Presence of muscarinic inhibitory and absence of nicotinic excitatory receptors at the terminal sympathetic nerves of chicken hearts

Summary Nicotine (2×10^–4 M) or acetylcholine (5.5×10^–4 M) in the presence of 3×10^–6 M atropine did not increase the rate or amplitude of contraction in isolated atria or ventricular strips of the chicken heart; both drugs also did not cause an output of noradrenaline or adrenaline and did not evoke antidromic discharges in the right sympathetic nerves of isolated perfused chicken hearts. In contrast, high K⁺-solutions evoked an output of noradrenaline and adrenaline and caused a burst of antidromic discharges. Dimethylphenylpiperazine (DMPP; 3.1×10^–4 M), by a tyramine-like action, elicited a small output of noradrenaline and increased rate and amplitude of contraction, but did not evoke antidromic discharges. The noradrenaline output caused by DMPP was not reduced by lowering the extracellular Ca²⁺ concentration from 1.8 to 0.1125 mM.-Acetylcholine (10^–5 and 10^–4 M) inhibited the noradrenaline and adrenaline outputs evoked by electrical stimulation of the right sympathetic nerves (3 Hz, 1 ms, 30 V); the inhibition was blocked by 3×10^–6 M atropine. —It is concluded that the terminal parts of sympathetic nerves of the chicken heart posses muscarinic inhibitory receptors but lack nicotinic excitatory receptors. Thus prejunctional nicotinic receptors are not an integral part of the terminal sympathetic neurone otherwise they would be present at this neurone in all species.This work was part of the M. D. thesis of U. Engel     

Inhibition of receptor-mediated noradrenaline release from the sympathetic nerves of the isolated rabbit heart by anaesthetics and alcohols in proportion to their hydrophobic property

The actions of anaesthetics (diethyl ether, enflurane, chloroform, methoxyflurane) and alcohols (ethanol, 1-propanol, 1-butanol, 1-pentanol) on the noradrenaline release from (and uptake into) the sympathetic nerve terminals were studied in isolated rabbit hearts perfused with Tyrode solution at constant flow rate. The noradrenaline in the perfusate was assayed spectrofluorimetrically.

Effect of phentolamine on noradrenaline uptake and release

Summary The influence of phentolamine on the uptake of exogenous noradrenaline infused into the aortic cannula and on the overflow of endogenous noradrenaline caused by sympathetic nerve stimulation was investigated in the isolated perfused rabbit heart. 10^–6 M phentolamine doubled the overflow of endogenous noradrenaline, but did not change noradrenaline uptake. 10^–5 M phentolamine increased the stimulation-induced overflow of noradrenaline 4-fold and inhibited amine uptake by about 50%. 10^–4 M phentolamine elevated the overflow of noradrenaline less than 10^–5 and 3×10^–5 M did. The augmentation of transmitter overflow was only partly reversed by 13 min perfusion with drug-free medium.Pretreatment of hearts with 1.5×10^–5 M cocaine or with 10^–7 or 10^–6 M desipramine did not change the effect of phentolamine on the overflow of noradrenaline evoked by nerve impulses. Pretreatment of hearts with 10^–5 M, but not with 10^–6 M, phentolamine prevented the increase of transmitter overflow by cocaine.It is concluded that low concentrations of phentolamine potentiate the overflow of noradrenaline during nerve stimulation by a mechanism different from that of cocaine, i.e. different from inhibition of neuronal re-uptake. The nature of this mechanism is discussed.This work was supported by the Deutsche Forsehungsgemeinschaft. We have the pleasure to thank Mrs. Ch. Arts, Miss B. Piel and Mr. E. Hagelskamp for skilful technical assistance.     

Effect of veratridine on the fluxes of 3H-noradrenaline and 3H-bretylium in the rat vas deferens in vitro

Summary The effect of the depolarizing agent veratridine on the accumulation and release of ³H-noradrenaline and ³H-bretylium in the rat vas deferens in vitro was examined. Veratridine produced inhibition of the accumulation and induced marked release of the amines. in vas deferens from non-reserpinized rats the release of noradrenaline evoked by veratridine was partially antagonized by omission of Ca²⁺ in the incubation medium and partially inhibited by low concentrations of desipramine. In reserpinized vas deferens the release of noradrenaline like that of bretylium in normal vas was not affected by omission of Ca²⁺ but inhibited by low concentrations (3–5×10^–7 M) of desipramine. Tetrodotoxin and the local anesthetics millicaine and lidocaine antagonized the effect of veratridine on the accumulation and release of the amines, probably due to prevention of the depolarization. High concentration (3×10^–5 M) of desipramine had a similar effect on The release of noradrenaline in normal tissue in presence of external Ca²⁺. It is concluded that noradrenaline is released by veratridine from normal vas deferens by two mechanisms: 1) an exocytosis release, 2) a carrier mediated desipramine sensitive outward transport. In reserpinized tissue the second mechanism is solely responsible for the release of noradrenaline. Bretylium is only released by the second mechanism. It is suggested that the inhibition of the amine accumulation by veratridine is due to an equilibrium of the influx at a low tissue to medium ratio.     

3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris–ciliary bodies. Tissue preparations were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris–ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked ₂-autoinhibition as indicated by the release-enhancing effect of the ₂-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free ³H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, ₂-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of ³H-noradrenaline release. Secondly, muscarinic inhibition of ³H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 M and rauwolscine 1 M throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC₅₀ of 0.33 M and maximal inhibition of 51%. The concentration–response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M₂ subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris–ciliary bodies are predominantly M₂. Finally, inhibition of ³H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of ³H-noradrenaline from mouse iris–ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris–ciliary body are predominantly M₂. Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.     

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices

Summary The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized ³H-5-HT were examined in rat hypothalamic slices. ³H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-³H-tryptophan in the presence of the various durgs tested. ACh (10^–5 M) in the presence of eserine (2×10^–4 M), and carbachol (10^–5 M) stimulated the release of ³H-5-HT. In contrast, oxotremorine (10^–5 M) reduced the ³H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10^–6 M) and d-tubocurarine (10^–6 M). It was not reduced by the muscarinic antagonists, atropine (10^–6 M) and scopolamine (10^–6 M). In fact, each of two antagonists added alone to the incubating medium enhanced ³H-5-HT release. The scopolamine (10^–6 M) stimulating effect on ³H-5-HT release was suppressed by d-tubocurarine (10^–6 M). Finally, the inhibiting effect of oxotremorine on ³H-5-HT release was not prevented by d-tubocurarine (10^–6 M) but was in the presence of atropine (10^–6 M) or scopolamine (10^–6 M).In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of ³H-5-HT and ³H-5-HIAA from l-³H-tryptophan and on the accumulation of l-³H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous ³H-5-HT in hypothalamic synaptosomes (P₂ fraction).These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of ³H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of ³H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.     

Alpha- and beta-adrenoceptor-mediated responses of the guinea-pig ileum and the effects of neuronal uptake inhibition

Summary The effects of noradrenaline and isoprenaline were examined on preparations of guinea-pig ileum, in which contractions were induced by three different methods; by transmural electrical stimulation, by exogenous carbachol and by potassium depolarization. Alpha- or beta-adrenoceptor-mediated responses were examined by construction of cumulative concentration-response curves in the presence of propranolol (10^–6 M) and phentolamine (5×10^–6 M) respectively. Stimulation of alpha-adrenoceptors by noradrenaline virtually abolished the twitches from transmural stimulation, but only partially inhibited the carbachol- and potassium-induced contractions. The effects on the last two preparations were attributed to a postsynaptic inhibition at alpha-adrenoceptors on the longitudinal smooth muscle. In the transmurally-stimulated preparation there was an additional pre-synaptic alpha-adrenoceptor-mediated inhibition of cholinergic transmission. The maximum beta-adrenoceptor-mediated inhibition of all three preparations to noradrenaline and isoprenaline was of the same magnitude and attributed only to a post-synaptic action on longitudinal smooth muscle.The predominant post-synaptic beta-adrenoceptor-mediated (carbachol-contracted ileum) and pre-synaptic alpha-adrenoceptor-mediated (transmurally-stimulated ileum) relaxations were significantly (P<0.05) potentiated by the neuronal uptake inhibitor desmethylimipramine. These receptors may therefore be considered to be closely associated with the sympathetic innervation. The effect on the post-synaptic alpha-adrenoceptor-mediated relaxation was equivocal. Additional minor excitatory responses were identified as a direct alpha-adrenoceptor-mediated contractile response to noradrenaline and as a beta-adrenoceptor-mediated potentiation of transmural stimulation by isoprenaline, possibly due to facilitation of cholinergic transmitter release.     

Modulation of coronary flow rate and cardiac contractility by the divalent cation ionophore A23187 and inhibitors of the cyclooxygenase and 5-lipoxygenase pathways: development of heterogeneous patterns of myocardial ischemia

Summary In the present studies, we demonstrate in buffer-perfused isolated working guinea pig hearts that indometacin reduces coronary flow rate in a dose-dependent manner (max 56.7 ± 5.5%, SEM, n = 6, of control at 5 × 10^–6 mol/l of indometacin, P < 0.01), and that this leads to a development of heterogeneous patterns of myocardial ischemia (elevated myocardial levels of reduced pyridine nucleotide, NADH) and depressed cardiac work (64.7 ± 11.7%, SEM, of control at 5 × 10^–6 mol/l of indometacin, P < 0.05). The effect of indometacin on coronary flow rate and consequently on myocardial tissue oxygenation was completely prevented by the preferential 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) (1 × 10^–6 mol/l), or the sulfidopeptide leukotriene receptor antagonist FPL 55712 (2 × 10^–5 mol/l), indicating that the isolated working guinea pig heart, even when deprived of blood, is able to produce vasoactive sulfidopeptide leukotrienes at significant levels. At higher concentrations of indometacin (5 × 10^–5 mol/l, 1 × 10^–4 mol/l), coronary flow rate returned to initial levels while cardiac work became further depressed despite normoxic levels of NADH. These data support that indometacin also has a direct suppressive effect on the myocardium independent of its coronary vascular effect. This conclusion is supported by the observation that addition of sodium arachidonate (6 × 10^–5 mol/l) completely inhibited the vascular effect of indometacin, but not the depressive effect on the myocardium. The divalent cation ionophore A23187 (6 × 10^–6 mol/l) had a strong positive chronotropic effect on the heart and a biphasic effect on coronary flow rate. After a brief period of increased coronary flow rate, presumably due to coronary vasodilatation, the ionophore caused a sustained reduction in coronary flow, and this was accompanied by high myocardial levels of NADH fluorescence of characteristically heterogeneous pattern. This is presumably caused by vasoconstrictory effects of elevated levels of intracellular Ca²⁺ of vascular smooth muscle, independent of stimulation of 5-lipoxygenase or cyclooxygenase pathways, since neither indometacin nor nordihydroguaiaretic acid modified the effect of A23187. Send offprint requests to: Ó. G. Björnsson     

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines

Summary In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5–20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the -adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca²⁺ or the application of tetrodotoxin (TTX, 2×10^–6 M). FS also resulted in a reversible increase in the fractional efflux of tritium (³H) from rat pancreatic tissues preincubated with either ³H-noradrenaline or ³H-adrenaline. The effects of FS (5–20 Hz) on ³H efflux were abolished by TTX (2×10^–6 M). TTX had no effect on the enhancement of ³H efflux caused by elevation of external potassium concentration (high K⁺, 75 mM). Removal of superfusate Ca²⁺ completely abolished both the FS- and high K⁺-induced increases in ³H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca²⁺-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.     

Adenosine analogs do not inhibit the potassium-stimulated release of substance P from rat spinal cord slices

Summary Adenosine agonists produce antinociception when injected directly onto the spinal cord of rats and mice. One mechanism to account for this effect could be inhibition of neurotransmitter release from nociceptive sensory neurons. Consequently, we studied whether these agents could inhibit the potassium stimulated release of one such transmitter, substance P, from rat spinal cord slices. A 2 cm section of lumbar spinal cord was dissected from male Sprague-Dawley rats, chopped into 0.5 × 0.5 mm sections and perfused at 37°C with a modified Krebs bicarbonate buffer containing either 3.5 mM, 30 mM, or 50 mM KCl in the presence and absence of various adenosine analogs. Perfusates, collected every 2 min, were assayed for substance P by radioimmunoassay. Exposure of tissue to 50 mM KC1 produced an approximate three-fold increase in the release of substance P over basal release. This increase in release was calcium dependent. Perfusion of spinal cord tissues with either adenosine (10^–3 M), N⁶-cyclohexyladenosine (10^–5 M or 5 × 10^–5 M), 5-N-ethylcarboxamide adenosine (10^–5 M) or L-N⁶-phenylisopropyladenosine (10^–5 M) did not significantly alter basal or potassium-stimulated release of SP when compared to controls. In contrast to the adenosine agonists, exposure of the spinal cord tissue to 10^–5 M morphine significantly reduced the potassium-stimulated release of substance P. Pretreatment of the slices with 10^–5 M theophylline or 8-phenyltheophylline did not significantly attenuate the inhibition of substance P release produced by morphine. Theophylline alone (10^–5 M) had no significant effect on either basal or potassium-stimulated release of SP. These studies demonstrate that adenosine does not inhibit the release of SP from spinal cord slices and does not appear to mediate the morphine-induced inhibition of SP release. The results suggest that the mechanism of the antinociceptive effects of adenosine at the level of the spinal cord is not via inhibition of substance P release. Send offprint requests to M. R. Vasko at the above address     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

The aziridinium derivative of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) accelerates the beating rate of isolated rat atria by enhancing the spontaneous release of noradrenaline

Summary The aziridinium derivative of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (az-DSP4) depletes endogenous noradrenaline stores and exerts neurotoxic actions on noradrenergic neurons. These effects are persistent in the central nervous system and transient in the periphery. To determine if transmitter release plays a role in the noradrenaline depletion caused by az-DSP4, the action of the compound was studied in isolated and spontaneously beating rat atria. 1. az-DSP4 enhanced atrial beating rate when present in the incubation medium at concentrations ranging from 10^–3 M to 10^–4 M but at 10^–3 s M decreased that rate below basal levels. 2. Preincubation of atria for 30 min with the noradrenaline uptake blocker desimipramine (DMI, 10^–6 M) or with the betablocker propranolol (10^–7 M), abolished the positive chronotropic action of az-DSP4. 3. The rate-accelerating effect of az-DSP4 could be prevented by pretreating the rats with reserpine (5 mg/kg i. p. 24 h) or enhanced by pargyline pretreatment (100 mg/kg i. p. 18 h). 4. az-DSP4 stimulated the spontaneous efflux of tritium from the isolated atria previously labeled with ³H-noradrenaline (4 × 10^–7 M), an increase that was mainly accounted for by DOPEG. 5. COMT and MAO activities in atria homogenates were inhibited by az-DSP4 in a concentration-dependent manner. However, MAO inhibition did not result in a change of the metabolic pattern as could be expected. 6. The results obtained indicate that az-DSP4 enhances the rate of spontaneous beating of isolated rat atria. The positive chronotropic effect of az-DSP4 requires the interaction of the compound with the noradrenaline uptake system. The mechanism of the accelerating effect of az-DSP4 most probably involves the release of noradrenaline from adrenergic nerve terminals in the atria and its subsequent interaction with adrenergic beta-receptors.Abbreviations DSP4 N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride - az-DSP4 aziridinium derivative of DSP4 - NA noradrenaline - DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - COMT catechol-O-methyltransferase - MAO monoamineoxidase Send offprint requests to M. E. Landa     

Endogenous adenosine inhibits lipolysis induced by nerve stimulation without inhibiting noradrenaline release in canine subcutaneous adipose tissue in vivo

Summary The influence of adenosine on blood flow, lipolysis and adrenergic neurotransmission was studied in canine subcutaneous adipose tissue in situ. Tissue concentrations of endogenous adenosine were elevated in 2 ways: (1) by inhibition of adenosine elimination by i. a. infusion of the uptake blocker dipyridamole (0.5–1.5 M in arterial plasma) + the deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA; 3–5M) or (2) by prolonged nerve stimulation (4 Hz for 30 min). Furthermore, effects of exogenous adenosine and the adenosine antagonist theophylline were studied. Dipyridamole+EHNA increased resting vascular conductance from 0.055 to 0.096 ml×min^–1×100 g^–1×mm Hg^–1, whereas lipolysis induced by sympathetic nerve stimulation (2,4 and 8 Hz during 2 min) was inhibited (by 91, 71 and 34%, respectively). Therapeutic concentrations of theophylline (5–15 g/ml plasma) completely reversed the effects of dipyridamole + EHNA on blood flow and significantly reversed the antilipolytic effect of these drugs. In untreated adipose tissue theophylline reduced resting vascular conductance by 40% and enhanced the lipolytic response to prolonged nerve stimulation but not that to brief nerve stimulation. Transmitter release, evaluated by the outflow of endogenous noradrenaline and/or by the outflow of radioactivity after ³H-noradrenaline labelling, was linearly related to the stimulation frequency (2,4 and 8 Hz). Dipyridamole + EHNA increased plasma noradrenaline by inhibiting uptake or binding of noradrenaline to blood cells. However, noradrenaline release induced by nerve stimulation was not significantly affected by dipyridamole + EHNA or by theophylline. It is concluded that a rise in endogenous adenosine concentration caused by drugs or by prolonged sympathetic nerve stimulation inhibits the lipolytic response to nerve stimulation without any effect on noradrenaline release. The results also show that theophylline in a therapeutic dose range is capable of enhancing lipolysis only if endogenous adenosine concentrations are raised.     

Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCl

Summary The isolated cat heart with intact sympathetic nerve supply was perfused with varying concentrations of acetylcholine, DMPP and KCl. The ensuing asynchronous discharge in cardiac sympathetic nerves was recorded and the noradrenaline liberated into the perfusate was measured.The infusion of acetylcholine for one minute at a relatively low concentration (5×10^–5 to 8×10^–5 g/ml) induced asynchronous firing over the entire period of infusion and an average liberation of 44 ng/min noradrenaline. Increasing the concentration of acetylcholine or adding atropine to the perfusion fluid greatly shortened the duration of the antidromic discharge but enhanced severalfold the amount of noradrenaline liberated.Within a narrow range of very low concentrations DMPP induced continuous firing. At higher concentrations antidromic discharges were restricted to the first few seconds of infusion. Muscarinic drugs such as pilocarpine and methacholine neither induced firing nor released noradrenaline. When added to acetylcholine, pilocarpine reduced the amplitude of acetylcholine-evoked firing and the amount of noradrenaline liberated.KCl in concentrations above 50 mM induced a very short-lasting firing and a considerable noradrenaline output which was concentration-dependent.It is concluded that the acetylcholine- and DMPP-induced action potentials by themselves contribute little to the noradrenaline-releasing activity of these drugs. As in the case of KCl, the sustained depolarization of the sympathetic nerve ending by acetylcholine and DMPP seems to enhance entry of Ca⁺⁺ into the ending, an essential factor for the liberation of noradrenaline.Preliminary results of this report have been communicated to the German Pharmacological Society at the Spring Meeting 1968 in Mainz.     

Phencyclidine and ketamine: Comparison with the effect of cocaine on the noradrenergic neurones of the rat brain cortex

Summary In slices of the rat occipital cortex, the influence of phencyclidine and ketamine on the accumulation of ³H-noradrenaline and the subsequent outflow of tritium was investigated, and was compared with the effect of cocaine.-All three drugs inhibited the accumulation of tritium during incubation of the slices with ³H-noradrenaline. Phencyclidine was slightly, whereas ketamine was much less effective than cocaine.-All three drugs accelerated the spontaneous outflow of tritium from slices preincubated with ³H-noradrenaline. The acceleration caused by low concentrations probably reflects an inhibition of the re-uptake of spontaneously released ³H-noradrenaline; in addition, high concentrations (10^–4M phencyclidine, 3×10^–4–10^–3M cocaine and 10^–3–3×10^–3M ketamine) appear to release tritiated compounds from the neurones. The distance between uptakeinhibiting and releasing concentrations was much greater for cocaine than for phencyclidine and ketamine.-All three drugs enhanced the overflow of tritium evoked by electrical field stimulation. The increase probably reflects an inhibition of the re-uptake of released ³H-noradrenaline; in addition, phencyclidine appears to enhance the release of noradrenaline per pulse.-The actions of phencyclidine and ketamine on central noradrenergic neurones may contribute to the characteristic psychotropic side-effects of these general anaesthetics.     

“Supramaximal” enhancement of the inotropic effect of noradrenaline by tyramine

Summary The positive inotropic effect of noradrenaline on the guinea-pig papillary muscle is potentiated in the presence of 1×10^–5 M tyramine, the concentration of noradrenaline that is necessary to produce a half maximal increase in force of contraction being reduced to about one third. There is no alteration of the maximal inotropic effect since the concentration-effect curve of noradrenaline is simply shifted to the left.In the presence of 3×10^–3 M tyramine, which by itself increases contractility by a dual mechanism (an indirect sympathomimetic and a direct postsynaptic one which is not induced by stimulation of adrenergic -receptors), noradrenaline (1×10^–5 M) produces an additional inotropic effect leading to a force of contraction which surmounts the maximum of the normal concentration-effect curve of noradrenaline by about 30%.The supramaximal isometric contraction curve of the papillary muscle produced by the combined effects of 3×10^–3 M tyramine and 1×10^–5 M noradrenaline differs from the contraction curve in the presence of 1×10^–5 M noradrenaline alone in having a steeper ascending slope and a slower relaxation phase. The mean velocity of force development (S ₁) exceeds the maximum value of the normal concentration-effect curve of noradrenaline by about 50%. There is no increase in the maximum of the mean velocity of relaxation (S ₂).The relaxation time of the supramaximal contraction curve as well as the duration of its action potential are the result of the opposing influences of the two substances, noradrenaline shortening and tyramine prolonging both action potential and relaxation time.     

Plasma normetanephrine for examination of extraneuronal uptake and metabolism of noradrenaline in rats

The importance of neuronal reuptake for terminating the actions of noradrenaline is well established, but the role of extraneuronal uptake is less clear. This study used plasma concentrations of the extraneuronal noradrenaline metabolite, normetanephrine, to estimate rates of extraneuronal removal of noradrenaline in rats. Animals received infusions of ³H-noradrenaline,. with and without inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), to examine the extraneuronal removal of noradrenaline and formation of normetanephrine from infused and endogenous noradrenaline. Infusions of ³H-normetanephrine were also carried out to examine the plasma kinetics of normetanephrine before and after inhibition of MAO.Normetanephrine was cleared rapidly from the circulation and had a short plasma halflife (1 min). Spillover of normetanephrine into plasma (79 pmol kg^–1min^–1) was a third that of noradrenaline, but increased 2.8-fold after inhibition of MAO; noradrenaline spillover remained unchanged. Combined inhibition of MAO and COMT decreased the plasma clearance of ³H-noradrenaline by 38070, reflecting removal of ³H-noradrenaline by extraneuronal uptake. Division of the rate of extraneuronal removal of ³H-noradrenaline by the specific activity of plasma ³H-normetanephrine during the ³H-noradrenaline infusion indicated that the rate of extraneuronal removal of endogenous noradrenaline was 250 pmol kg^–1min^–1; this was close to the spillover of normetanephrine into plasma after inhibition of MAO (219 pmol kg^–1 min^–1).Forty-five % of plasma normetanephrine was derived from circulating noradrenaline and 55% from noradrenaline before entry into the circulation. Assuming that these proportions reflected the sources of noradrenaline metabolized extraneuronally indicated that the rate of extraneuronal metabolism of noradrenaline before entry into the circulation was 138 pmol kg^–1min n^–1. Comparison of this with the rates at which noradrenaline was recaptured by sympathetic nerves (2540 pmol kg^–1min^–1) or spilled over into plasma (228 pmol kg^–1min^–1), indicated that 87% of the noradrenaline released by sympathetic nerves was recaptured, 5% was metabolized extraneuronally and 8% escaped into plasma. Thus, extraneuronal uptake removes much less of the noradrenaline released by sympathetic nerves than neuronal reuptake.     

Effects of phthalazinol on smooth muscle cells of the porcine coronary artery and on neuromuscular junction on the guinea-pig vas deferens

Summary In smooth muscle cells of the porcine coronary artery, phthalazinol (10^–5 M) did not modify the membrane potential and the membrane resistance. At a concentration of 10^–4 M or higher, it hyperpolarized the membrane, reduced the membrane resistance and enhanced the rectifying property of the membrane. At the concentration of 10^–5 M, phthalazinol raised the threshold for the induction of a contraction and suppressed nonselectively the amplitude of the contraction evoked by application of high [K]₀, acetylcholine or electrical depolarization of the membrane. Phthalazinol (10^–5–10^–4 M) did not modify the membrane properties of smooth muscle cells from the guinea-pig vas deferens. However, it suppressed the amplitude of the excitatory junction potentials and the facilitation phenomenon produced by repetitive stimulation at various rates. The action potential recorded from the adrenergic nerves was not affected in the presence of phthalazinol (10^–5 and 10^–4 M). The mean amplitude of the miniature excitatory junction potentials (m.e.j.p.s.) was not affected by treatment with phthalazinol (10^–5–10^–4 M), but the rate of which of m.e.j.p.s. appeared was reduced by phthalazinol (>5×10^–5 M). These results indicate that the vasodilator property of phthalazinol may result from a suppression of Ca-mobilization in both the smooth muscle cells and the adrenergic nerve terminals. The former affects the mechanical response directly and the latter leads to an inhibition of noradrenaline release.     

Effects of centrally applied drugs on pressor responses to hypothalamic stimulation

Summary The posterior hypothalamus of anaesthetized cats was superfused through a push-pull cannula with artificial cerebrospinal fluid and electrically stimulated with the non-insulated tip of the cannula. The hypothalamus was superfused with drugs, and their effects on the pressor responses to hypothalamic stimulation were studied.Superfusion with low concentrations of clonidine (1×10^–5 M or 5×10^–5 M) enhanced the pressor responses to hypothalamic stimulation, while the rise of blood pressure caused by stimulation of the hypothalamus was reduced during superfusion with high concentrations of clonidine (1×10^–3 M or 1×10^–2 M). A moderate concentration of clonidine (1×10^–4 M) was ineffective.Superfusion with carbachol or DMPP (1×10^–3 M or 1×10^–2 M) elicited a dose-dependent enhancement of the pressor responses to hypothalamic stimulation. Hexamethonium (1×10^–2 M) did not affect the rise of blood pressure but abolished the enhancing effects of carbachol and DMPP.In some experiments the nucleus of the solitary tract was superfused with the push-pull cannula and the posterior hypothalamus was stimulated electrically with a monopolar electrode. Superfusion of the nucleus of the solitary tract with tolazoline (1×10^–1 M) enhanced the pressor responses to hypothalamic stimulation.The present results are compatible with the following views: 1. Low concentrations of clonidine enhance the pressor responses to hypothalamic stimulation by activating the alpha-adrenoceptors of the hypothalamic area; high concentrations of clonidine inhibit the pressor responses by reducing the noradrenaline release via a feed-back mechanism and/or by inhibiting the release of acetylcholine from cholinergic nerve endings of the hypothalamus. 2. Nicotine agents enhance the rise of blood pressure caused by hypothalamic stimulation; their effects are mediated by nicotinic receptors. 3. More than one neurotransmitter seems to be involved in the central modulation of the arterial blood pressure.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

Evidence for two types of 5-hydroxytryptamine receptor on secretomotor neurons of the guinea-pig ileum

Summary Flat sheet preparations of the mucosa plus submucosa from the guinea-pig ileum were placed in Ussing chambers so that short circuit currrent (I _sc), an index of electrolyte movement across the mucosa, could be measured. In these preparations, 5-hydroxytryptamine (5-HT) increasesI _sc indirectly by stimulating both cholinergic and non-cholinergic secretomotor neurons. The 5-HT₃ receptor antagonist, ICS 205–930 (10^–13–10^–5 M), substantially depressed the secretory response due to 5-HT (10^–6 M), but not that produced by direct activation of muscarinic receptors on the mucosal epithelium with carbachol (10^–6 M), or by stimulation of secretomotor neurons with substance P (10^–8 M) or 1,1-dimethyl-4-phenylpiperazinium (10^–5 M). The residual response to 5-HT, after the addition of a maximally effective concentration of ICS 205–930 (10^–6 M), was further reduced by hyoscine (10^–7M). When that part of the 5-HT response attributable to the release of acetylcholine was blocked by hyoscine (10^–7M), ICS 205–930 did not further modify the response to 5-HT. The hyoscine-resistant component was, however, sustantially depressed by tetrodotoxin (3.5 × 10^–7 M). The response remaining after ICS 205–930 and hyoscine was not affected by methysergide (2 × 10^{– 5} M) or cyproheptadine (10^–7 M). We conclude that there are ICS 205–930 sensitive 5-HT receptors on cholinergic secretomotor neurons, and ICS 205–930, methysergide, and cyproheptadine insensitive 5-HT receptors on non-cholinergic secretomotor neurons.