Evidence for facilitatory and inhibitory muscarinic receptors on postganglionic sympathetic nerves in mouse isolated atria.

1. McNeil A 343 (10 microM-30 microM) enhanced the fractional stimulation-induced (S-I) outflow of radioactivity from mouse isolated atria which had been incubated with [3H]-noradrenaline. The enhancing effect of McNeil A 343 was not altered by hexamethonium (300 microM) suggesting that it was not due to an action at nicotinic receptors. It is also unlikely that McNeil A 343 enhanced the S-I outflow of radioactivity in mouse atria by blocking neuronal reuptake of noradrenaline since the effect persisted in the presence of cocaine (30 microM). 2. The facilitatory effect of McNeil A 343 on the S-I outflow of radioactivity was attenuated by atropine (0.3 microM), pirenzepine (0.2 microM or 1.0 microM), dicyclomine (1.0 microM) and methoctramine (1.0 microM) and was thus due to activation of muscarinic receptors. 3. In contrast to the effect of McNeil A 343, another muscarinic receptor agonist, carbachol (3.0 microM) significantly decreased the S-I outflow of radioactivity. The receptors through which McNeil A 343 acts to enhance the S-I outflow of radioactivity appear to be distinct from inhibitory prejunctional muscarinic receptors. The relatively M 1-selective antagonist, pirenzepine (0.2 microM), attenuated the facilitatory effect of McNeil A 343 whereas a higher concentration (1.0 microM) was required to block the inhibitory effect of carbachol. Conversely, the relatively M2-selective antagonist, methoctramine (0.1 microM), blocked the inhibitory effect of carbachol but a higher concentration of methoctramine (1.0 microM) was required to block the facilitatory effects of McNeil A 343.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of acetylcholine release in the guinea-pig trachea by the nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP)

The effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the NO synthase inhibitor L-N(G)-nitroarginine (L-NOARG) on the electrically evoked [(3)H]-acetylcholine release were studied in an epithelium-free preparation of guinea-pig trachea that had been preincubated with [(3)H]-choline. SNAP (100 and 300 microM) caused small but significant increases of the electrically evoked [(3)H]-acetylcholine release (121+/-4% and 124+/-10% of control). Resting outflow of [(3)H]-ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 1 microM). The facilitatory effect of SNAP (100 and 300 microM) was reversed into inhibition of release (to 74+/-4% and to 78+/-2%) after pretreatment of the trachea with capsaicin (3 microM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [(3)H]-acetylcholine. A significant inhibition by SNAP (100 microM) of [(3)H]-acetylcholine release (78+/-3%) was also seen in the presence of the NK(2) receptor antagonist SR 48968 (30 nM). L-NOARG (10 and 100 microM) significantly enhanced the electrically-evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies. It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK(2) receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release.     

The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [(3)H]-choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation.2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [(3)H]-ACh which was greatly enhanced by the addition of atropine.3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [(3)H]-ACh which declined asymptotically. This evoked release appeared to follow first-order kinetics with a rate constant which remained stable over the course of prolonged stimulation.4 The rate constant for the evoked release of [(3)H]-ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [(3)H]-ACh was released was nearly identical under these two conditions.5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [(3)H]-choline in the samples.6 Reduction of [(3)H]-ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [(3)H]-ACh without affecting the release of [(3)H]-choline.7 Results suggest that electrical stimulation of cortical slices which had been incubated with [(3)H]-choline causes the release of only [(3)H]-ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [(3)H]-ACh.     

Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the epithelium on acetylcholine release from parasympathetic nerves of the rat trachea

1. The present study was undertaken to investigate the influence of the airway epithelium on the release of acetylcholine (ACh) from parasympathetic nerves of the rat trachea. Epithelium-intact and epithelium-denuded preparations of rat trachea were incubated with [3H]-choline to incorporate [3H]-ACh into the cholinergic transmitter stores. Release of radiolabelled transmitter ACh was evoked by electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V). 2. Field stimulation both of epithelium-intact and epithelium-denuded radiolabelled tracheal preparations evoked an increase in the efflux of radioactivity; however, the mean stimulation-induced (S-I) efflux from epithelium-denuded preparations (2932 +/- 190 d.p.m., n = 9) was approximately 60% of that from epithelium-intact preparations (4802 +/- 820 d.p.m., n = 11). We have shown previously that, in epithelium-intact (but not epithelium-denuded) tracheal preparations, a substantial proportion of the S-I efflux is resistant to tetrodotoxin (1 microM) and to the removal of extracellular Ca2+, indicating that much of the S-I efflux is not caused by exocytotic release of neuronal [3H]-ACh. In epithelium-denuded tracheal preparations, superfused individually, phosphorylcholine (1 and 100 microM) did not alter S-I efflux. In epithelium-intact tracheal preparations, both in the absence and in the presence of atropine (1 microM), neither N(G)-nitro-L-arginine (100 microM), superoxide dismutase (100 units ml(-1)), indomethacin (10 microM), capsaicin (30 microM) nor alpha-chymotrypsin (1 unit ml(-1)) altered S-I efflux. 3. Experiments were also performed using two tracheal preparations superfused in series. When unlabelled epithelium-intact preparations were present in the upper chamber (superfused first), the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber (superfused second) did not differ significantly from radiolabelled epithelium-denuded preparations superfused individually. Moreover, there was no significant difference in the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber between experiments in which the upper chamber contained epithelium-intact or epithelium-denuded preparations. 4. Field stimulation of epithelium-intact tracheal preparations in the upper chamber with 90, 120 and 300-s periods (trains of 1 ms pulses, 5 Hz, 15 V) did not significantly alter the S-I efflux from radiolabelled epithelium-denuded tracheal preparations in the lower chamber. 5. When introduced into the upper (unlabelled epithelium-intact) and subsequently allowed to superfuse the lower (radiolabelled epithelium-denuded) tracheal preparations, the stable cholinomimetic carbachol (3 microM) markedly reduced the S-I efflux whereas ACh (0.1 and 1 microM) had no significant effect. However, in the presence of the anti-cholinesterase neostigmine (1 microM), ACh (1 microM) significantly reduced S-I efflux, indicating that ACh is subject to rapid hydrolysis by cholinesterase enzymes. When atropine (10 microM) was only exposed to radiolabelled epithelium-denuded preparations in the lower chamber, the inhibitory effects of ACh (1 microM) and carbachol (3 microM) on S-I efflux were prevented. 6. In conclusion, the findings of the present study do not support the notion that the airway epithelium exerts an inhibitory influence on ACh release from parasympathetic nerves of the rat trachea. Alternatively, if epithelium-dependent modulation of cholinergic transmission does occur in the rat trachea, then the mechanism does not appear to involve phosphorylcholine, nitric oxide, superoxide radicals, cyclo-oxygenase products of arachadonic acid, capsaicin-sensitive neuropeptides or vasoactive intestinal peptide. Moreover, the inhibitory effect of carbachol and ACh on transmitter ACh release in the rat trachea appears to be due solely to activation of prejunctional inhibitory muscarinic cholinoceptors on parasympathetic nerves and does not involve the liberation of a putative epithelium-derived inhibitory factor.     

Effects of endogenous and synthetic prostanoids, the thromboxane A2 receptor agonist U-46619 and arachidonic acid on [3H]-noradrenaline release and vascular tone in rat isolated kidney.

1. Rat kidneys were perfused with Krebs-Henseleit solution and the perfusion pressure was monitored. After incubation with [3H]-noradrenaline the renal nerves were stimulated. The stimulation-induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. The effect of prostaglandins on perfusion pressure, pressor responses to renal nerve stimulation (RNS) and S-I outflow of radioactivity was assessed. 2. Prostaglandin E2 (PGE2, 0.06 and 0.6 microM), PGF2 alpha (0.6 microM), PGI2 (0.6 and 3 microM) and iloprost (0.6 microM) increased perfusion pressure and enhanced pressor responses to RNS. These facilitatory effects of the prostaglandins were not a result of an enhanced transmitter release. In contrast, PGE2 dose-dependently inhibited, whereas the other prostaglandins failed to modulate S-I outflow of radioactivity. PGE2 (0.6 microM) also enhanced pressor responses to exogenous noradrenaline. 3. Arachidonic acid (1 microM) increased perfusion pressure and enhanced pressor responses to RNS. These effects were abolished in the presence of indomethacin (10 microM) suggesting that local production of prostaglandins from exogenous arachidonic acid was responsible for this facilitation. However, arachidonic acid (1 microM) did not modulate S-I outflow of radioactivity. Arachidonic acid (10 microM), despite causing a marked increase in perfusion pressure, failed to alter pressor responses to RNS and only slightly inhibited S-I outflow of radioactivity. 4. The thromboxane A2 (TxA2) receptor agonist U-46619 (0.1 microM) increased vascular tone and enhanced pressor responses to RNS. These effects were blocked by the newly developed selective TxA2 receptor antagonist, daltroban (BM 13505; 3 microM), suggesting that these facilitatory effects of U-46619 were due to activation of TxA2 receptors. However, U-46619 failed to alter the S-I outflow of radioactivity from rat isolated kidney. 5. The alpha 1-adrenoceptor agonist methoxamine (1 microM) also increased perfusion pressure and enhanced pressor responses to RNS without affecting the S-I outflow of radioactivity in the presence of the prostaglandin synthesis inhibitor indomethacin (10 microM). 6. The results suggest that PGE2 modulates noradrenaline release through an inhibitory prejunctional receptor mechanism. There is no evidence for prejunctional PGF2 alpha, PGI2 or TxA2 receptors in the rat isolated kidney. All prostaglandins increased vascular tone in the rat isolated kidney and this alone may provide a condition for enhanced pressor responses to RNS since methoxamine also enhanced pressor responses to RNS without affecting S-I outflow of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Poly-L-arginine-mediated release of acetylcholine from parasympathetic nerves in rat and guinea-pig airways.

1. The synthetic cationic polypeptide, poly-L-arginine (0.03-1 mg ml-1) induced concentration-dependent contraction of guinea-pig and rat isolated trachea. In guinea-pig isolated trachea, this response was attenuated in the presence of the muscarinic cholinoceptor antagonist, atropine (0.1 microM) and augmented by the acetylcholinesterase inhibitor, ecothiophate (0.1 microM). The neuronal sodium channel blocker, tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine and acetylcholine. 2. The contractile response to poly-L-arginine in rat isolated trachea was inhibited in the presence of atropine (0.1 microM) and the 5-hydroxytryptamine (5-HT) receptor antagonist, methysergide (1 microM). Treatment of rat tracheal preparations with capsaicin (100 microM) or tetrodotoxin (3 microM) failed to alter the contractile response to poly-L-arginine. In contrast, ecothiophate (0.1 microM) augmented the contractile response to poly-L-arginine in rat isolated trachea. 3. Electrical field stimulation (5 Hz, 2 min) of epithelium-denuded guinea-pig tracheal preparations preloaded with [3H]-choline resulted in a contractile response and the simultaneous efflux of radioactivity into the superfusate. Both these responses were abolished in the presence of tetrodotoxin (1.5 microM). Poly-L-arginine (1 mg ml-1) also increased the efflux of total radioactivity from epithelium-denuded guinea-pig isolated tracheal preparations preloaded with [3H]-choline, but this response was tetrodotoxin-insensitive. The negatively charged polyanion, heparin (1 mg ml-1) failed to increase significantly the efflux of radioactivity from epithelium-denuded preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of noradrenaline release in slices of rat kidney cortex through alpha 1- and alpha 2-adrenoceptors

Slices of rat kidney cortex were incubated in [3H]noradrenaline, then placed in a flow cell and subjected to electrical field stimulation. At a stimulation frequency of 5 Hz, both the alpha 2-adrenoceptor antagonist idazoxan (0.1 microM) and the alpha 1-adrenoceptor antagonist prazosin (0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity from the slice. However, neither clonidine (0.1 microM) nor methoxamine (10 microM), alpha 2- and alpha 1-agonists respectively, affected the S-I outflow of radioactivity at this stimulation frequency. At a lower stimulation frequency (1 Hz), the S-I outflow of radioactivity was not affected by idazoxan or prazosin, but was inhibited by both clonidine and methoxamine. The effect of clonidine was prevented by idazoxan (0.1 microM), but not by prazosin (0.1 microM). The effect of methoxamine was abolished by prazosin (0.1 microM), but not by idazoxan (0.1 microM). The inhibitory effect of methoxamine was not prevented by the prostaglandin synthesis inhibitor indomethacin (10 microM) or the adenosine receptor antagonist 8-phenyltheophylline (1 microM) and thus was not mediated by either prostaglandins or adenosine. The results suggest that both prejunctional alpha 1- and alpha 2-adrenoceptors are directly involved in modulation of noradrenaline release from the renal sympathetic nerves of the rat.     

Ketamine induces hyperactivity in rats and hypersensitivity to nicotine in rat striatal slices

The symptoms of schizophrenia can be modeled in rats through blockade of ionotropic glutamate receptors, which induces changes in central dopamine circuits. These circuits also contain nicotinic acetylcholine receptors that are activated by nicotine. A role for nicotine in the etiology of schizophrenia is supported by clinical observations of high tobacco use rates in individuals experiencing the psychopathology. The present study investigated the effect of the ionotropic glutamate receptor antagonist ketamine on the function of striatal nicotinic acetylcholine receptors to understand better the potential role of these receptors in schizophrenia. Ketamine (0.1-300 microM) was ineffective to evoke [(3)H] overflow from rat striatal slices preloaded with [(3)H]dopamine. Application of psychotomimetic ketamine concentrations (1-10 microM) to striatal slices augmented nicotine-evoked [(3)H] overflow. Finally, rats received ketamine (30-50 mg/kg) injections for 30 days, to model the development of the disorder, and hyperactivity was observed, although repeated ketamine treatment did not significantly alter nicotine-evoked [(3)H]dopamine overflow. These data indicate that the function of nicotinic acetylcholine receptors that mediate dopamine release are altered by ketamine, and support a role for nicotinic acetylcholine receptors in schizophrenia pathology.     

Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway

Summary In mouse atria previously incubated with [³H]-noradrenaline, carbachol (1.0 μmol/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the α-adrenoceptor antagonist phentolamine (1.0 μmol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 μmol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 μmol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,100 μmol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 μmol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 μmol/l) plus IBMX (100 μmol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 μmol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the postreceptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 μg). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 μg). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein. Send offprint requests to M. Costa at the above address     

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype

1. Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2. In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 microM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%). 3. Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 microM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM-1 microM), and enhanced by atropine (0.1-100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. 4. The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (-log EC50) orders at pre- and postjunctional level were: atropine > or = 4-diphenyl-acetoxy-N-piperidine (4-DAMP) > mamba toxin 3 (MT-3) > tripitramine > para-fluorohexahydrosiladiphenidol (pF-HHSiD) > or = methoctramine > or = pirenzepine > tripinamide, and atropine > or = 4-DAMP > pF-HHSiD > pirenzepine = tripitramine > tripinamide > methoctramine > MT-3, respectively. 5. The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.     

Prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are sensitive to N-ethylmaleimide, but not pertussis toxin.

1. The identity of the G-proteins involved in prejunctional alpha 2-adrenoceptor signal transduction in mouse atria was examined by use of the G-protein inactivators N-ethylmaleimide and pertussis toxin. 2. The alpha 2-adrenoceptor partial agonist clonidine (0.03 microM) inhibited the electrical stimulation-induced (S-I) outflow of radioactivity from mouse atria which were incubated with [3H]-noradrenaline and stimulated at 5 Hz. The partial alpha 2-adrenoceptor agonist St 363 (10 microM) inhibited the S-I outflow of radioactivity at the lower stimulation frequency of 2.5 Hz. The inhibitory effects of these compounds were not altered in mice pretreated with pertussis toxin (1.5 micrograms, i.v.). 3. The alpha 2-adrenoceptor antagonist, idazoxan (0.1 microM), increased the S-I outflow of radioactivity from mouse atria stimulated at 5 Hz, and this effect was not altered in atria from mice pretreated with pertussis toxin. 4. The inhibitory effects of clonidine and St 363 and the facilitatory effect of idazoxan on the S-I outflow of radioactivity from mouse atria were significantly less in atria incubated with N-ethylmaleimide (NEM, 3 microM) for 60 min before the [3H]-noradrenaline incubation. 5. The results suggest that prejunctional alpha 2-adrenoceptors in mouse atria function through G-proteins which are NEM-sensitive, but pertussis toxin insensitive.     

The effects of LSD in the guinea-pig ileum

The effects of lysergic acid diethylamide (LSD) on acetylcholine release and on smooth muscle tone were studied in the myenteric plexus-longitudinal muscle preparation of the guinea pig. LSD (0.01-10 microM) depressed in a concentration-dependent manner the electrically-evoked [3H]-acetylcholine outflow from strips preincubated with [3H]-choline. The maximal effect was a 45% inhibition by 1 microM LSD. The spontaneous outflow was not affected. Metitepine competitively antagonized (pA2 8.0) the LSD-induced reduction of the evoked outflow. Tolazoline and mepyramine did not affect the inhibitory action of LSD. The contractions in response to electrical stimulation were enhanced by 34% in the presence of 0.1 microM LSD. Other concentrations of LSD did not affect the twitches. LSD caused an increase in muscle tone which was antagonized non-competitively by mepyramine, metitepine and ketanserin. Ketanserin was a competitive antagonist against the histamine-induced contractions of the longitudinal muscle (pA2 8.49). The results suggest that LSD stimulates presynaptically located 5-HT receptors and thereby decreases the evoked acetylcholine release. In addition, LSD increases smooth muscle tone either directly through stimulation of H1 receptors or indirectly via histamine release.     

Comparison of the effects of bupropion on nicotinic receptor-evoked [(3)H]dopamine release from rat striatal synaptosomes and slices

Tobacco smoking is a nicotine addiction, mediated in part by the ability of nicotine to elicit dopamine release, as a result of the stimulation of nicotinic acetylcholine receptors associated with dopaminergic pathways. The smoking cessation agent bupropion is an inhibitor of the dopamine transporter, but has also been shown to inhibit nicotinic acetylcholine receptors. To assess the relative impact of its actions at these two targets, we have examined the effects of bupropion on nicotine-evoked [(3)H]dopamine release from rat striatal synaptosomes and slices, in the absence of any other transporter inhibitor. Bupropion (10 microM) significantly decreased nicotine-evoked [(3)H]dopamine release by approximately 50% in both preparations, consistent with the blockade of nicotinic receptors. In support of this interpretation, bupropion also selectively inhibited nicotine-evoked Ca(2+) increases in SH-SY5Y cells. In striatal slices (but not in synaptosomes) the concentration-response profile for bupropion has an inverted 'u' shape, with a decrease in nicotine-evoked [(3)H]dopamine release also observed in the presence of 0.1 microM bupropion. This effect of 0.1 microM bupropion (but not 10 microM bupropion) was reversed by the dopamine D(2) receptor antagonist raclopride. We propose that modest blockade of the dopamine transporter by low concentrations of bupropion results in feedback inhibition via dopamine D(2) autoreceptors. This is overcome at higher concentrations of bupropion, before inhibition of nicotinic receptors occurs. Therefore bupropion's inhibition of the dopamine transporter and nicotinic receptors appears to be separated with respect to concentration.     

Modafinil evokes striatal [(3)H]dopamine release and alters the subjective properties of stimulants

Modafinil is a mild psychostimulant used for the treatment of sleep and arousal-related disorders, and has been considered a pharmacotherapy for cocaine and amphetamine dependence; however, modafinil's mechanism of action is largely unclear. The present study investigated modafinil using drug discrimination and slice superfusion techniques. Rats were trained to discriminate cocaine (1.6 or 5 mg/kg) or amphetamine (0.3 mg/kg) from saline injection for food reinforcement. Modafinil (64-128 mg/kg) substituted partially for both cocaine doses and amphetamine. Pretreatment with a lower modafinil dose (32 mg/kg) augmented the discriminative stimulus properties of cocaine (1.6 mg/kg dose group) and amphetamine. In neurochemical experiments, modafinil (100-300 microM) evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine in a concentration-dependent manner; however, modafinil was less potent and efficacious than amphetamine and nicotine. The dopamine transporter inhibitor nomifensine (10 microM) blocked modafinil-evoked [(3)H]overflow, and concentrations of modafinil (<100 microM) that did not have intrinsic activity attenuated amphetamine (1 and 3 microM)-evoked [(3)H]overflow. Modafinil-evoked [(3)H]overflow was not altered by the nicotinic acetylcholine receptor antagonist mecamylamine, and modafinil did not alter nicotine-evoked [(3)H]overflow, indicating that nicotinic acetylcholine receptors likely are not important for modafinil's mechanism of action. The present results indicate that modafinil evokes dopamine release from striatal neurons and is a psychostimulant that is pharmacologically similar to, but much less potent and efficacious than, amphetamine.     

Multiple prejunctional actions of angiotensin II on noradrenergic transmission in the caudal artery of the rat.

1. Angiotensin II produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The threshold concentrations of angiotensin II for enhancement of S-I efflux (between 0.03 and 0.1 microM) and of the stimulation-evoked vasoconstrictor responses (about 0.3 microM) were 10-1000 times higher than those that have been found for several other vascular preparations. 2. The AT1 angiotensin II receptor antagonist losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of S-I efflux by 1 and 3 microM angiotensin II and the enhancement of vasoconstrictor responses by 1 microM angiotensin II. Surprisingly, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced S-I efflux to a much greater extent than did 0.1 microM angiotensin II alone. Moreover, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced stimulation-evoked vasoconstrictor responses, in contrast to the lack of effect of 0.1 microM angiotensin II alone. 3. In a concentration of 0.01 microM, the angiotensin II AT2 receptor antagonist PD 123319 did not affect the enhancement of either S-I efflux or vasoconstrictor responses by angiotensin II. However, in a higher concentration (0.1 microM), PD 123319 antagonized the enhancement of both the S-I efflux and vasoconstrictor responses by angiotensin II. 4. In concentrations of 0.01 and 0.1 microM, PD 123319 prevented the marked enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses produced by the combination of 0.1 microM angiotensin II and 0.01 microM losartan. 5. The potentiation by losartan (0.01 microM) of the facilitatory effect of 0.1 microM angiotensin II on S-I efflux and on stimulation-evoked vasoconstriction was still observed in the presence of either the cyclooxygenase inhibitor indomethacin (3 microM), or the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. The findings confirm our previous suggestion that, in the rat caudal artery, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 7. The synergistic prejunctional interaction of 0.01 microM losartan and 0.1 microM angiotensin II may be due to either the unmasking by losartan of a latent population of angiotensin II receptors also subserving facilitation of transmitter noradrenaline release, or alternatively, losartan may block an inhibitory action of angiotensin II on transmitter noradrenaline release which normally opposes its facilitatory effect.     

Enhanced acetylcholine release in the hippocampus of cannabinoid CB(1) receptor-deficient mice

We examined whether acetylcholine release in the hippocampus and striatum and noradrenaline release in the hippocampus is altered in CB(1) receptor-deficient mice. The electrically evoked tritium overflow from hippocampal slices preincubated with [(3)H]-choline was increased by about 100% in CB(1)(-/-) compared to CB(1)(+/+) mice whereas the electrically evoked tritium overflow from striatal slices preincubated with [(3)H]-choline and from hippocampal slices preincubated with [(3)H]-noradrenaline did not differ. The cannabinoid receptor agonist, WIN 55,212-2, inhibited, and the CB(1) receptor antagonist, SR 141716, facilitated, the evoked tritium overflow from hippocampal slices (preincubated with [(3)H]-choline) from CB(1)(+/+) as opposed to CB(1)(-/-) mice. Both drugs did not affect the evoked tritium overflow from striatal slices (preincubated with [(3)H]-choline) and from hippocampal slices (preincubated with [(3)H]-noradrenaline) from CB(1)(+/+) and CB(1)(-/-) mice. The selective increase in acetylcholine release in CB(1)(-/-) mice may indicate that the presynaptic CB(1) receptors on the cholinergic neurones of the mouse hippocampus are tonically activated and/or constitutively active in vivo.     

Alpha 2-autoreceptor subclassification in rat isolated kidney by use of short trains of electrical stimulation.

1 Rat kidneys were perfused with Krebs-Henseleit solution and incubated with [3H]-noradrenaline. The renal nerves were electrically stimulated at either 1 Hz for 30 s or 100 Hz for 0.06 s. The stimulation induced (S-I) outflow of radioactivity was taken as an index of endogenous noradrenaline release. 2 At a frequency of 1 Hz for 30 s the alpha-adrenoceptor antagonists BRL 44408 (0.01, 0.1 microM) and imiloxan (0.1, 1.0 microM) enhanced S-I outflow of radioactivity. However, at a frequency of 100 Hz for 0.06 s the alpha-adrenoceptor antagonists, idazoxan (0.1, 1.0 microM), imiloxan (0.1, 1.0 microM), BRL 44408 (0.1, 1.0 microM), BRL 41992 (0.1, 1.0 microM) and prazosin (0.01 microM) failed to enhance S-I outflow of radioactivity. 3 Thus, the rat isolated kidney stimulated at 100 Hz for 0.06 s, avoids autoinhibition by endogenous noradrenaline and alpha-adrenoceptor antagonist affinities (pKB) at the prejunctional alpha-autoreceptor were estimated without disturbance by the endogenous activator. 4 The alpha 2-adrenoceptor agonist, clonidine, inhibited the S-I outflow of radioactivity with a maximum of 90% and an EC50 of 7.2 nM. 5 All alpha-adrenoceptor antagonists used caused parallel shifts of the concentration-response curve for clonidine to the right. The rank order of potencies was: rauwolscine (alpha 2A/B) > idazoxan (alpha 2A/B) > phentolamine (alpha 2A/B) > WB 4101 (alpha 2A) > BRL 44408 (alpha 2A) > BRL 41992 (alpha 2B) > prazosin (alpha 2B) = imiloxan (alpha 2B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Facilitation of noradrenaline release from sympathetic nerves through activation of ACTH receptors, beta-adrenoceptors and angiotensin II receptors.

1. In rabbit pulmonary artery and left atrial strips previously incubated with [3H]-noradrenaline, the active fragment of adrenocorticotropic hormone (ACTH 1-24, 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity when a cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM) was present, but not in the absence of these drugs. In rabbit pulmonary artery a facilitatory effect of ACTH 1-24 (0.1 microM) was also observed when only cocaine (30 microM) was present. 2. ACTH 1-24 (0.1 microM) did not affect the S-I outflow of radioactivity from rat atria, rat pulmonary artery or guinea-pig pulmonary artery, either in the presence or in the absence of the cocktail containing corticosterone (40 microM), cocaine (30 microM) and propranolol (4 microM). These results suggest that the presence of facilitatory prejunctional ACTH receptors may be restricted to rabbit sympathetic nerve endings. 3. Angiotensin II (0.01 microM), but not isoprenaline (0.1 microM) or ACTH 1-24 (0.1 microM), significantly enhanced the S-I outflow of radioactivity from rabbit pulmonary artery. In the presence of phentolamine (1 microM) to block inhibitory alpha 2-adrenoceptors, the facilitatory effect of angiotensin II (0.01 microM) was significantly enhanced, and a significant facilitatory effect of isoprenaline (0.1 microM) and of ACTH 1-24 (0.1 microM) was then revealed. These results suggest that feedback inhibition of noradrenaline release, mediated through the prejunctional alpha 2-adrenoceptor mechanism, buffers increases in noradrenaline release during activation of facilitatory prejunctional receptors.(ABSTRACT TRUNCATED AT 250 WORDS)