation and inhibition by cisapride of spontaneous and evoked electrical responses in smooth muscles of guinea-pig stomach

In isolated circular smooth muscles of the guinea-pig stomach, cisapride depolarized the membrane, increased the amplitude and interval of slow waves, and enhanced the cholinergic excitatory junction potential, with no change in the nonadrenergic noncholinergic inhibitory junction potentials. Methysergide mimicked the excitatory actions of cisapride on junction potentials. Results showed that cisapride has dual actions on gastric muscles; excitation by facilitating the release of ACh possibly via 5-HT receptor blockade and also by depolarizing the smooth muscle membrane, and inhibition by reducing the frequency of slow waves, possibly by acting directly on the pacemakers.

Effects of vitamin E deficiency on autonomic neuroeffector mechanisms in the rat caecum, vas deferens and urinary bladder.

1. Modified sucrose-gap, standard organ-bath techniques and transmitter release studies were used to examine neuromuscular transmission in the caecum, vas deferens and urinary bladder in normal rats and in rats maintained for 12 months on a diet free of vitamin E. 2. In the caecum circular muscle, non-adrenergic, non-cholinergic inhibitory junction potentials were absent from 48 and 15% of preparations from vitamin E-deficient and control animals, respectively. Cholinergic excitatory junction potentials were absent from 83 and 8% of vitamin E-deficient and control preparations, respectively. Responses to applied noradrenaline (0.1-30 microM), alpha,beta-methylene ATP (3-100 microM) and acetylcholine (0.1-30 microM) were attenuated or absent in vitamin E-deficient tissues. Responses to applied KCl were similar in both groups. Release of [3H]noradrenaline or endogenous acetylcholine could not be evoked from vitamin E-deficient tissues. 3. In contrast, in isolated preparations of the vas deferens and urinary bladder, neuromuscular transmission by adrenergic, cholinergic and purinergic components were unaffected by long-term vitamin E deficiency. 4. In conclusion, vitamin E deficiency causes dysfunction of autonomic neuroeffector mechanisms in the smooth muscle of the rat caecum, at both a pre- and postjunctional level. The lesions in autonomic transmission mechanisms brought about by long-term vitamin E deficiency were found only in the caecum; no changes in sympathetic neuromuscular transmission were observed in the vas deferens, or in parasympathetic neuromuscular transmission in the urinary bladder.

Noradrenergic, noncholinergic inhibitory junction potentials in rat proximal colon: role of nitric oxide

Using a single sucrose gap apparatus, experiments were performed to determine the involvement of nitric oxide (NO) in the generation of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials in circular muscle of rat proximal colon. Inhibitors of NO synthase, N omega-nitro-L-arginine and its methyl ester, reduced the amplitude of the electrically evoked inhibitory junction potentials, without affecting membrane resting potential. Such an effect was stereospecific and it was prevented by L-arginine but not by D-arginine. Sodium nitroprusside induced a tetrodotoxin-resistant hyperpolarization, which was not affected by NO synthase inhibitors. Apamin reduced sodium nitroprusside induced hyperpolarization, as well as NANC inhibitory junction potentials, and alpha-chymotrypsin decreased the amplitude of electrical field stimulation evoked responses. Residual responses after NO synthase inhibitors or after alpha-chymotrypsin were further reduced by pretreatment with alpha-chymotrypsin or NO synthase inhibitors, respectively. These results suggest that, in rat colonic circular muscle, NO plays an important role in NANC inhibitory junction potential generation. However, another mechanism, peptidergic in nature, is also involved.

S10-2 - Are non - adrenergic, noncholinergic (NANC) inhibitory junction potentials mediated by NO ?

S10-2 - Are non - adrenergic, noncholinergic (NANC) inhibitory junction potentials mediated by NO ?

Suramin selectively inhibits the non-adrenergic non-cholinergic inhibitory junction potential in the guinea-pig stomach

In smooth muscle cells of the guinea-pig stomach fundus, transmural nerve stimulation evoked a cholinergic excitatory junction potential (e.j.p.) and, in the presence of atropine, a non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p.). Suramin (> 10 −5 M), a putative inhibitor of the P 2 purinoceptor, enhanced the e.j.p. amplitude and inhibited the i.j.p., with no significant effect on the membrane potential. Thus, a possible involvement of ATP in the generation of the NANC i.j.p. has to be considered.