Takio Kitazawa

5‐Hydroxytryptamine is a possible neurotransmitter of the non‐cholinergic excitatory nerves in the longitudinal muscle of rainbow trout stomach (Salmo gairdneri)

1 The neurotransmitter of the non‐cholinergic excitatory nerves in the rainbow trout stomach was identified on the basis of the pharmacological properties of the contractile responses to transmural stimulation (TMS) and nicotine. 2 TMS caused tetrodotoxin‐sensitive contractions of rainbow trout stomach strips in a frequency‐dependent manner (0.5–50 Hz). Atropine (1 μm) significantly decreased the contractile response to low‐frequency stimulation (0.5–2 Hz), but did not affect that to high‐frequency stimulation (3–20 Hz). 3 The atropine‐resistant contractile response to TMS (20 Hz) was unaffected by hexamethonium (100 μm), phentolamine (5.4 μm), pyrilamine (1 μm), naloxone (1 μm) or substance P‐induced desensitization. 4 5‐Hydroxytryptamine (5‐HT, 3 nm‐3 μm) caused atropine‐resistant contractions in a concentration‐dependent manner. In the presence of atropine, methysergide (1 μm) decreased the contractile responses to TMS and 5‐HT. 5 Nicotine (3 μm‐500 μm) induced atropine‐resistant contractions that were completely abolished by tetrodotoxin or hexamethonium. Also methysergide inhibited the contractile responses to nicotine. 6 An acid extract of rainbow trout stomach exhibited atropine‐resistant contractions that were decreased by methysergide, in both rainbow trout stomach and guinea‐pig ileum longitudinal smooth muscle preparations. 7 The present results indicate that, in longitudinal muscle strips of the rainbow trout stomach, 5‐HT is one of the mediators (neurotransmitters) of the non‐cholinergic excitatory contractions induced by TMS and nicotine.

Contractile response to substance p in isolated smooth muscle strips from the intestinal bulb of the carp (Cyprinus carpio)

1. The effect of substance P on the mechanical activity of carp intestinal bulb smooth muscle was investigated in vitro. 2. Bath-applied substance P (1 nM-1 microM) caused concentration-dependent contraction of the smooth muscle. The EC50 value was 20 +/- 3 nM (N = 13). 3. Pretreatment with tetrodotoxin (780 nM) or atropine (500 nM) partially decreased the contractile response to substance P, while methysergide (3 microM) did not decrease the response. 4. The contractile response to substance P was not decreased by [D-Pro2, D-Trp7.9]-substance P or [D-Pro4, D-Trp7.9]-substance P (4-11) pretreatment (10 microM for 5 min). 5. Exposure of the intestinal bulb to substance P (100 nM and 1 microM for 15 min) decreased the response to subsequent application of substance P, physalaemin and eledoisin in a concentration dependent manner, while the contractile response to acetylcholine or methionine-enkephalin was not affected. 6. Exposure of the intestinal bulb to physalaemin and eledoisin (100 nM for 15 min) decreased the response to subsequent application of substance P. 7. The above results indicate that substance P causes the contraction of the carp intestinal bulb smooth muscle through its direct action on the smooth muscle and its indirect action through enteric cholinergic nerves. Long-term exposure to substance P causes desensitization of the preparation to substance P, physalaemin and eledoisin at the receptor level.

Effects of morphine and methionine-enkephalin on the smooth muscle tonus and the contraction induced by transmural stimulation in the carp (Cyprinus carpio) intestinal bulb

The effects of morphine and methionine-enkephalin (met-enkephalin) on the smooth muscle tonus and the contraction induced by transmural stimulation were investigated in the isolated intestinal bulb of carp in vitro. Morphine (30 nM-3 microM) and met-enkephalin (3 nM-5 microM) caused dose-dependent non-sustained contraction. Naloxone (10 nM) inhibited the contraction induced by morphine or met-enkephalin in a competitive manner. Tetrodotoxin (400 nM) or atropine (500 nM) did not inhibit the contraction induced by morphine or met-enkephalin. Cooling of the bath fluid from 20 to 10 degrees C decreased nicotine- and transmural stimulation-induced contraction. But met-enkephalin-induced contraction was not affected. Transmural stimulation-induced contraction (3 Hz) was not affected by pretreatment with morphine, met-enkephalin or naloxone. The results demonstrated that morphine or met-enkephalin caused contraction of the smooth muscle directly through the activation of opiate receptors on the smooth muscle cells and neither morphine nor met-enkephalin regulated the cholinergic neurotransmission presynaptically.

α2‐Adrenoceptor‐mediated contractile response to catecholamines in smooth muscle strips isolated from rainbow trout stomach (Salmo gairdneri)

1 The type of adrenoceptor involved in the contractile response to catecholamines in smooth muscle strips isolated from rainbow trout stomach was determined. 2 Noradrenaline (10 nM‐10 μM) and adrenaline (10 nM‐3 μM) caused non‐sustained contractions which were markedly decreased by phentolamine (5.4 μM) but not by carteolol (5 μM). Phenylephrine (1 μM‐1 mM) was less effective in causing muscle contraction and methoxamine produced no contraction. Clonidine (100 nM‐300 μM) caused no mechanical response but inhibited the contraction to noradrenaline or adrenaline but not acetylcholine or 5‐hydroxytryptamine. 3 Yohimbine (10 nM‐1 μM) decreased the contraction induced by noradrenaline or adrenaline but prazosin (1 μM) did not. 4 Tetrodotoxin (780 nM) partially reduced the contraction induced by noradrenaline or adrenaline but atropine (500 nM) did not. 5 In the presence of atropine (1 μM), electrical transmural stimulation caused frequency‐dependent, tetrodotoxin‐sensitive contractions. 6 These results suggest that the contractile response induced by noradrenaline or adrenaline is mediated by α2‐adrenoceptors. It is also suggested that noradrenaline and adrenaline contract the smooth muscle by direct action and by indirect action through the non‐cholinergic excitatory nerve.

Effects of some autonomic drugs and neuropeptides on the mechanical activity of longitudinal and circular muscle strips isolated from the carp intestinal bulb (Cyprinus carpio)

1. The mechanical responses to some autonomic drugs and neuropeptides of longitudinal muscle (LM) and circular muscle (CM) strips isolated from the carp intestinal bulb were investigated in vitro. 2. Acetylcholine and carbamylcholine caused concentration-dependent transient contraction of both LM and CM strips. Tetrodotoxin had no effect, but atropine selectively decreased the contractile responses to acetylcholine and carbamylcholine. 3. Excitatory alpha-2 and inhibitory beta adrenoceptors were present in both LM and CM strips. 4. 5-Hydroxytryptamine (5-HT) caused concentration-dependent contraction of both LM and CM strips. Tetrodotoxin, atropine and methysergide decreased the contractile responses to 5-HT. 5. Some neuropeptides (angiotensin I, angiotensin II, bombesin, bradykinin, neurotensin, somatostatin and vasoactive intestinal polypeptide) did not cause any mechanical response (contraction or relaxation) in either smooth muscle strip. 6. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) caused contraction of both LM and CM strips. However, the time course of the contraction in LM was different from that in CM. The order of potency was NKA greater than SP greater than NKB in LM strips and NKA greater than SP much greater than NKB in CM strips. In LM strips, the contractile responses to tachykinins were unaffected by spantide and methysergide, but partly decreased by tetrodotoxin and atropine. On the other hand, the contractile responses of CM strips were unaffected by tetrodotoxin, atropine, methysergide and spantide. 7. Dynorphin (1-13) (DYN), leucine-enkephalin (L-Enk) and methionine-enkephalin (M-Enk) caused concentration-dependent contraction of both LM and CM strips. The order of potency was DYN greater than M-Enk greater than L-Enk. Naloxone selectively decreased the responses to opiate peptides. 8. The present results indicate that acetylcholine, carbamylcholine, catecholamines, 5-HT, tachykinins (SP, NKA and NKB) and opiate peptides (DYN, L-Enk and M-Enk) affect the mechanical activity of LM and CM strips isolated from the carp intestinal bulb through their specific receptors.

Beta-2 adrenergic receptors are not only for circulating catecholamines in ventricular muscles of carp heart (Cyprinus carpio)

Whether beta-2 adrenergic receptors are only for the circulating catecholamines in isolated ventricular muscles of carp heart was studied. Isoproterenol, epinephrine and norepinephrine had concentration dependent positive inotropic effects. The ED50 values for isoproterenol, epinephrine and norepinephrine were 6.23 +/- 1.9, 87.3 +/- 27.3 and 4500 +/- 580 nM, respectively. Phentolamine did not alter the inotropic effects. Tyramine increases the force of contraction and this action was completely blocked by propranolol and reserpine, but not by atenolol. Norepinephrine levels, in comparison with those of epinephrine, were similar in plasma and higher in ventricular muscles. However, the norepinephrine levels in ventricular muscles of carp heart were markedly lower than those in ventricular muscles of rat heart. These results suggest that ventricular muscles of carp heart contain adrenergic neurons, and that a catecholamine released from the nerve terminals, presumably epinephrine, may stimulate beta-2 adrenergic receptors.

Presynaptic alpha-adrenoceptor mediated inhibition of the neurogenic cholinergic contraction in the isolated intestinal bulb of the carp (Cyprinus carpio)

The effects of norepinephrine, epinephrine and clonidine on neurogenic cholinergic contraction were examined in the presence of a beta-adrenoceptor blocking agent, carteolol (5 X 10(-6) M), in the isolated intestinal bulb of the carp. Norepinephrine, epinephrine (10(-9)-10(-6) M) and clonidine (10(-8)-10(-5) M) inhibited the contraction induced by low frequency (2 or 5 Hz) transmural stimulation (TMS) without inhibiting the contraction induced by acetylcholine (ACh, 6 X 10(-8)-4 X 10(-7) M). Methoxamine (10(-4) M) and phenylephrine (10(-4) M) showed no such inhibitory effect on the TMS-induced contraction. The inhibitory effects of catecholamines and clonidine were decreased by phentolamine (5.4 X 10(-6) M) and yohimbine (10(-7)-10(-6) M) but not by prazosin (7 X 10(-7)-10(-6) M). Nicotine (10(-6)-10(-4) M) and serotonin (3 X 10(-8)-3 X 10(-6) M) caused contraction of the intestinal bulb indirectly by releasing endogenous ACh. This contraction was inhibited by norepinephrine, epinephrine and clonidine in a concentration-dependent manner. The present results suggest that catecholamines and clonidine inhibit cholinergic transmission via the activation of a presynaptic alpha-adrenoceptor (presumably of alpha-2 type) located on the cholinergic nerve terminals innervating the smooth muscle of the intestinal bulb of the carp.

Evidence that a substance P-like peptide mediates the non-cholinergic excitatory response of the carp intestinal bulb (Cyprinus carpio)

SummaryThe participation of substance P in the noncholinergic contraction induced by transmural stimulation (TMS) of the carp intestinal bulb was examined. In the presence of atropine, substance P caused the contraction of carp intestinal bulb smooth muscle in a concentration dependent manner (1 nmol/1 − 1 μmol/l). The EC50 value was 28 ± 7 nmol/l (n = 6). Substance P-induced desensitization (1 μmol/l for 15 min), decreased the response to substance P and the atropine-resistant contraction induced by TMS (20 Hz) selectively. In contrast, in the absence of atropine, the contraction induced by TMS (20 Hz) was slightly attenuated with the substance P-induced desensitization. The acid extract obtained from the carp intestinal bulb contained a smooth muscle excitatory material whose pharmacological properties were consistent with those of substance P. The present results indicate that a substance P-like peptide is present in the carp intestinal bulb which is involved in the non-cholinergic contraction induced by TMS.

Histamine directly acts on β-adrenoceptors as well as H1-histaminergic receptors, and causes positive inotropic effects in isolated ventricular muscles of carp heart (Cyprinus carpio)

1. The mechanism for positive and negative inotropic effects of histamine was studied in electrically stimulated ventricular strips of carp heart. 2. A high concentration of histamine (1 mM) caused a transient negative, and subsequent positive inotropic effects. The positive effect was significantly reduced by pyrilamine, diphenhydramine or dl-propranolol, but was not affected by cimetidine or d-propranolol. 3. Prior treatment with reserpine significantly decreased epinephrine and norepinephrine contents in ventricular muscles, and also almost completely abolished the positive inotropic effect caused by tyramine; however, this treatment failed to affect the positive inotropic effect of histamine. 4. The transient negative inotropic effect was reduced by neither atropine, diphenhydramine, pyrilamine nor cimetidine, and potentiated by pyrilamine. 5. These results suggest that the positive inotropic effect of histamine observed in the ventricular muscle of carp heart is mediated by a direct stimulation of both H1-receptors and beta-adrenoceptors. The negative inotropic effect is unrelated to either cholinergic or histaminergic receptor stimulation.

Isoproterenol-induced desensitization to the positive inotropic effect of isoproterenol in ventricular strips isolated from carp heart (Cyprinus carpio)

Whether the positive inotropic effect of isoproterenol in ventricular strips of carp heart is altered by previous exposure to the agonist was studied. Isoproterenol produced a concentration-dependent positive inotropic effect in these preparations which was competitively antagonized by propranolol. Isoproterenol dose-response curves were shifted significantly downward and to the right after previous treatment with and removal of isoproterenol. The desensitization could be demonstrated after 5 min incubation and near maximal desensitization was observed after 30 min exposure to 1000 nM isoproterenol. The responsiveness was only partially recovered after 180 min incubation in drug-free medium. Isoproterenol produces a rapidly developing desensitization to inotropic effects of the beta-adrenergic agonist. This phenomenon may be mediated by alterations in beta-adrenergic receptors, adenylate cyclase and/or the beta receptor-adenylate cyclase coupling mechanism.