Characterization of the facilitatory modulation of adrenergic neurotransmission via prejunctional purinoceptors in rabbit ear artery]

The purinergic modulation of the release of norepinephrine (NE) from sympathetic nerves in the rabbit ear artery was investigated. Methoxamine, an alpha 1-adrenoceptor agonist, enhanced the NE-release by electrical stimulation (ES) and released large amounts of adenyl purines (ATP, ADP, AMP and adenosine) from the endothelium. Both actions of methoxamine were blocked by prazosin. In addition, the enhancement of the NE-release by methoxamine was abolished by 8-sulfophenyl theophylline (8SPT), a P1-Purinoceptor antagonist. These findings indicated that the endogenous purines and purinoceptors participate in the facilitation of NE-release by alpha 1-adrenoceptor stimulation. P1-Purinoceptor agonists, such as adenosine and 2-chloroadenosine, and P2-purinoceptor agonists, such as ATP and beta,gamma-methylene ATP (beta gamma-mATP), enhanced the ES-evoked NE-release. This enhancement was antagonized not only by the P1-purinoceptor antagonist, 8SPT, but also by the P2-purinoceptor desensitizing agent, alpha,beta-methylene ATP. A phosphodiesterase inhibitor, Ro20-1724 potentiates the enhancement of NE-release by beta gamma-mATP. On the other hand, an adenylate cyclase inhibitor, SQ22536, inhibited the enhancement of NE-release by beta gamma-mATP. These findings suggested that prejunctional facilitatory purinoceptors exist on the adrenergic nerves of the rabbit ear artery. This receptor may be coupled to adenylate cyclase and is different from well-known P1 and P2 purinoceptors. In the rabbit ear artery, adrenergic neurotransmission may be regulated by endogenous ATP and its metabolites via prejunctional facilitatory purinoceptors, which were initiated by alpha 1-adrenoceptor stimulation; i.e. transsynaptic regulation of neurotransmission.     

Dysfunction of neurotransmitter modulation system on adrenergic nerves of caudal artery in type 2 diabetic Goto-Kakizaki rats

The Goto-Kakizaki (GK) rat is a non-obese and spontaneous model of mild Type 2 diabetes mellitus. In the present study, we compared the regulatory mechanisms of endogenous norepinephrine (NE) release from sympathetic nerves of caudal arteries of 12-week-old GK rats and age-matched normal Wistar rats. Electrical stimulation (ES) evoked significant NE release from caudal arteries of Wistar and GK rats. The amounts of NE released by ES were almost equal in Wistar and GK rats, although the NE content in caudal artery of GK rats was significantly lower than that of Wistar rats. We examined the effects of an α?-adrenoceptor agonist, clonidine (CLO), and an α?-adrenoceptor antagonist, yohimbine (YOH), on the release of endogenous NE evoked by ES. CLO significantly reduced NE release from caudal arteries of Wistar but not GK rats. On the other hand, YOH significantly increased NE release from both rats. Furthermore, we examined the effects of an A?-adenosine receptor agonist, 2-chloroadenosine (2CA), and an A?-adenosine receptor antagonist, 8-sulfophenyltheophylline (8SPT), on the release of endogenous NE evoked by ES. 2CA significantly reduced NE release from caudal arteries of Wistar but not GK rats. On the other hand, 8SPT did not affect NE release from both rats. These results suggest that the dysfunction of negative feedback regulation of NE release via presynaptic receptors on sympathetic nerves in GK rats may be involved in the autonomic nervous system dysfunction associated with diabetic autonomic neuropathy.     

Regional differences in the electrically stimulated release of endogenous and radioactive adenosine and purine derivatives from rat brain slices

Summary The release of both radioactive and endogenous purines was investigated in rat brain cortical, hippocampal and striatal slices at rest and following stimulation with electrical fields.Purities were labelled by incubating the slices with ³H-adenine. The purine efflux at rest and that evoked by electrical stimulation (10 Hz, 5 min) was analyzed by HPLC with ultraviolet absorbance detection. Both radio-active and endogenous purines in the effluent consisted mainly of hypoxanthine, xanthine, inosine and adenosine. No qualitative differences in the composition of the released purines were found in the three areas investigated. Electrical stimulation evoked a net increase in both radioactive and endogenous purine release. However the increase in ³H-adenosine following electrical stimulation was twice as large as that of endogenous adenosine. The electrically evoked release of both radioactive and endogenous purines was greatest in hippocampal slices and progressively smaller in cortical and striatal slices. In the three areas the addition of 0.5 M tetrodotoxin to the superfusing Krebs solution brought about a similar (83–100%) reduction in evoked ³H-purine and endogenous purine release. Superfusion of the slices with calcium-free Krebs solution containing 0.5 mM EGTA reduced evoked release of ³H-purines by 58–60% and that of endogenous purine components by 54–89%.The results demonstrate similar characteristics for both radioactive and endogenous purine release but indicate that the most recently synthetized adenosine is the most readily available for release. The features of the electrically evoked purine release support a neuronal origin of adenosine and derivatives and are consistent with the hypothesis of discrete regional differences in adenosine neuromodulation. Send offprint requests to F. Pedata at the above address     

Differential sensitivity to hydroxyl radicals of pre- and postjunctional neurovascular transmission in the isolated canine mesenteric vein

In some pathophysiological conditions, the first target of reactive oxygen intermediates is the vascular system. Superoxide anions, when generated in the vascular circulation, may then escape into the extracellular space via an anion channel and, following dismutation to hydrogen peroxide (H(2)O(2)), form hydroxyl radicals (HO(*)). In an attempt to understand the role of HO(*) in the regulation of transmission at the sympathetic neurovascular junction, the effect of HO(*) at nerve terminals was examined by measuring the amount of noradrenaline (NA) released from isolated, spirally cut, superfused canine mesenteric vein during basal and electrical stimulation (ES; 5Hz, 2ms, 9V); tension development evoked by ES was also recorded simultaneously. HO(*) was generated from Fenton's reagent (1. 5x10(-4)M H(2)O(2) plus 10(-4)M FeSO(4)); generation of HO(*) from H(2)O(2)/FeSO(4) in the superfusate was monitored by electron spin resonance spectroscopy using the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide throughout the experimental time course. Exposure to HO(*) of the helical strips produced an irreversible decrease in tension development evoked by ES with no effect on NA release, suggesting that the observed effect is elicited postjunctionally. The susceptibility of the processes of NA-mediated contraction to HO(*) may differ greatly from that of the NA release mechanism at the prejunctional site. Exposure of the strip preparation to HO(*) leads to a substantial stimulation of basal release of NA without affecting ES-evoked NA release, possibly due to enhanced non-exocytotic Ca(2+)-independent release elicited by HO(*). A direct demonstration of this concept was obtained by showing a significant increase in the basal response of NA release in Ca(2+)-free solution. The major conclusion of the present study is that HO(*) can damage NA-mediated contraction of the vascular preparations at the postjunctional site, and may selectively induce a non-exocytotic release of NA from the prejunctional site of sympathetic neurotransmission.     

REGIONAL DIFFERENCE OF ENDOGENOUS ATP RELEASE IN THE PULMONARY ARTERY OF RABBITS

1. The release of adenyl purines such as ATP, ADP, AMP and adenosine, from the pulmonary artery trunk (PAT), extra-pulmonary artery (EPA) and intrapulmonary artery (IPA) were compared. 2. The amount of basal overflow of adenyl purines from the PAT was significantly smaller than those from EPA and IPA. There was no significant difference between the amount of the overflow from EPA and PAT. 3. Methoxamine, an α₁-adrenoceptor agonist, significantly increased the overflow of adenine nucleotides from the PAT, EPA and IPA, but did not increase those of adenosine. Methoxamine-induced release of adenyl purines from IPA was significantly larger than those from EPA and PAT. 4. These results suggest that an α,-adrenoceptor-mediated mechanism for ATP-release is not homogeneously distributed in the pulmonary artery and a larger amount of ATP may be released in the peripheral part of the artery.     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

Presynaptic α2‐adrenoceptor‐mediated modulation of adenosine 5′ triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein

1 The modulatory effects of agonists and antagonists of prejunctional α₂‐adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)‐induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′ triphosphate (ATP) were measured in endothelium‐denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long‐duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2 The EFS‐evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 μM ) and guanethidine (10 μM ) in the artery and vein. Desipramine (10 μM ), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)‐evoked overflow of NA in both artery and vein. EFS‐evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS‐evoked overflow of ATP was equal in the artery and vein. 3 Stimulation of α₂‐adrenoceptors with clonidine (0.1 μM ) and oxymethazoline (0.3 μM ) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the α₂‐adrenoceptor agonists in the artery and vein. 4 In artery, blockade of α₂‐adrenoceptors with yohimbine at a concentration of 0.1 μM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 μM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 μM ) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 μM ) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan increased the NA overflow at both 4 and 16 Hz. No changes of EFS‐evoked ATP overflow were observed in the presence of 0.1 μM yohimbine in both artery and vein. Greater concentration of yohimbine (i.e. 1 μM ) increased the overflow of ATP in both the artery and vein only at 4 Hz EFS. Idazoxan (1 μM ) enhanced the ATP overflow only at 16 Hz in vein. The overflow of ADP was affected by both yohimbine and idazoxan in a similar manner to the ATP overflow so that the ATP:ADP ratios were not changed. 5 In conclusion, sympathetic nerves in both mesenteric arteries and veins appear to release ATP along with NA. Release of NA in veins exceeds release of NA in arteries, whereas both the canine artery and vein release equal amount of ATP. At long‐duration nerve stimulation (as might occur during stress) the α₂‐adrenoceptors appear to rather modulate release of NA than release of the cotransmitter ATP. The prejunctional autoinhibition of NA release is more effective at lower frequencies of nerve stimulation. The α₂‐adrenoceptor‐mediated neuromodulation plays a greater role in veins than arteries. Quantitative differences in α₂‐adrenoceptor‐mediated neuromodulation in the arteries and veins may participate to differing contributions of mesenteric blood vessels to the control of blood flow and volume distribution in splanchnic circulation.     

Endogenous adenosine release from hippocampal slices excitatory amino acid agonists stimulate release,antagonists reduce the electrically-evoked release

Summary The effect of excitatory amino acids and their antagonists on adenosine and inosine release has been investigated on unstimulated and electrically stimulated hippocampal slices.On unstimulated slices N-methyl-D-aspartate (NMDA), quisqualate and glutamate concentration-dependently evoked the release of adenosine and inosine. The effect of NMDA and quisqualate was antagonized by the NMDA receptor antagonist D(–)-2-amino-7-phosphonoheptanoic acid (D-AP7; 100 mol/1) and the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 mol/1) respectively. Glutamate (2 and 10 mmol/1)-evoked adenosine and inosine release was not antagonized by the NMDA and non-NMDA receptor antagonists indicating that the effect of glutamate is due to a metabolic rather than a receptor-mediated effect.Electrical field stimulation at 10 Hz also evoked a release of endogenous adenosine and inosine. Tetrodotoxin (0.5 gmol/1) abolished and absence of Ca²⁺ markedly reduced the electrically evoked release of adenosine and inosine. Adenosine and inosine release evoked by electrical stimulation at 20 Hz was significantly reduced in the presence of the NMDA receptor antagonist D-AP7, while at 10 Hz no consistent decrease was seen. In the presence of D-AP7 plus DNQX the 10 Hz-evoked adenosine and inosine release was reduced to about half. These data suggest that the electrically evoked release of adenosine and inosine is partly mediated by the release of excitatory amino acids which act at both non-NMDA and NMDA receptors. Send offprint requests to F. Pedata at the above address     

Comparison between radiolabelled and endogenous dopamine release from rat striatal slices: effects of electrical field stimulation and regulation by D2-autoreceptors

Summary Direct comparisons have been made between the release of radiolabelled and endogenous dopamine from superfused rat striatal slices prelabelled with ³H-dopamine. Both spontaneous release and release evoked by electrical field stimulation (3 Hz, 2 min) were measured using a high-sensitivity HPLC system with electrochemical (coulometric) detection, plus scintillation counting of chromatographically separated superfusate fractions. Two periods of electrical stimulation released similar amounts of endogenous dopamine, but the second stimulation released much less ³H-dopamine than did the first, although the levels of spontaneous release immediately before the two stimuli were similar. Substantial increases in endogenous 3,4-dihydroxy phenyl acetic acid (DOPAC) release but only minor increases in ³H-DOPAC release occurred following the two stimuli. The dopamine agonist pergolide (1 M) reduced the electrically-stimulated release of both ³H-dopamine and endogenous dopamine to a similar extent, whilst the D₂-selective antagonist sulpiride (1 M) produced large increases in both ³H-dopamine and endogenous dopamine electrically-stimulated release. In addition, spontaneous release of both ³H-dopamine and endogenous dopamine were decreased by pergolide and increased by sulpiride. Co-addition of sulpiride and pergolide produced lesser increases than those seen with sulpiride alone. These studies indicate that, despite major differences between ³H-dopamine and endogenous dopamine release in response to various stimuli, their regulation by D₂-autoreceptors appears similar; a novel finding being the modulation of spontaneous ³H-dopamine release by autoreceptors. This suggests that these autoreceptors do not selectively affect any specific intracellular pool contributing to dopamine release under these conditions, though it should be noted that the prelabelling process itself may alter the intracellular sources of subsequent release.This work was supported by Medical Research Council Send offprint requests to S. R. Nahorski at the above address     

Characterization of endogenous noradrenaline release from intact and epithelium-denuded rat isolated trachea.

1. Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2 at 3 or 15 Hz) or by high potassium (60 mM) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. In the presence of the neuronal uptake inhibitor desipramine, the alpha 2-adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. 3. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the second period of electrical stimulation (S2) was significantly smaller than that evoked by the first (S1). This decline of overflow was prevented when the NA precursor, tyrosine, was additionally present throughout the experiments. 4. After removal of the epithelium, the tissue content of NA was reduced by about 30%, suggesting that part of the NA may be present and released within the epithelium. However, the overflow of NA evoked by stimulation at 3 Hz or 15 Hz was reduced by 70-80%, indicating that the epithelium may additionally exert a permissive role on the release of NA within the airways, possibly by suppressing inhibitory factors. 5. Stimulation by high potassium (60 mM for 10 min) caused a large overflow of NA (about 45% of the tissue NA), both from epithelium-free and epithelium-denuded tracheae. Thus the 'endogenous inhibition' of NA release after removal of the epithelium is surmountable when a high potassium stimulus is applied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Participation by purines in the modulation of norepinephrine release by methoxamine

The alpha 1-receptor agonist methoxamine reduced, by a prazosin sensitive mechanism, the nerve stimulation evoked release of norepinephrine in the rat caudal artery. The effect of methoxamine was also antagonized by the purinoceptor antagonist 8-(p-sulfophenyl)theophylline suggesting an involvement of endogenous purines in this process. Indeed, methoxamine caused the release of adenine nucleotides and adenosine, an action which was blocked by prazosin. These results suggest that methoxamine releases ATP or a related purine which in turn decreases transmitter release by acting on prejunctional purinoceptors.     

Adenosine A2A receptor stimulation increases release of acetylcholine from rat hippocampus but not striatum,and does not affect catecholamine release

Rat striatal and hippocampal slices, preincubated with [³H] dopamine (DA) {or [³H] noradrenaline (NA)} and [¹⁴C] choline, were superfused continuously and stimulated electrically. 2-chloroadenosine (2-CADO 0.001–100 μM), a non-selective adenosine receptor agonist, produced a concentration-dependent inhibition of the electrically evoked DA and acetylcholine (ACh) release from the striatal slices and of the electrically evoked NA and ACh release from the hippocampal slices. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 3, 30 and 200 nM), a selective adenosine A₁ receptor antagonist, caused a concentration-dependent, parallel, rightward shift of the 2-CADO concentration-response curve, consistent with competitive antagonism. The pA₂ values ranged between 8.4 and 8.8. In the case of ACh release from the hippocampus, but in no other case, was there an increase in release of radioactivity at low concentrations of 2-CADO in the presence of DPCPX. The stimulation in the hippocampus could be blocked by a selective adenosine A_2A receptor antagonist KF 17837. By itself KF 17837 (0.1–100 μM) had no effect on electrically evoked NA release from hippocampal slices, but decreased electrically evoked ACh release. This inhibition was counteracted by DPCPX (1 μM). These results show that, under the conditions used, DA release in the striatum, and NA release in the hippocampus, as well as ACh release from the striatum are regulated by adenosine A₁ but not by adenosine A_2A receptors. By contrast, ACh release from the hippocampus is tonically regulated both by adenosine A₁ receptors, which inhibit release, and by adenosine A_2A receptors which stimulate release. Received: 25 April 1996 / Accepted: 30 August 1996     

Cultures of glial cells release purines under field electrical stimulation: the possible ionic mechanisms

Dissociated primary cultures of glial cells released a remarkable amount of purines, at rest and during field electrical stimulation. The HPLC identification of labelled compounds derived from 3H-Adenosine (3H-Ado) (employed to preload the cultures) indicated that nucleotides and nucleosides were represented in the superfusate in equivalent proportions (43.86% and 56.14% respectively). Very much higher amounts of unlabelled purines prevalently constituted by nucleotides compounds (91.10%) were also released and detectable in the superfusate. In all the experimental conditions their evoked release did not result frequency-dependent. Since: a linear increase related to the stimulation frequencies was found for the released labelled compounds; no labelled purines were assayed in 5 x 10-5M Dipyridamole-treated cultures; any significant presence of labelled nucleotides, inosine and hypoxantine was not found in cultures simultaneously treated with 1 x 10-5M 2'-deoxycoformycin and 1 x 10-4M 1-(-5-isoquinolinsulfonyl)-2-methylpiperizine (H7) (3H-Ado amounts resulted more than doubled in these experimental conditions); labelled compounds have been assumed as tracers of a glial purine rate whose release can be connected to electrically-evoked action potentials. Purine outflow from glial cells is not sodium dependent, in fact TTX (5 x 10-7M) did not affect their basal or electrically-evoked release. A remarkable calcium-dependence was also evidentiated by the 1 x 10-4M Verapamil-induced inhibition of basal and evoked release. TEA (1 x 10-2M), a specific inhibitor of potassium efflux throughout calcium-mediated specific channels, strongly reduced the evoked purine outflow and any additive effect of its was not detectable when administered simultaneously to the calcium antagonist. These findings indicate that the frequency-dependent purine release from cultured glial cells is linked to ionic mechanisms, which calcium and potassium are mainly involved in.     

Release of endogenous aspartate from rat cerebellum slices and synaptosomes: inhibition mediated by a 5-HT2 receptor and by a 5-HT1 receptor of a possibly novel subtype

Summary The effects of 5-hydroxytryptamine (5-HT) and of a number of 5-HT receptor agonists and antagonists on the release of endogenous aspartate were investigated in rat cerebellum slices and synaptosomes depolarized with high K⁺. The release of endogenous aspartate evoked from slices by 35 mmol/l KCl and from synaptosomes by 15 mmol/1 KCl was strongly (about 90%) calcium-dependent. In slices the release of aspartate was inhibited by exogenous 5-HT (0.1–100 nmol/1) in a concentration-dependent manner. The indoleamine was very potent, producing 30% inhibition at 0.1 nmol/l. The effect of 10 nmol/1 5-HT was partly but maximally counteracted by ketanserin (300–1000 nmol/1), a 5-HT² receptor antagonist, but fully blocked by 300 nmol/1 of the mixed 5-HT¹/5-HT² receptor antagonist methiothepin. The 5-HT^1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the 5-HT² receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) inhibited the K⁺-evoked release of endogenous aspartate in a concentration-dependent manner. The effect of 8-OH-DPAT was antagonized by methiothepin, but not by ketanserin which fully antagonized the inhibition produced by DOI. In cerebellar synaptosomes the release of endogenous aspartate evoked by 15 mmol/l K⁺ was inhibited by exogenous 5-HT and by 8-OH-DPAT, but not by DOI. Methiothepin (100–300 nmol/1) antagonized the inhibitory effects of 100 nmol/l 5-HT or 8-OH-DPAT. However, 1000 nmol/l of various 5-HT receptor antagonists [ketanserin, methysergide, (–)-propranolol, spiperone or ICS 205–930] did not counteract the effect of 100 nmol/15-HT.It is concluded that: (1) 5-HT projections to the rat cerebellum may exert a potent inhibitory action on the depolarization-evoked release of aspartate; (2) this inhi bition is mediated through receptors of both the 5-HT₁ and the 5-HT² type; (3) the 5-HT² receptors appear to be sited on structures which do not survive the standard preparation of synaptosomes, while the 5-HT₁ receptors are likely to be localized on Apartate-releasing nerve terminals; and (4) the 5-HT₁ receptors do not conform to the pharmacological criteria defining the known subtypes of the 5-HT₁-binding sites.     

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [³H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [³H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the α₄β₂^∗ subunit-selective RJR2403, provoked the release of [³H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and α₄β₂^∗ nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [³H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M₂/M₄ mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M₄ mAChR antagonists MT3, thus indicating the involvement of receptors of the M₄ subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate α₄β₂^∗ nAChRs mediating release of GABA; this evoked release can be negatively modulated by M₄ mAChRs coexpressed on the same GABAergic terminals.     

No evidence for presynaptic opioid receptors on cholinergic,but presence of κ-receptors on dopaminergic neurons in the rabbit caudate nucleus: involvement of endogeneous opioids

Summary The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [³H]dopamine or [³H] choline, superfused with medium (containing in most experiments the D₂ receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [³H]overflow from slices prelabeled with [³H]dopamine (evoked [³H]dopamine release) was significantly reduced by preferential -opioid receptor agonists, like U-50,488 H, but not by µ- or -opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA₂-value of the -selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors.On the other hand, the [³H]overflow from rabbit caudate nucleus slices prelabeled with [³H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D₂ heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential -opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release.From our data we conlude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic -opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids. Correspondence to R. Jackisch at the above address     

Release of [3H]-noradrenaline from rat hippocampal synaptosomes by nicotine: mediation by different nicotinic receptor subtypes from striatal [3H]-dopamine release.

1. The aim of the present experiment was to characterize nicotine-evoked [3H]-noradrenaline ([3H]-NA) release from rat superfused hippocampal synaptosomes, using striatal [3H]-dopamine release for comparison. 2. (-)-Nicotine, cytisine, DMPP and acetylcholine (ACh) (with esterase inhibitor and muscarinic receptor blocker) increased NA release in a concentration-dependent manner (EC50 6.5 microM, 8.2 microM, 9.3 microM, and 27 microM, respectively) with similar efficacy. 3. Nicotine released striatal dopamine more potently than hippocampal NA (EC50 0.16 microM vs. 6.5 microM). (+)-Anatoxin-a also increased dopamine more potently than NA (EC50 0.05 microM vs. 0.39 microM), and maximal effects were similar to those of nicotine. Isoarecolone (10-320 microM) released dopamine more effectively than NA but a maximal effect was not reached. (-)-Lobeline (10-320 microM) evoked dopamine release, but the effect was large and delayed with respect to nicotine; NA release was not increased but rather depressed at high concentrations of lobeline. High K+ (10 mM) released and NA to similar extents. 4. Addition of the 5-hydroxytryptamine (5-HT) reuptake blocker, citalopram (1 microM) to hippocampal synaptosomes affected neither basal NA release nor nicotine-evoked release. 5. The nicotinic antagonist, mecamylamine (10 microM), virtually abolished NA and dopamine release evoked by high concentrations of nicotine, ACh, cytisine, isoarecolone, and anatoxin-a. Although NA release evoked by DMPP (100 microM) was entirely mecamylamine-sensitive, DMPP-evoked dopamine release was only partially blocked. Dopamine release evoked by lobeline (320 microM) was completely mecamylamine-insensitive. 6. The nicotinic antagonists dihydro-beta-erythroidine and methyllycaconitine inhibited nicotine-evoked dopamine release approximately 30 fold more potently than NA release. In contrast, the antagonist chlorisondamine, displayed a reverse sensitivity, whereas trimetaphan and mecamylamine did not preferentially block either response. None of these antagonists, given at a high concentration, significantly altered release evoked by high K+. 7. Blockade of nicotine-evoked transmitter release by methyllycaconitine and dihydro-beta-erythroidine was surmounted by a high concentration of nicotine (100 microM), but blockade by mecamylamine, chlorisondamine, and trimetaphan was insurmountable. 8. Nicotine-evoked NA release was unaffected by tetrodotoxin, whereas veratridine-evoked NA release was virtually abolished. 9. We conclude that presynaptic nicotinic receptors associated with striatal dopamine and hippocampal NA terminals differ pharmacologically. In situ hybridization studies suggest that nigrostriatal dopaminergic neurones express mainly alpha 4, alpha 5, and beta 2 nicotinic cholinoceptor subunits, whereas hippocampal-projecting noradrenaline (NA) neurones express alpha 3, beta 2 and beta 4 subunits. Pharmacological comparisons of recombinant receptors suggest that release of hippocampal NA may be modulated by receptors containing alpha 3 and beta 4 subunits.     

Modulation of noradrenaline and neuropeptide Y (NPY) release in the pig kidney in vivo: involvement of alpha2, NPY and angiotensin II receptors

Summary The modulation of the release of noradrenaline (NA) and neuropeptide Y-like immunoreactivity (NPY-LI) was investigated in the pig kidney in vivo. Under control conditions a reproducible co-release of NA and NPY-LI was obtained upon stimulation of the renal nerves with 5 Hz for 1 min. Infusion of peptide YY (PYY, 1 g/kg/min i.v.), which binds to NPY receptors, caused renal vasoconstriction and reduced the stimulation-evoked overflow of NA and NPY-LI by 24 ± 4 and 33 ± 11%, respectively (P < 0.01). The PYY effect was reversible and was absent 1 h after the infusion. The alpha₂-adrenoceptor antagonist yohimbine (0.2 mg/kg i. v.) enhanced the overflow of NA and NPY-LI 2- to 3-fold. The angiotensin converting enzyme inhibitor captopril (5 mg/kg i. v.) did not significantly affect the overflow of NA or NPY-LI evoked by the nerve stimulation. Angiotensin II (0.5 g/kg/min i. v.), on the other hand, induced a reversible enhancement of the overflow of both NA and NPY-LI by 71 and 77%, respectively (P<0.01). Infusion of endothelin (0.2 g/kg/min i.v.), which reduced renal blood flow by a magnitude similar to that evoked by angiotensin II, did not significantly alter the nerve stimulation-evoked overflow of NA or NPY-LI. None of the administered drugs did significantly affect the percentage reduction in renal blood flow evoked by nerve stimulation.It is concluded that the release of NA and NPY-LI from sympathetic nerves in the pig kidney is inhibited in parallel via activation of NPY receptors by PYY and via alpha₂-adrenoceptors by endogenous NA. Furthermore, angiotensin II receptor stimulation facilitates the release whereas endothelin has no major effect on transmitter secretion.Send offprint requests to J. Pernow at the above address     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.     

DIRECT MEASUREMENT OF THE RELEASE OF ATP AND ITS MAJOR METABOLITES FROM THE NERVE FIBRES OF THE GUINEA-PIG TAENIA COLI

1. The release of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine from guinea-pig taenia coli in response to electrical stimulation of intramural nerves was measured directly using high performance liquid chromatography separation and fluorometric detection. 2. Purines were released in a frequency-dependent manner by trains of transmural electrical pulses at 1-30 Hz. 3. Electrically evoked release of ATP was abolished by tetrodotoxin (10^?6 mol/L) but was not affected by nicardipine (10^?6 mol/ L). 4. The release of purines was reduced in the presence of atropine. 5. Pituitary adenylyl cyclase-activating peptide did not evoke the release of any purines and did not modify the electrically evoked release of purines. 6. The results suggest that ATP and its major metabolites are released from a neuronal source, possibly the enteric inhibitory nerves, in the guinea-pig taenia coli.