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     

Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Summary Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [³H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission.Polymyxin B (7 and 21 mol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mol/l. Polymyxin B (21 mol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mol/l), 8-bromo-cyclic AMP (90 mol/l), tetraethylammonium chloride (300 mol/l), and idazoxan (0.1 mol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 pmol/1) by itself inhibited the S-I outflow of radioactivity.In the presence of a concentration of PMA (0.1 mol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mol/l) and 8-bromocyclic AMP (90 mol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mol/l). These results suggest that protein kinase C is not involved in a ₂-adrenoceptor mediated modulation of noradrenaline release. Send offprint requests to I. F. Musgrave at the above address     

Unlike β-endorphin,dynorphin1–13 does not cause retrograde amnesia for shuttle avoidance or inhibitory avoidance learning in rats

Posttraining administration of the opioid peptides, -endorphin or the enkephalins, is known to cause retrograde amnesia for a variety of tasks in rats. The present paper studies the effect of the posttraining administration of dynorphin_1–13 on retention of a step-down inhibitory avoidance task and of a shuttle avoidance task. For the purpose of comparison, the effect of human -endorphin was also studied. In confirmation of previous results, -endorphin (1.0 or 10.0 g/kg, IP) caused retrograde amnesia for the two tasks. Dynorphin_1–13 had no effect at doses between 0.008–125.0 g/kg IP or 1.25–125.0 ng/rat ICV in the inhibitory avoidance task, or at doses of 5.0, 25.0, or 125.0 g/kg in the shuttle avoidance paradigm. These findings suggest that, in contrast to -endorphin, dynorphin_1–13 may not be involved in memory regulation at the posttraining period in rats.     

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     

Adenosine modulation of non-adrenergic non-cholinergic neurotransmission in isolated guinea-pig atria

Summary Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1–10 M) concentration-dependently reduced the cardic response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 M 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 M) and by adenosine deaminase (1 g/ml), but was reduced by dipyridamole (1 M). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.Send offprint requests to A. Rubino at the above address     

Target sites for the inhibition of prostacyclin effect in guinea-pig ileum

Summary In the guinea-pig terminal ileum a maximally effective concentration of prostacyclin (PGI₂) (1 ol/l) induced contractions that were partially resistant to tetrodotoxin (TTX) 0.1 mol/l, to low temperature (20°C) and to atropine (30 nmol/l). Half maximum contractions evoked by PGI₂ (20 nmol/l) were abolished by TTX and by low temperature, which did not modify the response to exogenous acetylcholine (ACh), as well as by atropine. Procaine (5–500 ol/l) caused a concentration-dependent inhibition of contractions induced by PGI₂ (20 nmol/l and 1 mol/l) and by equieffective concentrations of ACh (20 nmol/l and 0.4 ol/l, respectively). The order of magnitude for this inhibition was ACh 20 nmol/l = PGI₂ 20 nmol/l > PGI₂1 mol/l > ACh 0.4 mol/l. In preparations exposed to TTX or to low temperature procaine (50 mol/l) did not affect the residual response to PGI₂ (1 mol/l). Quercetin (1 and 5 ol/l) inhibited the effect of PGI₂ and, at higher concentrations, it also caused partial depression of the responses to ACh. Quercetin did not alter TTX-resistant and low temperature-resistant contractions induced by PGI₂ 1 mol/l. Carbonyl cyanide-trifluoromethoxyphenyl hydrazone (FCCP) (0.1–1 ol/l) reduced the effect of PGI₂ and of ACh to approximately the same extent and inhibited the residual response to PGI₂ 1 mol/l in preparations treated with TTX or expressed to low temperature. The present results show that PGI₂, besides acting on cholinergic neurons, also exerts a direct effect on smooth muscle cells and FCCP can be used to block this effect. In contrast procaine and quercetin selectively inhibit the ACh-mediated component of PGI₂ action. Send offprint requests to R. M. Gaion     

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     

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     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

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     

SK&F 104078, a post-functionally selective α2-adrenoceptor antagonist in the human saphenous vein in vitro

Summary The present study investigated the effects of SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3methyl-1H,2,3,4,-tetrahydro-3-benzazapine) at pre- and post functional ₂-adrenoceptors in the human isolated saphenous vein. Noradrenaline (0.001–100 mol/l) produced concentration-dependent contractions of the human saphenous vein which were competitively antagonised by the ₁-adrenoceptor antagonist prazosin (0.01–1.0 mol/l) and the ₂-adrenoceptor antagonist, rauwolscine (0.01–1.0 mol/l), indicating the presence of both post functional ₁- and ₂-adrenoceptors in this preparation. The selective ₂-adrenoceptor agonist, UK-14,304 (0.01–100 mol/l) also produced concentration-dependent contractions of the human saphenous vein which were antagonised by both rauwolscine (0.1 mol/l) and prazosin (0.1 mol/l). In the presence of angiotensin II (0.05 mol/l), which itself produced a transient contraction, rauwolscine (0.1 mol/l) produced a rightward shift of the UK-14,304 concentration-response curve while prazosin (0.1 mol/l) had no effect. SK&F 104078 (10.0 mol/l) under these conditions also produced a rightward shift of the concentration-response curve to UK-14,304, but was at least 100-fold less potent than rauwolscine. At pre functional ₂-adrenoceptors, exogenous noradrenaline (0.01 and 0.1 gmol/l) induced a concentration-dependent inhibition of stimulation-evoked [7-³H]-noradrenaline release from the human saphenous vein in vitro, which was antagonised by rauwolscine (0:1 mol/l) and tolazoline (10.0 mol/l) but not by SK&F 104078 (10.0 gmol/l).Rauwolscine (0.1 mol/l) produced a small increase in stimulation-evoked [7-³H]-noradrenaline release while both tolazoline and SK&F 104078 failed to produce any enhancement in release in the absence of exogenous agonist atconcentrationsupto10 gmol/l.Insummary, noradrenaline and UK-14,304 contracted the human isolated saphenous vein by an action at both postfunctional ₁- and ₂-adrenoceptors. These data demonstrate that SK&F 104078 discriminates between post- and pre-junctional ₂-adrenoceptors in the human isolated saphenous vein. Send offprint requests to M. V. Sennitt at the above address     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Antagonism by γ-aminobutyric acid of the stimulant effect of angiotensin II on cardiac sympathetic ganglia in spinal dogs

Summary The effects of angiotensin II and neuro-aminoacids administered through the right subclavian artery (i. a.) to the cardiac sympathetic ganglia were investigated in spinal dogs. Angiotensin II (1–8 g) elicited a dose-dependent positive chronotropic effect which was reduced after i. a. injection of saralasin (100g). The effect of angiotensin II was not reduced after combined treatment with either hexamethonium (10 mg/kg) plus atropine (0.1 mg/kg) or hemicholinium-3 (5 mg/kg) plus preganglionic stimulation. The dosedependent response to angiotensin II of heart rate was inhibited by GABA (50, 500g), GABOB (500g) and muscimol (50, 100g). The inhibition of the response to angiotensin II by a small dose of GABA (50g), but not by a high one (500g), was antagonized by i. a. injection of picrotoxin (2 mg). The positive chronotropism induced by bethanechol (25, 50g) and a small dose of acetylcholine (25g) were significantly inhibited by a high dose (500g) but not by a low dose (50g) of GABA. These results confirm that angiotensin II stimulates cardiac chronotropism by acting on the angiotensin II receptor located at the cardiac ganglia and show that this stimulant effect is antagonized by GABA.     

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     

Methoxamine inhibits noradrenaline release through activation of α1- and α2-adrenoceptors in rat isolated kidney: involvement of purines and prostaglandins

Summary The effects of the at-adrenoceptor agonist methoxamine and the ₂-adrenoceptor agonist bromoxidine (UK 14034) on the stimulation induced (S-1) outflow of radioactivity at 100 Hz/6 pulses from rat isolated kidney preincubated with ³H-noradrenaline were investigated. Methoxamine (0.3 – 30 mol/l) inhibited S-1 outflow of radioactivity to a maximum of 83% with a pEC₅₀ of 5.85 (5.71–5.94). UK 14304 (0.0003-0.3 mol/l) inhibited S-I outflow of radioactivity to a maximum of 99% with a pEC₅₀ of 8.35 (8.26–8.47). a Adrenoceptor antagonist affinities (pK_D) against methoxamine and UK 14304 at prejunctional -adrenoceptors were determined. The concentration response curve of methoxamine was shifted to the right by the 1/_2B-adrenoceptor antagonist prazosin (0.1 mol/l) with a pK_D of 7.41 and that of UK 14304 by prazosin (0.3 mol/l) with a pK_D of 6.24. The ₂-adrenoceptor antagonist rauwolscine (0.1 mol/l) shifted the concentration response curve of UK 14304 potently to the right with a p_KD of 8.34. The concentration response curve of methoxamine was shifted also to the right by rauwolscine (0.1 mol/l) and the ₂-adrenoceptor antagonist idazoxan (0.1 mol/l), however, both antagonists suppressed the maximum response of methoxamine to 466% and 56%, respectively. A ten times lower concentration of rauwolscine (0.01 mol/l) did not shift the concentration response curve of methoxamine but the inhibitory effect of methoxamine still reached only a maximum of 59%. The concentration response curve of methoxamine obtained in the presence of rauwolscine (0.01 mol/l) was shifted to the right by further addition of prazosin (0.1 mol/l) with a pK_D of 8.80 but was also shifted to the right by either the purinoceptor antagonist 8-(p-sulfophenyl) theophylline (8-SPT; 100 mol/l) or the prostaglandin synthesis inhibitor indomethacin (20 mol/l). These results suggest that methoxamine inhibits S-1 outflow of radioactivity in rat isolated kidney probably through three different mechanisms. 1. Activation of postjunctional 1-adrenoceptors and prostaglandin mediated transjunctional inhibition. 2. Activation of postjunctional ₂-adrenoceptors and purine mediated transjunctional inhibition. 3. Activation of prejunctional inhibitory ₂-adrenoceptors at which methoxamine seems to be a partial agonist. Correspondence to L. C. Rump at the above address     

Distinct distribution of opioid receptor types in rat lumbar spinal cord

Summary The distribution of opiate binding sites was studied in sections of rat lumbar spinal cord under conditions selective for , and receptors. While the levels of binding sites were highest in the substantia gelatinosa, elevated levels were also observed in laminae III, IV, V and VIII. In contrast, binding was notable only in lamina I. The levels of typical sites were low, and were concentrated in the substantia gelatinosa. An additional, atypical site was detected using ³H-diprenorphine in the presence of , and receptor blocking agents, and this site was also concentrated in the substantia gelatinosa. Send offprint requests to B. J. Morris at the above address     

Abolition by calcium antagonists of the autoregulation of renal blood flow

Summary Intra-arterial infusions of Ca antagonistic vasodilator substances such as nifedipine (1–10 g/min) and verapamil (30–100 g/min) inhibited renal autoregulation in the perfused dog kidney. Simultaneous infusion of 30 mg/min of CaCl₂ with nifedipine (3 g/min) or with verapamil (30 g/min) abolished the effects of these Ca antagonists. It is suggested that Ca ions play an important role in establishing renal autoregulation.     

5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well.Antagonists to block the contractile response to 5-HT via 5-HT₂, 5-HT₃ or 5-HT₄ receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) strongly inhibited the relaxations to ATP and VIP. l-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of l-NNA (100 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70–100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -Chymotrypsin (0.015 mg·ml^–1) and trypsin (0.005 mg·ml^–1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-d-Phe⁶, Leu¹⁷]VIP (1 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.     

Biphasic effect of 5′-guanylylimidodiphosphate on fat cell adenylate cyclase

Summary 5-Guanylylimidodiphosphate (GMP-PNP) had a biphasic effect on basal adenylate cyclase activity of rat fat cell ghosts, being inhibitory and stimulatory while GTP was mainly inhibitory. At low concentrations of GMP-PNP a transient inhibitory phase preceded the onset of activation. This initial inhibition was overcome by higher concentrations of GMP-PNP, ATP or magnesium.The stimulatory effects of GMP-PNP were increased by high concentrations of ATP or magnesium, the apparent K _m for activation being a function of time. After 5 min of incubation half-maximal activation was obtained at 3 M GMP-PNP, after 20 min of incubation the K _m for GMP-PNP was found to be between 0.1 and 0.3 M. After 20 min of incubation a 15fold increase of cyclase activity above basal level was observed in the presence of 1 M GMP-PNP. GTP competitively inhibited the stimulant effect of GMP-PNP. On the other hand, it activated basal activity only under carefully selected conditions.GMP-PNP and noradrenaline had a synergistic action on cyclase activity. At high substrate concentrations (1 mM ATP) GMP-PNP shifted the apparent K _m for activation by noradrenaline from 3 M to 0.1 M. At low substrate and high magnesium concentrations 1 M noradrenaline was unable to stimulate adenylate cyclase. Under these conditions GMP-PNP facilitated the stimulation by the hormone, although GMP-PNP itself inhibited basal activity.It is suggested that GMP-PNP activates the adenylate cyclase by competing at a common nucleotide binding site with inhibitory agents such as free ATP or GTP. Moreover, the guanyl nucleotide analogue may initiate conformational changes of the enzyme system which facilitate the response to hormones.