Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary Segments of the rabbit ear artery were preincubated with (–)-³H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of ³H-noradrenaline and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.     

Neural ATP release and its α2-adrenoceptor-mediated modulation in guinea-pig vas deferens

Summary Contractions, release of previously stored [³H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined.Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03–3 M) and suramin (30–300 M) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 M combined with suramin 300 M. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 M and suramin 300 M, bromoxidine (0.01–1 M) decreased and rauwolscine (0.1–10 M) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP.It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 M and suramin 300 M reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional ₂-adrenoceptors. The ₂-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP. Correspondence to B. Driessen at the above address     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

Activation of β2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats

Summary The aim of the present study was to investigate -adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with ³H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25–20 Hz) increase in the S-1 outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mol/l). The non-selective -adrenoceptor agonists isoprenaline (0.l gmol/1) and adrenaline (0.01 and 0.1 mol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) were blocked by the selective ₂-adrenoceptor antagonist ICI 118551 (0.1 mol/l) but not by the selective ₁-adrenoceptor antagonist atenolol (0.3 mol/l). The cell-permeable CAMP analogue 8-bromo-cAMP (300 mol/l) enhanced S-1 outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mol/l) and adrenaline (0.1 mol/l) did not enhance S-1 outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mol/l) alone in both strains. However, the facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) but not that of adrenaline (0.01 mol/l) were significantly greater in SHR than in WKY. The results suggest that stimulation of prejunctional ₂-adrenoceptors by adrenaline even in the absence of a-adrenoceptor blockade enhances noradrenaline release in kidney cortex of young SHR and WKY. This ₂-adrenoceptor mediated effect may possibly be dependent on cAMP formation. The greater facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) in SHR as compared to WKY are in accord with receptor binding studies which show a higher density of ₂-adrenoceptors in SHR than in WKY kidney cortex.Abbreviations SHR Spontaneously hypertensive rats - WKY WistarKyoto rats - cAMP 3-5-cyclic adenosine monophosphate - S-I stimulation induced Send offprint requests to: L. C. Rump     

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

Summary Rat hippocampal synaptosomes preloaded with [³H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mol/l whereas the minimal effective concentration of fluoxetine was 2.5 mol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 mol/l (EC₅₀ about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 mol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 mol/l) consisted of true [³H]serotonin, compared with 7001o when 0.5 mol/l d-fenfluramine was used; 4) the releasing action of 0.5 mol/l d-fenfluramine was completely Ca⁺⁺-dependent, while at higher dfenfluramine concentrations the Ca⁺⁺-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca⁺⁺-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mol/l) and at this concentration its mechanism of action was Ca⁺⁺-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. displacement of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 mol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced displacement of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity. Send offprint requests to M. Gobbi at the above address     

Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane

Summary After loading the isolated nerve-muscle preparation of the cat nictitating membrane with ³H-(±)-noradrenaline the effects of exogenous dopamine and (-)-noradrenaline were determined on ³H-transmitter overflow elicited by nerve stimulation in the presence of cocaine, 29 M. Dopamine, 0.20 M, and (-)-noradrenaline, 0.18 M, inhibited ³H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 M. Chlorpromazine, 1 M, or pimozide, 1 M, antagonized selectively the reduction in ³H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 M, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 M, prevented the inhibition of ³H-transmitter overflow obtained with (-)-noradrenaline, dopamine or apomorphine. In the absence of cocaine neither chlorpromazine nor pimozide were able to increase ³H-transmitter overflow during nerve stimulation. In contrast to these results, block of -adrenoceptors by phentolamine or phenoxybenzamine resulted in an increase ³H-transmitter overflow during nerve stimulation. Inhibition by dopamine of ³H-transmitter overflow appears to be mediated through dopamine receptors probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional -adrenoceptors that mediate the negative feed-back regulatory mechanism for noradrenaline release by nerve stimulation. The prejunctional inhibitory dopamine receptors are not involved in an endogenously mediated regulatory mechanism for noradrenaline release by nerve stimulation under normal conditions. The possibility that these dopamine receptors are involved in the hypotension commonly observed in patients with chronic l-Dopa treatment is discussed.     

Influence of neuronal uptake on the presynaptic α-adrenergic modulation of noradrenaline release

Summary Conditions required for the inhibitory feedback modulation of noradrenergic neurotransmission were studied in isolated atria of the rat.The alpha adrenergic antagonist, yohimbine, 0.8 M, or phentolamine, 1 M, did not affect the chronotropic response to 4 or 8 shocks at 0.8 Hz but increased it when a higher number of shocks was applied. When neuronal uptake was inhibited by cocaine, 2.9 M, or desipramine, 0.1 M, the enhancement of neurotransmission by yohimbine or phentolamine was higher than that observed in the presence of -adrenergic antagonists alone.In atria preincubated with ³H-noradrenaline, the effect of the drugs on the ³H-overflow evoked by 240 shocks at 2.0 Hz was studied. Cocaine 2.9 M, did not increase the evoked overflow but yohimbine, 0.8 M, did. The ³H-overflow obtained in the group of yohimbine plus cocaine was significantly higher than was expected from the effects of both drugs alone.It is concluded that yohimbine or phentolamine enhance the chronotropic response in rat atria only when the concentration of noradrenaline in the biophase is sufficiently high to activate presynaptic receptors. In this tissue, the efficiency of the neuronal uptake influences the degree of -adrenergic autoinhibition.     

Effect of nicotine and tacrine on acetylcholine release from rat cerebral cortical slices

Summary The effect of nicotine (1–10 M) and tacrine (9-amino-1,2,3,4-tetrahydroacridine; THA) on stimulation evoked release of [³H]acetylcholine from the rat brain slice preparation preincubated with [³H]choline was investigated.In these preparations, nicotine enhanced while tacrine inhibited evoked [³H]acetylcholine release. These effects were blocked by (+)tubocurarine (1 M) and atropine (0.1 M) respectively. In the presence of idazoxan (0.3 M) plus atropine (0.1 M), nicotine (3 M) continued to enhance evoked [³H]acetylcholine release while the inhibitory effect of tacrine (1 M) on evoked [³H]acetylcholine release was reversed to an enhancement. Under these circumstances the effects of both nicotine and tacrine were blocked by (+)tubocurarine (1 M).These findings demonstrate that tacrine can both inhibit or enhance [³H]acetylcholine release, most likely through its activity as a cholinesterase inhibitor. Under normal circumstances following tacrine the predominant effect of the elevated levels of acetylcholine will be activation of inhibitory presynaptic muscarine receptors on cholinergic nerves and an inhibition of evoked [³H]acetylcholine release. Under conditions where both presynaptic inhibitory muscarine and ₂-adrenoceptors are blocked, the elevated levels of acetylcholine produced by tacrine will lead to the activation of facilitatory presynaptic nicotine cholinoceptors on cholinergic nerves and an enhancement of evoked [³H]acetylcholine release. Send offprint requests to R. Loiacono at the above address     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

The steady-state concentration gradient for 3H-noradrenaline generated by uptake1 in the extracellular space of the rat vas deferens incubated with this amine

Summary The rat vas deferens was incubated with 0.2 mol/l ³H-noradrenaline for 60 min, washed out with amine-free solution for 100 min and then prepared for autoradiography (same tissues as presented by Azevedo et al. (1990) Naunyn-Schmiedeberg's Arch Pharmacol 342: 245 – 248). The autoradiography images were then digitized, and grain density was determined as a function of the distance from the surface of the tissue. When neither monoamine oxidase nor vesicular uptake was impaired, i. e. under control conditions, grain density declined monophasically exponentially towards the centre of the tissue. This decline amounted to 0.017 m^–1 or 0.124 varicosity^–1, since the average distance between varicosities was calculated to be 7.4 m. After inhibition of monoamine oxidase and vesicular uptake the rate constant was significantly reduced, and the grain density in close proximity of the surface of the tissue was also reduced.It is proposed that the distribution of grain density observed in controls reflects the steady-state concentration gradient that is generated by uptake₁ during the incubation with ³H-noradrenaline.During spontaneous efflux of ³H-noradrenaline one has to distinguish between re-uptake of the ³H-amine into the leaking varicosity and uptake en passant (during diffusion through the extracellular space). On the basis of the present results, the extent of uptake en passant was calculated (with a computer-assisted model) for the spontaneous efflux of heterogeneously distributed ³H-noradrenaline (after wash-out). Uptake en passant into varicosities located between the source of efflux and the medium amounted to about 55% of the net leakage of ³H-noradrenaline from all varicosities. Earlier experiments had indicated that the sum of the two uptake processes was responsible for the neuronal uptake of 90% of the gross leakage of ³H-noradrenaline from varicosities. Hence, the following appears to take place: about 78% of the gross leakage of noradrenaline from a varicosity are subject to re-uptake into the same varicosity. During diffusion to the medium, about 55% of the ³H-noradrenaline escaping re-uptake is then subject to neuronal uptake en passant. Send offprint requests to E. Schömig at the above address     

Possible involvement of both N- and L-type voltage-dependent Ca channels in adrenergic neurotransmission of canine saphenous veins in low Ca2+ plus tetraethylammonium medium

Summary The involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca²⁺ + 20 mM tetraethylammonium (low Ca²⁺ + TEA) medium has been studied by examining the effects of -conotoxin GVIA (-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked ³H overflow from canine saphenous veins preloaded with [³H]-noradrenaline. Nisoldipine (10 and 30 M) and nifedipine (30 M) reduced significantly the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 M nisoldipine and 10 tM nifedipine. -CTX inhibited markedly the TNS-evoked ³H overflow in both normal Krebs and low Ca²⁺ + TEA media, the inhibition by -CTX being ten times more potent in low Ca²⁺ + TEA medium. Nisoldipine (30 M), when combined with 1 nM -CTX, produced a further significant inhibition of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. However, no additional inhibition by 30 M nisoldipine was observed when -CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 M) did not affect the inhibitory effect of 10 nM -CTX on the evoked ³H overflow. The low Ca²⁺ + TEA medium increased the spontaneous ³H overflow, which was not influenced by -CTX and dihydropyridines. These results suggest that in low Ca²⁺ + TEA medium but not normal Krebs one, Ca²⁺ entry via both N- and L-type VDCCs may be involved in adrenergic neurotransmission in the canine saphenous veins. Correspondence to Y. Takata at the above address     

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [³H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline (0.05 M), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 LM-3 mM), 2-methylthio-ATP (3–100 M), as well as DMPP (10 and 100 M) induced a significant overflow of tritium. The EC₅₀-value of ATP was 20 M. Both the ATP-induced and the DMPP-induced tritium overflow was Ca²⁺-dependent and sensitive to tetrodotoxin (0.3 M) and -conotoxin (0.1 M); in addition, it was inhibited by the ₂-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 M). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P₂-purinoceptor antagonists suramin (300 M) and reactive blue 2 (3 M); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 M) and markedly inhibited by hexamethonium (100 M). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [³H]-choline.The results suggest that ATP at molar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.     

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine

Summary To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mo1/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mol/ 1) an -adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 ol/l), nor phenylephrine (100 mol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mol/l) or adrenaline (30 mol/l), in the presence of a -adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pH₁ was prevented by hexamethyleneamiloride (10 mol/l). Consistent with phenylephrine's ability to stimulate Na⁺ influx via the Na/H antiporter, phenylephrine (100 mol/l) increased intracellular Na⁺ activity by about 3 mmol/l in ouabain (650 mol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial -adrenoceptors may aggravate digitalis toxicity.This work was conducted in part under the auspices of the Association for US/French Biomedical Cooperation Send offprint requests to S. M. Vogel at the above address     

GABAergic modulation of D-1 dopamine receptor-mediated 3H-acetylcholine release from rabbit retina

Summary Dopamine evokes calcium-dependent release of ³H-acetylcholine from superfused rabbit retina labeled in vitro with ³H-choline, through activation of a D-1 dopamine receptor. This study investigates the activation of this receptor by endogenous dopamine and the modulation of the spontaneous and dopamine-evoked release of ³H-acetylcholine from rabbit retina labeled with ³H-choline by GABAergic agonists and antagonists. Endogenous dopamine, released from dopaminergic amacrine neurons by the indirect amines tyramine or D-amphetamine evoked the calcium-dependent release of ³H-acetylcholine from rabbit retina. The release of ³H-acetylcholine elicited by tyramine (10 M) or D-amphetamine (10 M) was attenuated by the selective D-1 antagonist SCH 23390 (0.1 M) and by the dopamine uptake inhibitor nomifensine (3 M). At concentrations of 1 mM and 1 M respectively, GABA and muscimol inhibited the spontaneous release of tritium from rabbit retina labeled in vitro with ³H-choline. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium. GABA and the GABA agonist muscimol (0.01–100 M) inhibited in a concentration-dependent manner the release of ³H-acetylcholine elicited by 100 M dopamine with IC₅₀ values of 4.5 M and 0.02 M respectively. The inhibition of dopamine-evoked ³H-acetylcholine release by GABA (10 M) and muscimol (0.1 M) was antagonized by the GABA antagonists bicuculline and picrotoxin. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium, and potentiated the release of ³H-acetylcholine evoked by 100 M dopamine consistant with a tonic, inhibitory GABAergic input to the cholinergic amacrine neurons in rabbit retina. Dopamine-evoked acetylcholine release in rabbit retina may be of physiological importance as D-1 dopamine receptor-mediated increases in ³H-acetylcholine release from rabbit retina can be elicited by endogenous dopamine. In addition, activation of GABA receptor sites modulates the spontaneous and dopamine-evoked acetylcholine release from rabbit retina. Send offprint requests to M. L. Dubocovich at the above address     

Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via α2-adrenoceptors in chick sympathetic neurons

Summary Effects of ₂-adrenoceptor agonists on whole-cell Ca²⁺ currents and ³H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the 2-adrenoceptor-mediated inhibition of Ca ²⁺ currents and the modulation of noradrenaline release caused by noradrenaline (1 mol/l; in the presence of 10 mol/l cocaine) or the ₂-adrenoceptor agonists 5-bromo-6-(2imidazolin-2-ylamino)quinoxaline (UK 14,304, 10 mol/ l) and clonidine (10 mol/l). These results suggest that the ₂-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca²⁺ channels via pertussistoxin-sensitive GTP-binding-proteins. Send offprint requests to S. Boehm at the above address     

Modulation of noradrenaline release by presynaptic alpha-2 and beta adrenoceptors in rat atria

Summary The effect of pretreatment with the beta-2-selective adrenoceptor agonist, (+-)-clenbuterol (0.3 mg/kg, twice daily, 14 days) on prejunctional alpha-2- and beta-adrenoceptors was studied in rat atria. When atria from non-pre-treated rats had been preincubated with (³H)-noradrenaline, (-)-isoprenaline (0.02 to 4.0 M) did not affect tritium overflow evoked by stimulation of the cardioaccelerant nerves, but a higher concentration (40 M) decreased it. Blockade of prejunctional inhibitory alpha-2-adrenoceptors by yohimbine (0.03, 0.3 and 0.8 M) enhanced the overflow of tritium. In the presence of yohimbine, isoprenaline (1.2 M) significantly increased stimulation-induced transmitter overflow, suggesting that in rat atria the facilitatory effect of isoprenaline mediated via prejunctional beta-adrenoceptors, is masked by the dominant influence of inhibitory alpha-2-adrenoceptors. (-)-Propranolol (0.1 M) prevented the isoprenaline-induced increase in atrial rate and the isoprenaline-induced enhancement of transmitter release in the presence of yohimbine (0.3 M), but did not modify by itself the stimulation-induced efflux of tritium, suggesting that neuronally released noradrenaline failed to activate facilitatory prejunctional beta-adrenoceptors. When atria from clenbuterol-pretreated rats had been preincubated with ³H-noradrenaline, the facilitatory effect of yohimbine 0.03 and 0.3 M was markedly enhanced and, in this case, isoprenaline (1.2 and 12.0 M) failed to cause its facilitatory effect in the presence of the alpha-2-adrenoceptor antagonist. Propranolol did not modify the facilitatory effect of yohimbine. No changes in the isoprenaline-induced increase in atrial rate were observed in clenbuterol-treated rats. In addition, the treatment reduced the positive chronotropic effect of nerve stimulation without affecting the response to exogenous noradrenaline, suggesting a reduction in the transmitter release induced by nerve stimulation in clenbuterol-treated rats. These results suggest that chronic treatment with clenbuterol desensitizes facilitatory prejunctional beta-adrenoceptors, without affecting the postsynaptic beta-adrenoceptors, thus implying that prejunctional beta-adrenoceptors possess properties of the beta-2-subtype. Send offprint requests to M. A. Enero at the above address     

The novel cardioprotective agent BMS-180448 activates a potassium conductance in cardiac and vascular smooth muscle

The goal of the present study was to further characterize the effects of the novel cardioprotective agent BMS-180448 on potassium fluxes in cardiac and vascular smooth muscle. Exposure of voltage-clamped guinea pig ventricular myocytes to BMS-180448 (300 M) produced an inhibition of IK followed by the delayed (5.5 ± 0.5 min) activation of a large time-independent potassium current. At 100 M, BMS-180448 produced only inhibition of I_K. The BMS-180448 activated current was refractory to block by 30 M, glyburide but was largely inhibited by 100 M alinidine (84 ± 6% inhibition at + 40 mV). Cromakalim (100 M)-activated currents were fully inhibited by 3 M, glyburide and 79 ± 4% blocked by 100 M alinidine. The current responses to BMS-180448 were unaffected by the inclusion of 10 mM UDP (100 M, ATP) in the pipette. BMS-180448 also produced a concentrationdependent increase in ⁸⁶Rb efflux from aortic strips; efflux responses were increased in low calcium medium and fully antagonized by 3 M, glyburide. Thus, BMS-180448 activates a potassium conductance in both cardiac and smooth muscle. The glyburide sensitivity of the BMS-180448-induced increase in ⁸⁶Rb efflux from the aortic preparations suggests that this drug activates I_KATP in vascular smooth muscle. Moreover, the observation that BMS-180448 (100 M) partially inhibits the effects of cromakalim in ventricular muscle cells suggests that these drugs interact, directly or indirectly, with a common site in cardiac muscle.     

Phorbol 12,13-dibutyrate,an activator of protein kinase C,stimulates both contraction and Ca2+ fluxes in dog saphenous vein

Summary The vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 M) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 mol/kg wet wt.) which were unaffected by 10 M phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca²⁺-free solution. 15 Ca efflux measurements performed in Ca²⁺-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 M) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca²⁺ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 M. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 M PDBu did not cause contractions in the presence of 0.1 M Ca²⁺. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca²⁺-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca²⁺. Send offprint requests to P. J. S. Chin at the above address     

Inhibition by ethanol of excitatory amino acid receptors and nicotinic acetylcholine receptors at rat locus coeruleus neurons

The frequency of spontaneous action potentials of locus coeruleus (LC) neurons was recorded extracellularly in pontine slices of the rat brain. Ethanol (1–100 mM) elevated the firing rate in most neurons; this effect was concentration-dependent. (S)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 0.03–1 M), kainate ( 0.1–3 M), N-methyl-D-aspartate (NMDA; 1–30 M), substance P 0.01–1 M, nicotine 0.1–10 M and ,-methylene ATP ,-meATP; 0.3–30 M, all increased the firing. Application of ethanol g10–100 mM to the superfusion medium for 10 min, reproducibly and concentration-dependently inhibited the facilitatory effect of NMDA g10 M. However, the inhibitory effect of ethanol (100 mM) decreased during a 30-min superfusion period and after the washout of ethanol the sensitivity of LC neurons to NMDA (10 M) tended to overshoot above their initial level. Although NMDA was more potent in the absence than in the presence of external Mg²⁺, ethanol (100 mM) continued to depress the facilitatory effect of a low concentration of NMDA (EM) in a Mg²⁺-free medium. By contrast, in a medium containing normal Mg²⁺, ethanol (100 mM) failed to significantly interfere with the increase in firing rate induced by a high concentration of NMDA (30 M). The effects of kainate (0.5 M), AMPA (0.3 M) and nicotine (1 M) were also depressed by ethanol (100 M), while the effects of substance P (0.03 M) and ,-meATP (30 M) were not changed. In conclusion, ethanol selectively counteracts the opening of cationic channels caused by excitatory amino acid (EAA) receptor agonists and nicotinic acetylcholine receptor agonists. During a longer lasting incubation with ethanol, the inhibition of the NMDA-induced excitatory effect declines, indicating the development of tolerance. Correspondence to: P. Illes at the above address     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address