Effects of the bispyridinium compounds HGG12, HGG42, and obidoxime on synaptic transmission and NAD(P)H-fluorescence in the superior cervical ganglion of the rat in vitro

Postganglionic compound action potential (AP) and intracellular NAD(P)H-fluorescence were recorded simultaneously in the perifused superior cervical ganglion of the rat (SCG) to study the effects of the bispyridinium oximes HGG12, HGG42 and obidoxime.HGG12 and HGG42 inhibit the compound action potential (AP) (ID₅₀: 70 M) and the reductive part of NAD(P)H changes (ID₅₀: 75 M) recorded upon stimulation of the SCG, while obidoxime has no ganglion blocking effects in concentrations up to 1 mM.The effects of inhibitors of cholinergic transmission were also studied in order to understand the mechanism of action of the oximes. Hexamethonium (C6) and atropine, competitive inhibitors of receptors of nicotinic and muscarinic cholinergic transmission respectively, were found to block synaptic transmission (C6 ID₅₀: 150 M, atropine ID₅₀: 70 M) and the reductive part of the NAD(P)H response (C6 ID₅₀: 70 M, atropine ID₅₀: 50 M) in a quantitatively similar way.Comparison of the ganglionic action of HGG12 and HGG42 with that of the inhibitory agents characterises them as inhibitors of receptors of nicotinic ganglionic transmission. Furthermore at concentrations of about 10 M, HGG12 behaves like atropine and leads to an increase in AP and reductive fluorescence response. It is therefore probable that HGG12 has in addition an affinity for ganglionic muscarinic receptors which HGG42 does not have.Abreviations SCG superior cervical ganglion - AP fast postganglionic action potential - pAP action potential of presynaptic nerve endings - EPSP excitatory postsynaptic potential - mR metabolic response as indicated by the change of NAD(P)H-fluorescence after electrical stimulation - C6 hexamethonium bromide - OP organophosphates - ID50 inhibitory concentration for 50% of AP or integrated mR     

Antagonism by calcium of the inhibitory effect of methylmercury on sympathetic ganglia

Electrophysiological investigation of the characteristics of ganglionic block by methylmercury (mHg) was carried out on the isolated superior cervical ganglion of the rabbit. A low concentration (5 M) of mHg consistently produced a small increase in the amplitude of the compound action potential whereas an inhibitory effect was observed with higher concentrations (10–20 M). The inhibitory effect was temperature-dependent and was not reversible by washing. Raising the external calcium concentration antagonized the inhibitory effect of mHg by markedly slowing down the progression of ganglionic block. The evidence indicates that the blocking effect of mHg is exerted principally on the presynaptic nerve terminals, and may be due to inhibition of acetylcholine release by interfering with the function of calcium at these terminals.     

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     

Reactive red 2: a P2y-selective purinoceptor antagonist and an inhibitor of ecto-nucleotidase

Effects of reactive red 2 and its parent compound acid red 33 were studied in rat vas deferens and guinea-pig taenia coli. In rat vas deferens, reactive red 2 (1 to 10 M) shifted the concentration-response curve for the P_Zx-purinoceptor-mediated contraction effect of , \-methylene ATP slightly to the right and progressively decreased the maximum (apparent antagonist K_d value 0.42 M). Acid red 33 (1000 M) shifted the curve to the right without changing the maximum (apparent K_d 386 M). The concentration-contraction curve of noradrenaline was not altered by reactive red 2. In the carbachol-precontracted guinea-pig taenia coli, reactive red 2 (0.1 to 10 M) shifted the concentration-response curve for the P_2Y-purinoceptor-mediated relaxation effect of adenosine 5-O-(2-thiodiphosphate) (ADP&S) progressively to the right; only at the highest concentration of antagonist (10 M) was the maximum slightly depressed; a pA₂ value of 7.55 (K_d 0.028 M) was derived from the shift. Acid red 33 (1000 M) shifted the concentration-relaxation curve of ADP\S to the right without changing the maximum (apparent K_d 171 M). Reactive red 2 (1 to 10 LM) also shifted the concentration-response curve for the relaxation effect of , \-methylene ATP, which is mediated by an unclassified P₂-purinoceptor, progressively to the right but simultaneously decreased the maximum (apparent K_d1.6 M). The concentration-relaxation curve of 2-chloroadenosine was not altered by reactive red 2. Pieces of vas deferens and taenia coli degraded 76 and 66 % of added ATP (10 M) within 30 min, respectively. Reactive red 2 (0.1 to 100 M) progressively reduced this degradation by up to 95%, with IC₅₀values of 3.9 ± 0.6 and 3.9 ± 2.3 M, respectively. Acid red 33 (1000 M) reduced the degradation by 30 and 20%, respectively.The results indicate that reactive red 2 is a relatively potent antagonist at both P_Zx-purinoceptors in rat vas deferens and P_2Y-purinoceptors in guinea-pig taenia coli, with a 15 fold selectivity for the P_2Y-purinoceptor. It inhibits ecto-nucleotidase in both tissues. The dichloro-triazine residue that distinguishes the compound from acid red 33 greatly enhances the potency at both receptor subtypes as well as at the nucleotidase. As regards P₂-purinoceptor subtypes, the results confirm the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli.     

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     

Diethylstilbestrol potentiation of the dopamine-elicited formation of adenosine 3′,5′-monophosphate in incubated male rat hypothalamus

Summary Dopamine (DA) stimulates the cAMP-generating system in the male rat hypothalamus only to a very low extent (25% above control). Diethylstilbestrol (DES), a synthetic estrogen, was found to be extremely potent (a 4- and 16-fold stimulation at 20 M and 100 M, respectively). Addition of either one to an incubation medium containing varying concentrations of the other resulted in a synergistic response. The potentiation by 20 M DES of the effect elicited by 100 M DA was the most remarkable, namely, a 3-fold stimulation of the combined response. A 4- and 7.5-fold stimulation of cAMP accumulation was observed when adenosine (100 M) or adenosine (100 M)+DA (100 M) were present in the incubation medium. Theophylline (0.5 mM), an adenosine antagonist, could effectively reduce this effect, as did adenosine deaminase (10 g/ml). Clomiphene (50 M), an estrogen antagonist, exhibited a marked decrease in DES+DA-elicited cAMP formation. Pimozide (40 M) had the ability to significantly block the stimulatory effects of DES and DA.     

The effect of some antidepressants on prejunctional muscarinic receptors on the sympathetic nerves of the isolated rabbit ear artery

Summary The antimuscarinic activity of amitriptyline, desipramine, iprindole, mianserin and viloxazine on prejunctional sympathetic nerve endings were compared in the isolated rabbit ear artery. In the presence of cocaine (10 M) and yohimbine (1 M), amitriptyline (0.5–1 M), desipramine (1–3 M) and iprindole (5–10 M), desipramine (1–3 M) and iprindole (5–10 M) produced parallel rightward shifts of the concentration-response curve for the inhibitory effect of carbachol (CCh) on responses to electrical stimulation of the preparation at 3 Hz. Mianserin (3 M) produced some inhibition but altered the slope of the concentration-responses curve to CCh while viloxazine (10 M) produced no inhibition.The depression of tritium efflux by CCh from arteries preincubated in ³H-noradrenaline was inhibited significantly (P<0.05) by amitriptyline (0.1 M) and desipramine (1 M) and not by iprindole (17 M), mianserin (3 M) or viloxazine (10 M). Amitriptyline was 10-fold more active than desipramine and at least 30-fold more active than the other antidepressants as a muscarine receptor blocking drug in this preparation.Thus, mianserin, viloxazine and iprindole exhibit much weaker antimuscarinic activity relative to amitriptyline on prejunctional muscarine receptors on sympathetic nerve endings compared with that observed by others for excitatory muscarine receptors in sympathetic ganglia. The findings support an earlier suggestion that these receptors differ.     

The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 g) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 g; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 g; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 g; i.c.v.). Atropine (10 g; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 g; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 g; i.c.v.) attenuated the pressor effect of U-46619 (1 g; i.c.v.). Higher doses of mecamylamine (75 and 100 g; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 g; i.c.v.) or -bungarotoxin (10 g; i.c.v.), selective antagonists of 7 subtype of nicotinic acetylcholine receptors (7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 g). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 g; i.c.v.) produced the same magnitude of blockade that was observed after the 10 g methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 g; i.c.v.) at the dose of 25 g. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly 7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

The site of action of muscle relaxant purine nucleosides

Summary The effects of a series of purine nucleosides, including the novel marine natural product 1-methylisoguanosine, have been examined on muscle relaxation in conscious animals and on spinal reflexes and neuromuscular transmission in mice anaesthetized with sodium pentobarbitone. 1-Methylisoguanosine (5–15 mol kg^–1) and 2-chloroadenosine (1–5 mol kg^–1), both of which cause muscle relaxation in conscious animals, depressed both mono- and polysynaptic spinal reflexes but did not affect neuromuscular transmission. At much higher doses (300 mol kg^–1) both compounds did depress neuromuscular transmission. Adenosine and 1-methyladenosine did not produce muscle relaxation in conscious animals and only slightly depressed polysynaptic reflexes at the highest doses tested (300 mol kg^–1). Theophylline 50 mol kg^–1 enhanced polysynaptic reflexes and antagonized the depression of these reflexes by 1-methylisoguanosine. Neither adenosine nor 1-methylisoguanosine affected the development of tension by isolated diaphragm muscles in vitro. It is concluded that the muscle relaxant purine nucleosides 2-chloroadenosine and 1-methylisoguanosine produce their effects primarily by depressing activity in the central nervous system. Transmission at the neuromuscular junction is not affected at doses in the range of those producing muscle relaxation.     

β-methyl-digoxin

Summary The tissue/plasma ratio of -methyl-digoxin for cardiac muscle in cats was about the same 24 h after a single dose of 30 g/kg as after a loading dose of 30 g/kg followed by 3 maintenance doses of 7.5 g/kg at 24 h intervals. The ratio for the brain increased 2-fold during that time.After the i.v. injection of a toxic loading dose of 70 g/kg -methyl-digoxin or digoxin, maintenance doses of as little as 15 g/kg at 48 h intervals sufficed to maintain the minimum plasma glycoside concentrations determined by RIA at about 3 ng/ml. There was no difference in the plasma concentrations or in the severity of intoxication produced by both glycosides.Cats vomited within 3 h after i.v. injection of 100 g/kg -methyl-digoxin, whereas a loading dose of 30 g/kg followed by 3 injections of 7.5 g/kg at 24 h intervals were well tolerated. The concentration of radioactivity in the brain 3 h after 100 g/kg was less than 24 h after the last injection of 7.5 g/kg in the experiments with repeated dosage.Cerebral side-effects such as vomiting, loss of appetite and weight were better correlated with the glycoside concentrations in the plasma than with those in the brain.     

Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro

Previous reports on a series of benzoylthiophenes, including PD 81,723 {2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl)thiophene}, have shown specific enhancement of agonist binding at the adenosine A₁ receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 {thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester} and RS-74513-000 {2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene} on response elicited by adenosine A₁ receptors in isolated guinea pig left atrium and ileum.In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA {N⁶-cyclopentyladenosine} with a pK_B value of 6.2 ± 0.2 (n = 4) . At a low concentration which had no antagonistic effect (0.1 M), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4–11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 M) did not enhance or antagonize effects of CPA. At concentrations above 1 M, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A₁ antagonist CPX {8-cyclopentyl-1,3-dipropyl-xanthine} (1 M).Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 M) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 M). Maximum enhancement was observed at 3 M; the concentration-response curve to CPA was shifted leftward by 3.2 fold (95% confidence limits 2.4–4.2). Higher concentrations of RS-74513-000 (10 and 30 M) decreased electrically induced contraction, this effect was not reversed by CPX. These findings confirmed that functional effects of A₁ adenosine receptor may be enhanced by substituted benzoylthiophenes in vitro. The differential effect of PD 78,416 and RS-74513-000 on cardiac and ileal A₁ receptors suggests that it may be possible to design selective enhancers for cardiac and neural functions.     

Effects of intrathecal or intracerebroventricular pretreatment with pertussis toxin on antinociception induced by β-endorphin or morphine administered intracerebroventricularly in mice

We have previously demonstrated that -endorphin and morphine, when administered supraspinally, produce antinociception by activating different descending pain inhibitory systems in both rats and mice. However, the signal transduction mechanisms involved in the descending pain-inhibitory systems that are activated by -endorphin and morphine administered intracerebroventricularly (i.c.v.) have not been characterized. Therefore, in the present study, the effects of intrathecal (i.t.) and i.c.v. pretreatments with pertussis toxin (PTX) on antinociception induced by -endorphin or by morphine administered i.c.v. were studied in ICR mice. Antinociception was assessed by the tail-flick assay and by the hotplate assay. Intrathecal pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick response induced by -endorphin (1 g) or by morphine (1 g) administered i.c.v. However, i.t. pretreatment with PTX was not effective in reducing the inhibition of the hot-plate response induced by -endorphin or by morphine administered i.c.v. Intracerebroventricular pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick and hot-plate responses induced by morphine (1 g), but not that induced by -endorphin (1 g), administered i.cv. Our results suggest that there are PTX-sensitive G proteins coupled to the spinal descending pain inhibitory systems that are activated by -endorphin and morphine administered i.c.v. At a supraspinal level, i.cv. morphine- but not -endorphin-induced antinociception is mediated by PTX-sensitive G proteins. Correspondence to: Hong W. Suh at the above address     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Inhibition of dopamine-sensitive adenylate cyclase by opioids: possible involvement of physically associated µ- and δ-opioid receptors

Summary D-1 dopamine receptor-stimulated cyclic AMP efflux from rat neostriatal slices (induced by 30 M dopamine + 10 M (–)sulpiride) was concentration-dependently reduced by morphine, [abetd-Ala-abetd-Leu]-enkaphalin (DADLE), [d-Pen-abetd-Pen]enkephalin (DPDPE) and bremazocine. Naloxone (0.1 M) selectively antagonized the inhibitory effect of (a submaximally effective concentration of) morphine, whereas ICI 174864 (0.75 M) completely blocked the inhibitory effects of DADLE, DPDPE and bremazocine without affecting that of morphine, indicating a role of µ- as well as -opioid receptors. Upon simultaneous activation of D-1 dopamine receptors and b-opioid receptors the (-receptor-mediated) inhibitory effect of morphine was abolished, while it was not changed following simultaneous activation of D-1 and (inhibitory) D-2 dopamine receptors. Cyclic AMP efflux induced by isoprenaline or adenosine was not affected by the opioids and that induced by vasoactive intestinal peptide (VIP) was inhibited by morphine and DADLE only. In the latter case naloxone, but not ICI 174864, antagonized the inhibitory effects.These data show that D-1 dopamine receptor-stimulated adenylate cyclase activity in rat neostriatum, but not that stimulated through other receptors, is inhibited by two pharmacologically distinct opioid receptor subtypes. It is speculated that these - and -opioid receptors share a common inhibitory guanine nucleotide binding protein and may represent closely associated recognition sites of a functional opioid receptor complex. Send offprint requests to A. N. M. Schoffelmeer at the above address     

P2-purinoceptor-mediated autoinhibition of sympathetic transmitter release in mouse and rat vas deferens

Effects of drugs acting at P₂-purinoceptors on the release of newly taken up [³H]-noradrenaline were studied in slices of mouse and rat vas deferens. The slices were superfused and stimulated electrically, in most experiments by trains of 60 pulses/8 Hz.In mouse vas deferens, the P₂-purinoceptor antagonists reactive blue 2 (1.8–100 M) and brilliant blue G (10–300 M) increased the stimulation-evoked overflow of tritium in a concentration-dependent manner as shown previously for suramin. Reactive blue 2, which preferentially blocks the P_2Y-subtype, was the most potent compound and the compound with highest maximal effect, an increase by 104%. Pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), in contrast, caused a small increase only at a single concentration (30 M). The effects of reactive blue 2, brilliant blue G and suramin were not additive. The P₂ agonist adenosine 5-O-(3-thio)-triphosphate (ATPS) reduced the evoked overflow of tritium. As shown previously for suramin, reactive blue 2 30 M and brilliant blue G 100 M antagonized the effect of ATPS. From the shift of the ATPS concentration-response curve to the right, an apparent pK_B value of 5.3 was estimated for reactive blue 2 and an apparent pKB of 4.5 for brilliant blue G. In rat vas deferens, reactive blue 2 (3–30 M), brilliant blue G (10 M) and suramin (30–300 M) also increased the evoked overflow of tritium. As in the mouse, reactive blue 2 was the most potent compound and the compound with highest maximal effect, an increase by 9001o. As previously demonstrated in the mouse, suramin (300 M) increased the evoked overflow of tritium only when rat vas deferens slices were stimulated by trains of 60 pulses at 1 or 8 Hz, but not when they were stimulated by trains of 6 pulses/100 Hz.The results confirm the operation of a P₂-purinoceptor-mediated prejunctional negative feedback controlling the release of noradrenaline in mouse vas deferens and demonstrate the same mechanism in rat vas deferens. The prejunctional P₂-purinoceptors are P_2Y-like in both species. They are a novel kind of autoreceptors, operating in parallel to prejunctional ₂-autoreceptors. Correspondence to: I. von Kügelgen at the above address     

Inhibitory adenosine A1-receptors on rat locus coeruleus neurones

Summary Intracellular recordings were performed in ₁-pontine slice preparation of the rat brain containing the locus coeruleus (LC). Adenosine (100, 300 mol/l) and its structural analogues, namely (–)-N⁶-(R-phenyliso-propyl)-adenosine (R-PIA; 3 – 30 mol/l) and S-PIA (10, 30 mol/l), as well as 5-N-ethylcarboxamido-adenosine (NECA; 3–30 mol/l) inhibited the firing rate of spontaneous action potentials and produced hyperpolarization; their rank order of potency was RPIA - NECA > S-PIA > adenosine. When applied by superfusion, all agonists strongly desensitized the LC cells; the hyperpolarization never surmounted 6 mV. Upon pressure ejection of adenosine 10 mmol/l from ₁- micropipette positioned close to an LC neurone, the membrane potential was raised by 14 mV and the apparent input resistance decreased by 20%. When the membrane potential was hyperpolarized by current injection to ₁- similar extent as adenosine did, the fall in input resistance was only 7%. The adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) 30 mol/l decreased the frequency of action potentials alone; on simultaneous bath-application with adenosine 300 mol/l it potentiated the hyperpolarization caused by the purine derivative. 8-Cyclopentyl-1,3-dipropylxanthine (CPDPX) 0.1 mol/l had no effect on its own, but it antagonized both R-PIA 30 mol/l and NBTG 30 mol/l. A higher concentration of CPDPX (1 mol/l) facilitated the spontaneous firing. In conclusion, both exogenous and endogenous adenosine activates somatic and/or dendritic A₁-receptors of LC neurones leading to an enhancement of potassium conductance and thereby to ₁- decreased firing rate and ₁- hyperpolarization. Send offprint requests to P. Illes at the above address     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

Trifluoperazine and calmidazolium have multiple actions on the release of noradrenaline from sympathetic nerves of mouse atria

The aim of this study was to determine whether the calmodulin inhibitors trifluoperazine (TFP) and calmidazolium (CMZ) could decrease the action-potential-evoked release of noradrenaline from mouse isolated atria incubated with [³H]-noradrenaline in support of the hypothesis that calmodulin is involved in neurotransmitter release.TFP (10 M and 30 M) significantly enhanced stimulation-induced (S-1) outflow of radioactivity from mouse atria but had no effect at 1.0 M or 70 M. TFP (70 M) also significantly increased the spontaneous outflow of radioactivity. The facilitatory effect of TFP (10 M) on S-I outflow of radioactivity persisted in either the presence of 3-isobutyl-1-methylxanthine (100 M) or atropine (0.3 M) indicating that this effect of TFP was not mediated through either inhibition of phosphodiesterases or through interference with presynaptic muscarinic receptors, respectively. In the presence of phentolamine, the facilitatory effect of TFP (10 M) on S-I outflow was reduced but there was no effect on S-I outflow at 70 M. However, in the presence of a combination of both phentolamine (l.0 M) and the neuronal uptake blocker desipramine (1.0 M) a significant inhibitory effect of TFP (70 M) on the S-I outflow of radioactivity was observed, indicating that effects of TFP on presynaptic a-adrenoceptors and neuronal uptake had disguised an inhibitory effect on S-1 noradrenaline release. Another inhibitor of the Ca²⁺-calmodulin complex, calmidazolium (CMZ, 10 M) inhibited the S-1 outflow of radioactivity but had no effect at 1.0 M. However, CMZ (10 M) also induced a concomitant increase in the spontaneous outflow of radioactivity. In the presence of both phentolamine (1.0 M) and desipramine (1.0 M), CMZ (10 M) also decreased S-1 outflow of radioactivity. The spontaneous outflow of radioactivity by calmidazolium (10 M) was mainly attributable to a rise in unmetabolized noradrenaline.Since concentrations of both TFP and CMZ, which inhibited S-1 noradrenaline release, also caused an increase in the spontaneous outflow of radioactivity, it is possible that the inhibitory effects on S-1 noradrenaline release may be secondary to changes in spontaneous outflow. This suggests that these drugs have complex effects on transmitter release dynamics which are perhaps due to multiple roles for calmodulin within the sympathetic nerve terminal. Correspondence to: M. Barrington at the above address     

The cardiovascular effects of intraventricularly administered histamine in the anaesthetised rat

Summary In urethane-anaesthetised rats intraventricular (i.c.v.) injections of histamine (0.1–10.0 g) elicited dose-related rises in both the resting blood pressure and heart rate. These cardiovascular effects of histamine were antagonised in a dose-dependent manner by i.c.v. pretreatments with the histamine H₁-receptor antagonists mepyramine (10, 50 and 100 g) and diphenylpyraline (100 and 200 g). Pretreatment with the histamine H₂-receptor antagonist metiamide (100 and 200 g i.c.v.) failed to modify either of the responses. A dose-related antagonism of the hypertensive response to histamine i.c.v. was elicited by phentolamine (100 and 200 g i.c.v.) but the positive chronotropic effect was not modified by this pretreatment. The cardiovascular responses to histamine i.c.v. were abolished by mecamylamine (5.0 mg/kg i.v.) and greatly reduced by 6-hydroxydopamine (3×250 g i.c.v.), but only the tachycardia was significantly modified by atropine (100 g i.c.v.) and propranolol (1 mg/kg i.v.). Propranolol (100 g i.c.v.), bilateral vagotomy, or acute bilateral adrenal demedullation failed to modify the cardiovascular responses to histamine i.c.v. The results suggest that histamine is able to modify the resting blood pressure and heart rate by independent central modes of action, which involve central adrenergic and cholinergic mechanisms.Preliminary findings of this study were presented at the Autumn meeting of the British Pharmacological Society (Finch and Hicks, 1975).