Akira Ohga

The effect of veratridine on the release of catecholamines from the perfused adrenal gland.

1 Experiments on perfused adrenal glands of guinea‐pigs were carried out to study the catecholamine output induced by veratridine in the presence of hexamethonium and atropine. 2 Veratridine (10 μm to 200 μm) caused a dose‐dependent increase in catecholamine output. 3 The addition of veratridine to the perfusion medium for a period of 3 min caused an increase in catecholamine output which reached a maximum 5 min to 10 min after withdrawal of the drug. The catecholamine output then gradually declined and reached near resting values within 30 minutes. It was never sustained for a longer period, even when veratridine was infused for 1 hour. 4 Veratridine failed to increase the catecholamine output in the absence of extracellular Ca2+. However, the addition of Ca2+ after an infusion of veratridine (100 μm) in the absence of Ca2+ caused an increase in the catecholamine output which was proportional to the concentration of Ca2+ (0.55 mm to 8.8 him) used. 5 Veratridine did not increase the catecholamine output in the absence of extracellular Na+ ions, NaCl being replaced by equimolar choline chloride or LiCl. Veratridine also failed to evoke catecholamine output in a Na+‐ffee solution in which Na+ was replaced by sucrose; this was the case even in the presence of a high concentration of Ca2+ (8.8 mm). 6 Tetrodotoxin (0.1 μm) and excess Mg2+ (20 mm) reversibly inhibited the catecholamine output induced by veratridine. 7 Ouabain (10 μm) significantly potentiated the veratridine‐induced catecholamine output. 8 It is suggested that Na+‐dependent Ca2+ influx as well as voltage‐dependent Ca2+ influx mechanisms may be involved in the catecholamine output induced by veratridine.

Voltage-independent catecholamine release mediated by the activation of muscarinic receptors in guinea-pig adrenal glands

1 The differences between the mechanisms of muscarinic and nicotinic receptor‐mediated catecholamine secretion with respect to their dependence on voltage changes and extracellular Ca were examined using perfused adrenal glands of the guinea‐pig. 2 Acetylcholine (ACh, 10−6 to 10−3 m) caused a dose‐dependent increase in catecholamine secretion. The ED50 value for ACh was 7 × 10−5 m. In the presence of atropine (10−5 m), the dose‐response curve for ACh was shifted to the right. Hexamethonium (5 × 10−4 m) preferentially reduced the responses to higher concentrations of ACh (> 10−5 m). Pilocarpine (5 × 10−4 m) and nicotine (3 × 10−5 m) also stimulated catecholamine release. 3 During perfusion with isotonic KCl solution, ACh and pilocarpine, but not nicotine, evoked catecholamine secretion. These responses were abolished by atropine (10−6 m). Pilocarpine‐stimulated catecholamine secretion was enhanced during perfusion with isotonic KCl solution. Under these conditions, hexamethonium (10−3 m) significantly augmented ACh‐evoked catecholamine release. 4 During perfusion with either Ca‐free isotonic KCl or Ca‐free Locke solution, ACh and pilocarpine caused a partial increase in catecholamine secretion whereas nicotine and high K solution (56 mm) did not. The responses to ACh and pilocarpine were completely inhibited by atropine but not by hexamethonium. 5 When guinea‐pig adrenal glands were perfused with isotonic KCl solution containing 2.2 mm Ca which was subsequently removed and replaced with EGTA, ACh‐induced catecholamine secretion was similar in magnitude to that observed during perfusion with Locke solution. 6 We conclude that both nicotinic and muscarinic receptors are involved in ACh‐induced catecholamine secretion from guinea‐pig adrenal chromaffin cells. Activation of muscarinic or nicotinic receptors appears to stimulate catecholamine release through different mechanisms with respect to both voltage‐dependence and Ca requirements.

DISSIMILARITY BETWEEN THE RESPONSES TO ADENOSINE TRIPHOSPHATE OR ITS RELATED COMPOUNDS AND NON‐ADRENERGIC INHIBITORY NERVE STIMULATION IN THE LONGITUDINAL SMOOTH MUSCLE OF PIG STOMACH

1 Transmural electrical stimulation (TMS) of longitudinal smooth muscle strips taken from the cardiac portion of the pig stomach produced biphasic responses consisting of initial contractions followed by relaxations. The excitatory component was enhanced by neostigmine and abolished by atropine. After atropine treatment, TMS and nicotine or 1,1‐dimethyl‐4‐phenyl‐piperazinium, caused a relaxation or a relaxation followed by an after‐contraction. All of these responses were abolished or reduced reversibly with tetrodotoxin and cocaine, while hexamethonium only abolished the response to ganglion‐stimulating agents. 2 The relaxation caused by TMS reached a maximum amplitude at 5–10 Hz, and was entirely resistant to the effects of α‐ and β‐adrenoceptor blocking agents, or a combination of them, and also to guanethidine. These results strongly suggested that the relaxation was elicited by stimulation of intramural non‐adrenergic inhibitory neurones. 3 In the presence of atropine and guanethidine, adenosine triphosphate (ATP, 5–20 μM) caused only a tonic contraction, and ATP (25–200 μM) or adenosine diphosphate (25–200 μM) produced a contractile response or a biphasic one (tonic contraction preceded by a slight relaxation). Adenosine monophosphate and adenosine caused only the tonic contraction over the range of concentrations (25–200 μM). 4 Stimulation of the intramural inhibitory neurones of the tissue consistently evoked an inhibitory junction potential, which showed a summation during repetitive stimulation. One the other hand, ATP elicited mainly a small depolarization of a few mV. 5 When the desensitization to ATP of the muscle was achieved in the presence of atropine and guanethidine, the relaxation induced by stimulation of the non‐adrenergic inhibitory neurones could be evoked without any modification. 6 Dipyridamole neither potentiated the inhibitory responses due to stimulation of the intramural inhibitory neurones nor showed any consistent effect on the ATP‐induced response. 7 From these results, it is unlikely that ATP, or any related compound, is the transmitter substance of the intramural inhibitory neurones in the longitudinal smooth muscle of the pig stomach.

Presynaptic inhibitory effects of catecholamines on cholinergic transmission in the smooth muscle of the chick stomach.

In the vagus nerve--smooth muscle preparation isolated from the chick proventriculus, adrenaline, clonidine (10(-8) - 2.5 x 10(-7) M), noradrenaline (10(-7) - 2.5 x 10(-6) M) and dopamine (10(-5) - 10(-4) M) inhibited the contraction induced by low frequency (0.5 Hz) stimulation of the vagus nerve, but they did not inhibit the contraction elicited by acetylcholine (5 x 10(-8) - 5 x 10(-7) M). The concentration producing 50% inhibition was 10(-7) M for adrenaline and clonidine, 10(-6) M for noradrenaline, and 5 x 10(-5) M for dopamine. Isoproterenol (5 x 10(-8) - 5 x 10(-7) M) inhibited the responses induced by both stimulation of the vagus nerve and acetylcholine. The inhibitory effects of the catecholamine and clonidine were blocked by phentolamine (2.7 x 10(-6) M) but not by 5-(3-tert-Butylamino-2-hydroxy)-propoxy-3, 4-dihydrocarbostyril hydrochloride (OPC 1085) which blocked the effect of isoproterenol. It is suggested that presynaptic alpha-receptors are present in the myenteric plexus of the chick proventriculus, and that the catecholamines and clonidine exert their inhibitory effects on cholinergic transmission via these receptors.

Mechanical, electrical and cyclic nucleotide responses to peptide VIP and inhibitory nerve stimulation in rat stomach.

1. Effects of apamin on electrical and mechanical activities and cyclic nucleotide accumulation in response to vasoactive intestinal peptide (VIP) and intramural nerve stimulation were investigated in isolated circular strips of the rat stomach in the presence of atropine and guanethidine. 2. Circular muscles generated rhythmic contractions and slow waves in the antrum but not in the fundus. Intramural nerve stimulation and VIP caused frequency‐ and dose‐dependent relaxation of fundic strips and inhibition of spontaneous contractions of antral strips. Apamin partly reduced the responses to intramural nerve stimulation but not those to VIP. 3. In the antrum, apamin reduced inhibitory junction potentials (IJPs) evoked at the nadir of slow waves but not at their zenith. In the fundus, apamin partly decreased the amplitude of IJPs. Repetitive nerve stimulation was associated with an apamin‐sensitive hyperpolarization and apamin‐resistant decrease in the slow wave amplitude in the antrum. 4. VIP caused a dose‐dependent hyperpolarization of fundic circular muscle membrane. In the antrum, VIP inhibited spike potentials superimposed on slow waves and it decreased the slow wave amplitude in about half of the preparations. These electrical responses to VIP were resistant to apamin. 5. Intramural nerve stimulation evoked an apamin‐resistant output of VIP from muscle strips, which no longer occurred after tetrodotoxin or removal of extracellular Ca2+. 6. Intramural nerve stimulation and VIP elicited apamin‐resistant increases in cyclic AMP and cyclic GMP accumulations. The effects of VIP on cyclic AMP were greater than those on cyclic GMP. The effects of intramural nerve stimulation on cyclic GMP were faster in onset than those of cyclic AMP. 7. It is suggested that VIP is a neurotransmitter of the intramural inhibitory nerves concerned in the apamin‐resistant relaxation of the rat stomach.

Intracellular Ca2+ antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca2+

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca2+ than in its presence in perfused cat adrenal glands. The intracellular Ca2+ antagonist, TMB-8 (10(-4) M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10(-4) M) in the presence of hexamethonium (10(-3) M) during perfusion with Ca2+-free Locke solution containing EGTA (10(-5) M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca2+ from an intracellular pool just as caffeine does.

The mode of action of caffeine on catecholamine release from perfused adrenal glands of cat

1 Adrenaline and noradrenaline secretion induced by caffeine was investigated in the perfused cat adrenal glands. 2 Caffeine (10–80 mm) caused a dose‐dependent increase in both adrenaline and noradrenaline secretion when applied for 1 min and 10 min after replacing Ca2+ with 10−5m EGTA in the perfusion solution. The ratio of adrenaline to noradrenaline was about 1:1. Mg2+ and/or Ca2+ inhibited the response to caffeine. 3 When caffeine (40 mm) was repeatedly applied in the absence of extracellular Ca2+, the secretory response almost disappeared but only at the second challenge with caffeine. However, the response was partially restored after readmission of Ca2+ (2.2 mm) and was augmented after the readmission of Ca2+ with ouabain (10−5m). 4 Caffeine‐induced secretion of adrenaline and noradrenaline increased with the increase in the preloaded concentration of Ca2+ and attained a maximum at 16 mM Ca2+. 5 During perfusion with Ca2+‐free Locke solution containing hexamethonium (10−3m), acetylcholine (10−4m) caused increases in both adrenaline and noradrenaline secretions with a ratio of about 1:2. The secretory responses were partially inhibited by preceding stimulation with exposure to caffeine (80 mm). 6 These results suggest that caffeine mobilizes Ca2+ from an intracellular storage site that may not be entirely the same as that linked to muscarinic receptors, and causes an increase in both adrenaline and noradrenaline secretion from cat adrenal chromaffin cells.

Ryanodine inhibits caffeine-evoked Ca2+ mobilization and catecholamine secretion from cultured bovine adrenal chromaffin cells.

The effects of ryanodine, a selective inhibitor of the Ca2+‐induced Ca2+ release mechanism, on caffeine‐evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and cate‐cholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5–40 mM) caused a concentration‐dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg2+‐free medium containing 0.2 mM EGTA. Ryanodine (5 × 10–5M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 × 10–7–5 × 10–5M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2/Mg2+‐free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin‐permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.

Pharmacological evidence for the involvement of Na+ channels in the release of catecholamines from perfused adrenal glands.

Veratridine (0.1 mM) was found to be effective in producing an increase in the catecholamine output from perfused guinea‐pig adrenal glands in the presence of high concentrations of hexamethonium (1.83 mM) and atropine (28.8 μM). The response to veratridine was abolished by removal of either Na+ or Ca2+ from perfusion media and by the addition of tetrodotoxin (0.1 μM). It is suggested that the response to veratridine may be due to an increase in the tetrodotoxin‐sensitive Na+ permeability of chromaffin cell membranes.

Gastric vasodilatation and vasoactive intestinal peptide output in response to vagal stimulation in the dog.

1. Gastric vasodilatation, relaxation, and vasoactive intestinal peptide (VIP) output in response to vagal stimulation were studied in anaesthetized dogs. 2. Stimulation of the peripheral end of the vagus nerve (10 Hz, 40 V, 0.5 ms) normally evoked a gastric contraction, but caused relaxation in atropinized dogs. There was no detectable difference between the electrical thresholds for activation of the vagal preganglionic excitatory and inhibitory motor fibres. 3. Vagal stimulation also evoked gastric vasodilatation, which was blocked by hexamethonium but not by combined muscarinic and adrenergic blockade. Vagal fibres evoking vasodilatation had higher thresholds to electrical stimulation than those evoking motor responses. 4. Both gastric motor responses to vagal stimulation increased with increasing frequency up to 10 Hz and a plateau between 10 and 40 Hz, but the vasodilator response was significantly reduced above 20 Hz. Vagal stimulation at 10 Hz caused an increase in gastric venous VIP output which was significantly reduced at 40 Hz. 5. Low‐intensity vagal stimulation (10 Hz, 40 V, 0.05 ms) elicited gastric relaxation (40% of a maximum), with no release of VIP or gastric vasodilatation. 6. It is concluded that release of VIP in response to stimulation of the vagal innervation to the stomach in the dog is primarily vasodilating in action.