Effects of histamine on the resting and stimulation-induced release of [3H]-noradrenaline in guinea-pig isolated atria.

1 The sympathetic transmitter stores of guinea-pig isolated atria were labelled with [3H]-noradrenaline. The effects of histamine (0.3 to 100 mumol/l) on resting and stimulation-induced (S-I, 2 Hz for 10 s) release of radioactivity were investigated. 2 Histamine, in low concentrations (0.3 and 1 mumol/l) had no effect on resting release but inhibited S-I release of radioactivity. The inhibition was abolished by the H2-receptor antagonist, cimetidine (10 mumol/l) and also by the H1-receptor antagonist, mepyramine (1 mumol/l). 3 The inhibitory actions of histamine on S-I release were not due to indirect effects involving alpha-adrenoceptors, beta-adrenoceptors, muscarinic cholinoceptors or prostaglandin synthesis. 4 Histamine in a high concentration (100 mumol/l) increased the resting and S-I release of radioactivity. The increase in resting release was abolished by the neuronal uptake blocking drug cocaine (30 mumol/l) but the increase in S-I release was only partially blocked by cocaine.     

Accumulation and overflow of 3H following incubation of the guinea-pig gall bladder with [3H]-noradrenaline

1 Strips of guinea-pig gall bladder readily accumulate ³H following incubation in the presence of 5 × 10^-8 M (-)-[³H]-noradrenaline. This accumulation was reduced by lowering the incubation temperature (from 37° to 23°C), by cocaine (10^-6 M), by nortriptyline (10^-8, 10^-6 and 10^-4 M) and following incubation of the tissues with 6-hydroxydopamine (10^-3 M for 3 h). At 10^-6 M, (-)-noradrenaline and (-)-adrenaline, but not (-)-isoprenaline, inhibited the accumulation of ³H.

2 Following preloading of strips of guinea-pig gall bladder with 3.6 × 10^-7 M (-)-[³H]-noradrenaline for 1 h, the spontaneous overflow of ³H was observed. Cocaine (10^-4 M), nortriptyline (10^-6 M), (-)-isoprenaline (10^-5 M), acetylcholine (10^-5 M) and adenosine 5′-triphosphate (ATP, 10^-4 M) had no effect on the spontaneous overflow of ³H. KCl (10^-1 M), (-)-noradrenaline (10^-5 M), (-)-adrenaline (10^-5 M), and tyramine (10^-5 M) increased the overflow of ³H. These results illustrate similar characteristics of the guinea-pig gall bladder to other noradrenergically-innervated tissues in accumulating and releasing ³H following incubation in the presence of [³H]-noradrenaline.

3 Following incubation in the presence of 3.6 × 10^-7 M (-)-[³H]-noradrenaline, field stimulation, at 5 Hz, of strips of gall bladder, in the absence or presence of 10^-6 M atropine, increased the overflow of ³H and, simultaneously, induced contractions. The contractile responses to 5 Hz were smaller in the presence than in the absence of 10^-4 M lignocaine. Lignocaine (10^-4 M) reduced the overflow of ³H evoked by field stimulation at 5 Hz. It is suggested that the contractile responses to 5 Hz are due to nerve stimulation and that the increased overflow of ³H is due to the stimulation of noradrenergic nerves.

4 The overflow of ³H evoked by field stimulation at 5 Hz was unaltered and increased by propranolol (10^-6 M) and phentolamine (10^-6 M), respectively. Clonidine (5 × 10^-5 M) had no effect in the absence but reduced the amount of ³H which overflowed in response to field stimulation at 5 Hz in the presence of 10^-6 M atropine. The contractile responses to field stimulation at 5 Hz were reduced by phentolamine (10^-6 M) or clonidine (5 × 10^-6, 10^-5 and 5 × 10^-5 M) whether or not atropine (10^-6 M) was present. These results illustrate the presence of postsynaptic α-adrenoceptors and suggest the presence of presynaptic α-adrenoceptors in the gall bladder of the guinea-pig.

     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado at the above address     

Uptake of [3H]-nicotine and [3H]-noradrenaline by cultured chromaffin cells.

Three day-old cultured bovine adrenal chromaffin cells incubated at room temperature with Krebs-HEPES solution containing different concentrations of [3H]-nicotine, took up and retained increasing amounts of the drug by a mechanism that did not saturate. Concentrations of cold nicotine as high as 100 microM did not alter the amount of [3H]-nicotine retained by cells. Imipramine, cocaine, tetracaine or mecamylamine, at concentrations (10 microM) that blocked the catecholamine secretory effects of nicotine completely, did not modify the uptake of [3H]-nicotine. Both imipramine and cocaine drastically inhibited [3H]-noradrenaline uptake by cells in a concentration-dependent manner (IC50S of 0.08 and 1 microM, respectively). These data indicate that the secretory effects of nicotine are not coupled to its previous uptake into cells, and are evidence in favour of a site of action for nicotine located in or at the surface of the chromaffin cell membrane.     

Cholinesterase activity and exposure time to acetylcholine as factors influencing the muscarinic inhibition of [3H]-noradrenaline overflow from guinea-pig isolated atria.

Guinea-pig isolated atria were incubated and loaded with [3H]-noradrenaline. The release of 3H and of [3H]-noradrenaline was induced by field stimulation (6-9 trains of 150 pulses at 5 Hz). The stimulation-evoked overflows of 3H and of [3H]-noradrenaline were determined. In the absence of an inhibitor of acetylcholinesterase, acetylcholine (12 min preincubation before nerve stimulation, up to 10 microM) failed to inhibit the evoked [3H]-noradrenaline overflow. In the presence of atropine, an increase by acetylcholine of evoked release was observed in the same atria. In contrast, the selective muscarinic agonist methacholine significantly decreased the evoked overflow. The inhibition was antagonized by atropine. Methacholine did not enhance release in the presence of atropine. When present for only 2 min, acetylcholine 10 microM inhibited the evoked overflow and no facilitation of release was observed in the presence of atropine. In the presence of physostigmine, acetylcholine (12 min preincubation, 1 and 10 microM) inhibited evoked [3H]-noradrenaline overflow, but the overflow was increased by acetylcholine 10 microM in the presence of atropine. In the presence of cocaine, corticosterone, phentolamine, propranolol and hexamethonium together, acetylcholine 1 microM inhibited the evoked [3H]-noradrenaline overflow. The inhibition was significantly enhanced in the presence of physostigmine. It decreased with preincubation time of the agonist, despite the presence of physostigmine and constant replacement by new drug. Neither inhibition nor facilitation of evoked release was observed in the presence of atropine. It is concluded that a muscarinic inhibition by acetylcholine (upon prolonged exposure time) may be masked by a concomitant facilitation of release and/or desensitization of the muscarinic inhibitory mechanism. Furthermore, degradation by acetylcholinesterase contributes in part to the ineffectiveness of acetylcholine as a presynaptic inhibitor. When a distortion of the overflow/release ratio was excluded, adrenergic and nicotinic effects were prevented, and acetylcholinesterase was inhibited, the fading of muscarinic inhibition by acetylcholine may have been exclusively due to a slow and moderate desensitization of the presynaptic muscarinic mechanism.     

Prejunctional alpha-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra

We evaluated the effects of prejunctional alpha-adrenoceptors on nitric oxide (NO)-mediated urethral relaxation in rabbits using a muscle bath technique and high-performance liquid chromatography coupled with a microdialysis procedure. The amount of NO(2)(-)/NO(3)(-) released during electrical field stimulation was measured by an NO(2)(-)/NO(3)(-) analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with alpha-adrenoceptor agonists and antagonists. The amount of NO(2)(-)/NO(3)(-) released by electrical field stimulation increased after pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM), but decreased after pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional alpha(1)- and alpha(2)-adrenoceptors, respectively.     

Binding of [3H]-prazosin and [3H]-dihydroergocryptine to rat cardiac alpha-adrenoceptors

1 [3H]-prazosin binds specifically to a single class of alpha-adrenoceptors in rat cardiac membranes (KD25 degrees C = 0.2 nM). 2 That these receptors are of the alpha 1-type was indicated by competition studies, i.e. alpha 1-antagonists such as prazosin and (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1, 4-benzodioxane (WB 4101) were more potent than the alpha 2-antagonists, yohimbine and piperoxan in inhibiting [3H]-prazosin binding. 3 A comparative study of [3H]-prazosin binding and [3H]-dihydroergocryptine binding to cardiac membranes showed that both [3H]-prazosin and [3H]-dihydroergocryptine (at low concentrations) bind to alpha 2-adrenoceptors, while [3H]-dihydroergocryptine (at higher concentrations) also binds to another class of sites.     

Evidence for the presynaptic location of the alpha-adrenoceptors which regulate noradrenaline release in the rat submaxillary gland

Summary Fifteen days after duct ligation, the wet weight of the rat submaxillary gland was reduced to 40% of the contralateral control. Under these experimental conditions, the noradrenaline (NA) content expressed as g/g was 1.2±0.1 in the control glands and 1.9±0.2 in the atrophied glands.The accumulation of ³H-NA in the tissue expressed as Ci/gland, did not differ when the atrophied glands were compared with the corresponding controls. Consequently, the uptake and retention of ³H-NA was not modified by the atrophy of the secretory cells of the gland.The spontaneous efflux of radioactivity from normal and atrophied submaxillary glands prelabelled with ³H-NA was similar. The analysis of the metabolic pattern in both experimental groups revealed that in the spontaneous outflow and also during potassium-induced depolarization, the formation of the O-methylated metabolite, ³H-normetanephrine (NMN) was reduced by more than 50% in the atrophied glands. During depolarization induced by K⁺, a 2-fold increase in the outflow of the deaminated glycol ³H-3,4-dihydroxyphenylglycol (DOPEG) was observed.The effect of phentolamine on the release of radioactivity induced by 60 mM K⁺ in normal and in atrophied submaxillary gland slices prelabelled with ³H-NA was also investigated. In both experimental groups, the fractional release of total radioactivity induced by K⁺ was similar. Phentolamine, 3.1 M, produced a 3-fold increase in the fractional release of radioactivity both in the control and the atrophied glands. These results indicate that the increase of K⁺-induced release of ³H-NA induced by phentolamine was independent of the presence or absence of the postsynaptic structures. It is concluded that phentolamine increases transmitter release by blocking alpha-adrenoceptors located in the noradrenergic nerve endings of the rat submaxillary gland.     

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

Summary To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [³H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue.During the incubation of the tracheae with [³H]choline a significant synthesis of [³H]acetylcholine (35,000 dpm/preparation) and [³H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [³H]phosphorylcholine was enhanced, whereas the content of [³H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [³H]phosphorylcholine (900 dpm/3 min) and [³H]choline (800 dpm/3 min); [³H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [³H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [³H]phosphorylcholine (1,900 dpm) without affecting the outflow of [³H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [³H]acetylcholine (2,400 dpm), but only a small outflow of [³H]phosphorylcholine. Chemical stimulation (30 mol/1 veratridine) also caused a large release of [³H]acetylcholine (1,700 dpm) without affecting the outflow of [³H]phosphorylcholine or [³H]choline. Indomethacin (3 mol/1) enhanced the electrically-evoked release of [³H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [³H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release. Send offprint requests to I. Wessler at the above address     

Clonidine and morphine increase [3H]-noradrenaline overflow in mouse vas deferens.

1. Field stimulation of mouse isolated vas deferens produced a biphasic contraction that consisted of an initial brief non-adrenergic, non-cholinergic (NANC) twitch, followed by a more prolonged noradrenergic component. 2. Field stimulation of vasa, previously loaded with [3H]-noradrenaline ([3H]-NA), increased the amount of radioactivity in the Krebs bathing solution; 77% of this radioactivity was derived from [3H]-NA. 3. Tetrodotoxin (3 x 10(-6) M) abolished the biphasic motor response to field stimulation and the accompanying increased overflow of [3H]-NA. 4. Morphine (10(-7)-10(-5) M) inhibited the initial NANC component but potentiated the secondary noradrenergic component of the motor response to field stimulation. Morphine also increased the field stimulation-induced overflow of radioactivity. Naloxone (10(-6) M) antagonized the effects of morphine on the motor response and also on the overflow of radioactivity. 5. Clonidine (10(-9)-10(-7) M) inhibited the initial NANC component but potentiated the secondary noradrenergic component of the motor response to field stimulation. Clonidine also increased the field stimulation-induced overflow of radioactivity. 6. The ability of morphine (10(-7) M) and of clonidine (10(-9) M) to potentiate the field stimulation-induced overflow of radioactivity persisted in the presence of a combination of tranylcypromine (10(-5) M), desmethylimipramine (10(-5) M) and 17-beta-oestradiol (10(-5) M). 7. The inhibition of the initial NANC component of the motor response to field stimulation produced by morphine and clonidine may be related to the ability of these drugs to potentiate both the secondary noradrenergic component and the overflow of radioactivity, if the NANC transmitter is involved in regulating NA release.     

Effect of the dihydropyridine Bay K 8644 on the release of [3H]-noradrenaline from the rat isolated vas deferens.

The effects of Bay K 8644 on the release of [3H]-noradrenaline evoked by potassium, electrical stimulation or tyramine from the rat isolated vas deferens labelled with [3H]-noradrenaline were investigated. Bay K 8644 (1 microM) by itself did not affect the spontaneous release of tritium from the rat isolated vas deferens. However, it increased the calcium-dependent release of tritium elicited by both high potassium (59 mM) and electrical field stimulation. The exposure of rat vas deferens to phentolamine (10 microM) increased the release of tritium induced by potassium (59 mM) and electrical field stimulation. Bay K 8644 (1 microM) failed to increase further the release of tritium elicited by both stimuli in preparations previously treated with phentolamine (10 microM). The calcium-independent release of [3H]-noradrenaline evoked by tyramine (10 microM) was not affected by Bay K 8644 (1 microM). The results of our study support the view that alpha2-adrenoceptors modulate noradrenaline release by restricting calcium influx into sympathetic nerve terminals through voltage-dependent channels.     

Methiothepin enhances the potassium-evoked release of [3H]-noradrenaline in rat pineal gland

Summary The 5-hydroxytryptamine (5-HT) autoreceptor antagonist methiothepin increased in a concentration-dependent manner the K⁺-evoked release of [³H]-noradrenaline in pineal glands from normal and parachlorophenylalanine (PCPA)-treated rats. However, 5-HT and the 5-HT receptor agonists, LSD and 5-methoxytryptamine, were inactive at modulating the K⁺-evoked release of [³H]-noradrenaline in pineal glands from normal and PCPA-treated rats. When tested on the uptake of [³H]-noradrenaline in the pineal gland, methiothepin was found to be a potent inhibitor (IC₅₀ = 10.6 nmol/l). Exposure to methiothepin failed to increase the K⁺-evoked release of [³H]-noradrenaline when tested in the presence of cocaine. While the K⁺-evoked release of [³H]-noradrenaline was shown to be modulated through inhibitory presynaptic ₂-adrenoceptors in pineal glands from normal and PCPA-treated rats, no evidence was obtained for a presynaptic modulation through 5-HT receptors of [³H]-noradrenaline release. The facilitation by methiothepin of the K⁺-evoked release of [³H]-noradrenaline in rat pineal gland appears to be due to the inhibition of noradrenaline uptake by this compound.Some of the results were presented at the Meeting of the British Pharmacological Society (Galzin et al. 1986) Send offprint requests to S. Z. Langer