Osamu Yagasaki

Mediators of nonadrenergic, noncholinergic inhibition in the proximal, middle and distal regions of rat colon.

1 The mediators of non‐adrenergic non‐cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. 2 Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 μm), but not by atropine (1 μm) or guanethidine (4 μm). 3 In the proximal colon, l‐nitro‐arginine (N5‐nitroamidino‐l‐2,5‐diaminopentanoic acid) inhibited the TMS‐induced NANC relaxation and l‐arginine (1 mm) reversed this inhibition. Nitric oxide (0.3–10 μm) induced relaxation of the proximal segment. 4 NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10–28, 10 μm) had no effect on the TMS‐induced NANC relaxation, which was also resistant to α‐chymotrypsin (2 units ml−1) and a substance P antagonist ([d‐Pro2, d‐Trp7,9]substance P, 1 μm). 5 In the middle colon, l‐nitro‐arginine did not inhibit the TMS‐induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. l‐Arginine did not reverse the partial inhibition. 6 Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS‐induced NANC relaxation. After α‐chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS‐induced NANC relaxation was augmented. 7 In the distal colon, l‐nitro‐arginine did not have any significant effect on the TMS‐induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS‐induced NANC relaxation. α‐Chymotrypsin‐treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. 8 These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results also suggest that VIP is the most likely candidate as a NANC transmitter in the distal colon.

Acetylcholine release from the myenteric plexus of guinea-pig ileum by prostaglandin E1

Release of acetylcholine (ACh) by prostaglandin E1 from the nerve terminals of the guinea‐pig longitudinal muscle strip was studied in order to reveal the effect of PGE1 on myenteric plexus activity. The ACh released was collected in the presence of physostigmine (2·1 μg ml−1) and choline (0·1 μg ml−1) at 38° C. Five to 100 ng ml−1 PGE1 enhanced the release dose‐dependently. The effect was maintained during the presence of PGE1 in the organ bath, while rapid tachyphylaxis was observed with the ACh‐releasing action of nicotine. Tetrodotoxin or morphine almost completely inhibited the effect of PGE1 on ACh release. Hexamethonium, in a concentration which completely blocked the effect of nicotine, partially inhibited the effect of PGE1. In the late phase of nicotine action, the tissue was still sensitive to PGE1 despite the continued exposure to nicotine. These data suggest the presence in the myenteric plexus of PG receptors which can increase ACh release.

The differential contribution of endogenous prostaglandins to the release of acetylcholine from the myenteric plexus of the guinea‐pig ileum

1 Prostaglandin E (PGE) may be essential for maintaining the sensitivity of the myenteric plexus of guinea‐pig ileum to nicotine. The contributions of prostaglandins to nervous activity evoked by different stimuli have now been investigated by measuring the amount of acetylcholine (ACh) released from the myenteric plexus of the guinea‐pig ileum. 2 The amount of ACh released in response to dimethylphenylpiperazinium (DMPP) or substance P was depressed to about 40% of control by 2.8 μm indomethacin (Ind), whereas the release of ACh induced by 5‐hydroxytryptamine (5‐HT) was not affected. The inhibitory effects of Ind were oyercome by 14.3 nm PGE2. 3 Mepacrine 5 μm, an inhibitor of phospholipase A2, depressed the release of ACh in response to DMPP and substance P to the same extent as Ind. These inhibitory effects of mepacrine were overcome by arachidonic acid (10 μm), but not by arachidonic acid plus Ind. The release of ACh evoked by 5‐HT or electrical field stimulation (EFS) was also inhibited to about 60% of control by mepacrine but these inhibitions were overcome by arachidonic acid (10 μm) either in the absence or the presence of Ind. 4 The results suggest that endogenous prostaglandins and arachidonic acid contribute to the maintenance of the excitability of the myenteric plexus by DMPP and substance P. By contrast, the release of ACh induced by 5‐HT and EFS may be regulated by arachidonic acid and not by prostaglandins.

Contribution of endogenous prostaglandins to excitation of the myenteric plexus of guinea-pig ileum: Are adrenergic factors involved?

The role of endogenous prostaglandins (PGs) in the nicotine-induced contraction and release of ACh was investigated in the isolated guinea-pig ileum. A low concentration of indomethacin (IND 2.8 microM) inhibited the contraction and ACh release induced by nicotine. These inhibitory effects of IND were reversed by PGE2 at concentrations which are thought to be released spontaneously. SC-19220, a PG receptors antagonist, also inhibited the contraction and ACh release induced by nicotine. Unlike the nicotine-induced release of ACh, the potassium-induced release of ACh was unaffected by IND and SC-19220. IND was as potent in inhibiting the responses to cholinergic nerve stimulation by nicotine after treatment of the preparations with antiadrenergic agents. It is concluded that the inhibitory effect of IND does not depend on the functional integrity of adrenergic neurons and that endogenous PGs contribute directly to the modulation of myenteric plexus excitability by nicotine.

Differences in control of descending inhibition in the proximal and distal regions of rat colon

1 Descending inhibition in the proximal and distal portions of rat colon was studied separately, in vitro. 2 In the proximal colon, localized distension with a small balloon caused three types of response (contraction; relaxation; relaxation, then contraction) of the circular muscle on the anal side of the distended region. 3 Distension caused descending relaxation of circular muscle in all segments of the proximal colon, although for this prostaglandin F2α (PGF2α) was necessary in some segments to increase muscle tone. 4 Atropine and guanethidine did not inhibit this descending relaxation, but tetrodotoxin did. 5 Hexamethonium inhibited the descending relaxation in 14 of 17 preparations of proximal colon tested, but not in the others. 6 In the distal colon, distension consistently caused an increase in the tone of the circular muscles. Descending relaxation was observed only after development of higher tone. Atropine and guanethidine did not inhibit the relaxation, but tetrodotoxin did. 7 Hexamethonium did not inhibit the descending relaxation in most of the preparations of distal colon examined. 8 AF64A, an inhibitor of choline uptake, inhibited the response mediated by cholinergic neurones in vitro to electrical transmural stimulation of the longitudinal muscle of proximal colon. 9 Treatment of colonic preparations with AF64A in vitro resulted in inhibition of descending relaxation in those of proximal, but not those of distal, colon. 10 The participation of intrinsic cholinergic neurones in the descending neuronal pathway is strongly suggested by the results in the proximal colon, but less so in the distal colon. 11 The tone and spontaneous contractile activity of colonic circular muscles are discussed in relation to their neuronal control.

Effects of desensitization to adenosine 5‘‐triphosphate and vasoactive intestinal polypeptide on non‐adrenergic inhibitory responses of longitudinal and circular muscles in the rat ileum

Non-cholinergic, non-adrenergic inhibitory nerves are widely distributed in peripheral tissues, particularly within the gastrointestinal tracts in various species of animals (Holman et al 1965). Although there is an abundance of evidence and observations supporting a role for ATP as an inhibitory transmitter (reviewed by Burnstock 1975) many experimental data argue against such a role for ATP (Ohga & Taneike 1977; Ambach et al 1977; Crema et al 1982). Immunohistochemical techniques have demonstrated the occurrence of several biologically active polypeptides within gastrointestinal nerves, and their localization in the myenteric and submucous plexus (Besson et a1 1978; Hokfelt et all980; Schultzberg et all980). In particular, vasoactive intestinal polypeptide (VIP) has been found to have potent inhibitory effects on a variety of smooth muscles including intestine (Piper et al 1970) and so it has been claimed that VIP, rather than ATP, may function as a non-adrenergic inhibitory neurotransmitter in some tissues (Fahrenkrug et a1 1978; Goyal et a1 1980; Matsuzaki et al 1980). The present experiments were carried out to examine ATP and VIP as possible candidates for the neurotransmitter substance of non-adrenergic inhibitory nerve fibres in rat ileum. Because of the lack of specific and reliable antagonists to ATP (Baer & Frew 1979; Small & Weston 1979; Jenkinson 1981) and VIP (Bolton et al 1981) desensitization to these substances was achieved by means of continuous or repeated exposure of the preparations to the respective drugs. We assumed that desensitization to exogeneous agents may be associated with the simultaneous development of desensitization to the corresponding substances when they are released by nerve excitation.

Role of prostacyclin in acetylcholine release from myenteric plexus of guinea-pig ileum

The roles of metabolites of arachidonic acid in spontaneous and agonist-induced acetylcholine release from a longitudinal muscle preparation with myenteric plexus of guinea-pig ileum were studied. Indomethacin significantly decreased both spontaneous acetylcholine release and its release induced by nicotine and substance P. We had found that prostaglandin E2 (PGE2) partly reversed this inhibition. We now found that a stable prostacyclin analog, OP-41483 at 100 nM, completely reversed the inhibition of acetylcholine release by indomethacin. On the other hand, PGD2, PGF2 alpha and ONO-11113, a thromboxane A2 analog, did not have any significant effect on the inhibition by indomethacin. OP-41483 had no effect on acetylcholine release induced by nicotine or substance P in the absence of indomethacin. To confirm the modulatory role of endogenous prostaglandins on acetylcholine release, we also studied the release of 6-keto-PGF1 alpha, a metabolite of prostacyclin, and PGE2 from longitudinal muscle preparations. The preparations released appreciable amounts of 6-keto-PGF1 alpha continuously during the experiments. Indomethacin inhibited release, while nicotine did not affect it so significantly. Our results suggest that endogenous prostacyclin modulates acetylcholine release from cholinergic nerve terminals in the myenteric plexus of guinea-pig ileum.

The role of sialic acid in the nerve terminal for the release of transmitter

The role of sialic acid in the frequency of miniature endplate potentials (MEPPs) was examined using neuraminidase and gangliosides in the mouse diaphragm. Neuraminidase increased and decreased MEPP frequency in normal K+ and high K+ solution, respectively. The effects were dependent on the presence of Ca2+ in extracellular medium. Neuraminidase liberated sialic acid from and lowered Ca2+-binding capacity of synaptosomal membrane. Gangliosides treatment of the tissue partially restored the effects of neuraminidase on the frequency of MEPP and Ca2+-binding capacity. It is possible that sialic acid in the nerve endings provides a functional storage site which supply intracellular Ca2+ to cause a transmitter release.

The effect of a reduction in temperature on the quantal release of transmitter at the mouse neuromuscular junction

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.

Sodium salicylate facilitates calcium-dependent release of transmitter at mouse neuromuscular junctions.

1 The effects of sodium salicylate on the frequency of miniature endplate potentials (m.e.p.ps) and on the quantal content of endplate potentials (e.p.ps) in mouse diaphragm muscles at 36°C and 24°C were studied by conventional microelectrode techniques. 2 At 36°C, salicylate (10 mm) elevated the frequency of m.e.p.ps in a manner which was insensitive to [Mg2+]o and independent of the presence of [Ca2+]o. Lowering the temperature to 24°C abolished the stimulating effect. 2,4‐Dinitrophenol (10 μm) had similar effects at 36°C and these disappeared at 24°C. All subsequent experiments were performed at 24°C. 3 Salicylate (10 mm) further increased the frequency (F) of m.e.p.ps stimulated by [Ca2+]o in a depolarizing solution. When [Ca2+]o was varied in the absence of salicylate, a linear relationship between ln(F) and ln([Ca2+]o) was obtained. Salicylate shifted this relationship to the left, with respect to the control, without altering the slope. 4 Salicylate (5 mm) also increased the quantal content (m) of e.p.ps in a solution that contained 5 mM Mg2+, in a concentration‐dependent fashion. As [Ca2+]o was varied in the absence of salicylate, a linear relationship between ln(m) and ln([Ca2+]o) was observed. Salicylate shifted this linear relationship to the left, with respect to the control, without altering the slope. Doubling the concentration of [Mg2+]o antagonized this effect of salicylate on the quantal content of e.p.ps. 5 These results indicate that salicylate enhances the effect of changing [Ca2+]o. Salicylate probably facilitates the entry of Ca2+ into the nerve terminal or sensitizes the process that is regulated by Ca2+, thereby stimulating the release of transmitter. Surface negative charges may have an important role in the effect of [Ca2+]o.