Tritium Overflow Research Articles

Histamine inhibits dopamine release in the mouse striatum via presynaptic H3 receptors.

In superfused mouse striatal slices preincubated with [3H]dopamine 25 nmol/l, the electrically (3 Hz) evoked tritium overflow was inhibited by histamine 10 mumol/l by 18%. The degree of inhibition was increased to 38% by haloperidol but not affected by (1) atropine, (2) reducing the stimulation frequency to 0.3 Hz or (3) increasing the concentration of [3H]dopamine (used for preincubation) to 100 nmol/l. The effect of histamine was mimicked by the H3 agonist R-(-)-alpha-methylhistamine; it was not affected by the H1 antagonist dimetindene and the H2 antagonist ranitidine but abolished by the H3 antagonist thioperamide. Tritium overflow evoked by Ca2+ ions (introduced into Ca(2+)-free, K(+)-rich medium containing tetrodotoxin) was not affected by histamine 10 mumol/l in the absence, but inhibited (by 30%) in the presence of haloperidol; the effect of histamine was abolished by thioperamide. In conclusion, the dopaminergic nerve terminals in the mouse striatum are endowed with presynaptic H3 receptors. Simultaneous blockade of dopamine autoreceptors increases the extent of the H3 receptor-mediated inhibition of dopamine release.

Effect of opioid receptor subtype‐selective agonists on purinergic and adrenergic components of neurogenic contractions of mouse vas deferens

1. Effects of opioid agonists on the purinergic and adrenergic components of neurogenic contractions and in some experiments on transmitter overflow were studied in the mouse isolated vas deferens. 2. When the vas deferens was stimulated every 2 min by pairs of pulses 2 s apart in the presence of prazosin 0.3 microM (to isolate the purinergic component) or alpha,beta-methylene-ATP 3 microM (to isolate the adrenergic component), each pulse elicited a separate twitch. The opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, mu-receptor-selective), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-selective) and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide (U-50488, kappa-selective) concentration-dependently reduced both purinergic and adrenergic contractions. For each agonist, maximal effects and concentrations causing half-maximal effects were very similar for inhibition of the purinergic component on the one hand and for inhibition of the adrenergic component on the other hand, although the adrenergic component was inhibited with a slight preference. Moreover, effects on contractions elicited by the first and the second pulse of the pairs were very similar. 3. When vasa deferentia preincubated with [3H]-noradrenaline were stimulated with trains of 100 pulses delivered at 20 Hz, morphine 10 microM reduced significantly both evoked tritium overflow and evoked contractions. Its effect was antagonized by naloxone. 4. It is concluded that, in contrast to drugs acting at some other presynaptic receptors, opioid mu-, delta- and kappa-agonists inhibit purinergic and adrenergic neurogenic contractions of the mouse vas deferens in a similar manner. In contrast to a previous report, no enhancement by morphine of the release of noradrenaline elicited by high frequency pulse trains was observed.

Mechanism of Pindolol-Induced Vasoconstriction in Isolated and Perfused Dog Coronary Arteries

By using rat brain cortical slices preloaded with [3H]norepinephrine, we examined whether ATP-sensitive K+ channels are involved in altered adrenergic neurotransmission during hypoxia. The tritium overflow evoked by transmural nerve stimulation (TNS) was significantly inhibited at 5 min of hypoxia and reached the maximum inhibition at 20 min. The inhibition of the TNS-evoked tritium overflow under a 20-min hypoxia was reversed by subsequent reoxygenation and was concentration-dependently antagonized by glibenclamide (0.1 and 1 microM). 86Rb+ efflux was increased after introduction of hypoxia and reached the peak value at about 20 min, which was concentration-dependently antagonized by glibenclamide (0.1-10 microM). Hypoxia decreased cortical ATP content. Linear correlations were mutually observed among the changes by hypoxia in the TNS-evoked tritium overflow, tissue ATP content and 86Rb+ efflux. The spontaneous tritium outflow was inhibited only after hypoxic periods of more than 16 min, the inhibition being reversed by reoxygenation and antagonized by 1 microM glibenclamide. These results suggest that the inhibition of rat central adrenergic neurotransmission during hypoxia may be associated with an activation of ATP-sensitive K+ channels.

Cyclic AMP modulates but does not mediate the inhibition of [ 3H]norepinephrine release by activation of alpha-2 adrenergic receptors in cultured rat ganglion cells

The purpose of this study was to determine whether a decrease in cyclic AMP accumulation mediates the inhibition of norepinephrine release in response to alpha-2 adrenergic receptor activation in cultured rat superior cervical ganglion cells. Superior cervical ganglia from neonatal rats were dissociated and cultured on collagen-coated plastic strips. Neurotransmitter release was assessed by measuring the fractional overflow of tritium in superfused cells prelabeled with [ 3H]norepinephrine. Intracellular cyclic AMP accumulation was measured using radioimmunoassay. Electrical field stimulation at 1 Hz, 30 pulses, 1 ms duration at 20 min intervals produced an increase in the fractional overflow of tritium that was composed predominantly of intact [ 3H]norepinephrine. The alpha-2 adrenergic receptor agonist UK-14,304 dose-dependently attenuated the increase in fractional tritium overflow elicited by electrical field stimulation. The adenylyl cyclase activator, forskolin, increased cyclic AMP accumulation in superior cervical ganglion cells and UK-14,304 dose-dependently inhibited forskolin-stimulated cyclic AMP accumulation. UK-14,304 had no effect on basal cyclic AMP accumulation or cyclic AMP accumulation during electrical field stimulation. Forskolin (1–10 μM) or the non-hydrolysable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (1–100 μM), slightly increased basal and dose-dependently potentiated the increase in fractional tritium overflow in response to electrical stimulation. Despite enhancement by forskolin and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate of fractional tritium overflow caused by electrical field stimulation, UK-14304 (1–10 μM) reduced release to a similar degree as that observed in the absence of forskolin or 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate. These data suggest that in cultured rat superior cervical ganglion cells, stimulation of alpha-2 adrenergic receptors decreases cyclic AMP accumulation when adenyl cyclase is activated. A decrease in cyclic AMP does not mediate the inhibition of [ 3H]norepinephrine release in response to alpha-2 adrenergic receptor activation, although elevations in cyclic AMP can enhance electrically-stimulated release of [ 3H]norepinephrine.

Release of Sympathetic Neurotransmitter Evoked by Electrical Stimulation Is Increased in the Chronically Decentralized Artery

The present study was performed to confirm our previous proposal that the increase in neurotransmitter release is responsible for the supersensitivity of chronically decentralized artery to transmural nerve stimulation (TNS) in the rabbit. The ear artery was decentralized unilaterally by removing the preganglionic fiber proximal to the superior cervical ganglion (SCG), and the ear arteries and SCG were dissected 8 weeks after the operation. The increase in the tritium overflow induced by TNS from the chronically decentralized artery, which had been incubated with 3H-noradrenaline (NA) for 1 hr, was markedly increased at lower frequencies (0.1 and 0.2 Hz) than that from the control artery, whereas there was no difference at higher frequencies (> 0.5 Hz). No difference was observed in the neuronal uptake of 3H-NA during incubation for 1 hr between the control and decentralized arteries. There was also no change in the contents of catecholamines in both the artery and SCG after chronic decentralization, when assayed by a radioenzymatic procedure. In conclusion, the results obtained indicate that the supersensitivity to TNS after chronic decentralization is not due to the deranged catecholamine uptake and storage mechanisms in adrenergic nerve terminals and augmented transmitter biosynthesis, but due to the increased release of transmitter in response to low frequencies of TNS.

CGP 35348 blocks noradrenaline-release-inhibiting GABAB receptors in the pig retina, rat vena cava and pithed rat vasculature.

In superfused pig retina discs, preincubated with 3H-noradrenaline, the electrically (3 Hz) evoked tritium overflow was inhibited by gamma-aminobutyric acid (GABA) and the GABAB receptor agonist R-(-)-baclofen but was not affected by the GABAA receptor agonist 3-amino-1-propanesulphonic acid. The effect of GABA was counteracted by the GABAB receptor antagonist CGP 35348 [P-(3-aminopropyl)-P-diethoxymethylphosphinic acid] but was not influenced by the GABAA receptor antagonist SR 95531 [2-(3-carboxypropyl)-3-amino-6-paramethoxyphenylpyridazinium bromide]. Furthermore, CGP 35348 antagonized the inhibitory effect of R-(-)-baclofen on the electrically (0.66 Hz) evoked tritium overflow in superfused rat vena cava segments, preincubated with 3H-noradrenaline, and on the electrically (0.5 Hz) induced rise in diastolic blood pressure in pithed rats. The present results suggest that GABA is capable of inhibiting noradrenaline release (most likely from vascular sympathetic nerve endings) in the pig retina via GABAB receptors and that CGP 35348 is a valuable tool for the characterization of GABAB receptors in vitro and in situ.

Allicin-induced hypotension in rabbit eyes.

The intent of this work was to examine the actions of allicin on 1) intraocular pressure (IOP) in normal and unilaterally sympathectomized (SX) rabbits; 2) cAMP accumulation in the rabbit iris-ciliary body (ICB) and cultured nonpigmented epithelial (NPE) ciliary body cells; and 3) 3H-norepinephrine (NE) release by calculating fractional tritium overflow in response to electrical field stimulation (EFS, 5 Hz, 12 V/cm) in isolated, perfused rabbit ICBs. Allicin, one of the active compounds produced by garlic, was evaluated on IOP and it was determined that allicin (1, 2.5, or 10 micrograms), topically, but not the precursor, alliin (10 micrograms), lowered the IOP unilaterally in normal rabbits. Allicin (10 micrograms) reduced the IOP by 6 +/- 1 mmHg (n = 4) in normal rabbits at 2 hrs (maximum response) whereas no change occurred in sympathectomized rabbit eyes. Moreover, allicin (0.01, 0.1, or 1 microM) caused 40, 40, or 52% inhibition, respectively, of 3H-NE overflow in response to EFS. Isoproterenol (ISO, 1 microM) stimulated cAMP accumulation by 3.6 and 9 fold in isolated rabbit ICB and cultured NPE cells, respectively. Allicin (1 microM) had no effect on basal cAMP level while it inhibited ISO-stimulated cAMP accumulation by 40% and 23% in ICB and NPE cells, respectively. This study suggests that allicin lowered IOP, in part, by dual actions at the neuroeffector junction.

Subclassification of release-regulating alpha 2-autoreceptors in human brain cortex.

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices. The effect of clonidine (0.01- 1 g1M) on the evoked overflow of tritium was reduced in presence of 1 muM desipramine.9. It is proposed that autoregulation of NA release can occur in human cerebral cortex. The process involves activation of alpha 2-adrenoceptors which may be either the alpha2X or the alpha2D subtype.

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition, as well as inhibition by glutamate, of noradrenaline release in rabbit brain cortex slices.

A possible contribution of adenine nucleotides to the endogenous purinergic, A1-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1-100 nmol/l) as well as the enzyme adenosine deaminase (0.1-10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The alpha 1-adrenoceptor-selective agonist methoxamine (10 but not 1 mumol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mumol/l but then not attenuated further by DPCPX 100 nmol/l. L-Glutamate (300 mumol/l-2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mumol/l but greatly, and to the same extent, attenuated by DPCPX 100 nmol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg++ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mumol/l and 1 mmol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg++).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the L-arginine-NO pathway and of cyclic GMP in electrical field-induced noradrenaline release and vasoconstriction in the rat tail artery.

1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction. Dibutyryl-cyclic GMP (100 JM), a weak activator of cyclic GMP-dependent protein kinase, affected neither the pre- nor the postjunctional response to electrical field stimulation.5. These data show that an NO-like substance of endothelial origin, derived from L-arginine, attenuates vasoconstriction in the rat tail artery, whether neurally-induced or evoked by exogenous noradrenaline.Since noradrenaline release was unaltered by compounds modifying NO production, this NO-like compound acted through a postjunctional mechanism. The lack of prejunctional effects of both soluble and membrane-associated guanylate cyclase activators, despite a large effect of 8-bromo-cyclic GMP,suggests that endogenous cyclic GMP production, if present in sympathetic nerves, may not be involved in the regulation of noradrenaline release in the rat tail artery.

Inhibition of noradrenaline release in the rat vena cava via prostanoid receptors of the EP3-subtype.

1. In segments of the rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution (containing desipramine and corticosterone), we studied the effects of prostaglandins of the D, E and F series, of a prostacyclin analogue and a thromboxane-mimetic and of subtype-selective prostaglandin E-receptor (EP-receptor) ligands on the electrically (0.66 Hz)-evoked tritium overflow. 2. The electrically-evoked tritium overflow was inhibited by prostaglandin E2 (maximum inhibition by about 80%; pIC40 7.49). The effect of prostaglandin E2 was not affected by rauwolscine, which, by itself, increased the evoked overflow; the alpha 2-adrenoceptor antagonist was added to the superfusion medium in all subsequent experiments. Indomethacin failed to affect either the evoked tritium overflow or its inhibition by prostaglandin E2. 3. The inhibitory effect of prostaglandin E2 on the electrically-evoked tritium overflow was not altered by the EP1-receptor antagonist. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) at a concentration at least 30 fold higher than its pA2 value at EP1-receptors. The following compounds mimicked the effect of prostaglandin E2 showing the following rank order of potencies: misoprostol (EP2-/EP3-receptor agonist) congruent to sulprostone (EP1-/EP3-receptor agonist) congruent to prostaglandin E1 = prostaglandin E2 >> iloprost (EP1-/IP-receptor agonist) = prostaglandin F2 alpha. The evoked overflow was not affected by high concentrations of prostaglandin D2 or the thromboxane-mimetic U46619 (9,11-dideoxy-11 alpha, 9 alpha-epoxy-methano-prostaglandin F2 alpha). 4. The present results suggest that the postganglionic sympathetic nerve fibres innervating the rat vena cava are endowed with presynaptic EP3-receptors.They are not tonically activated by endogenously formed products of cyclo-oxygenase and do not interact with the presynaptic M2-adrenoceptors.

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery.

The muscarinic agonists acetylcholine (1-50 mumol/l), carbachol (1-10 mumol/l) and McN-A-343 (1-50 mumol/l, selective for M1 receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [3H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mumol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, non-selective), pirenzepine (1 and 5 nmol/l, M1-selective), methoctramine (1 and 5 mumol/l, M2-selective) and p-fluoro-hexahydro-sila-difenidol (0.1-1 mumol/l, M3-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methyl-atropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) > or = methoctramine (6.20). These results demonstrate the existence of facilitatory M1 receptors modulating noradrenaline release in blood vessels.

Effects of S14001 on adrenergic neuroeffector interaction in isolated canine saphenous veins

1. 1. The effects of ( S) fluoro-6 (morpholinyl-2 methoxy)-8-tetrahydro-1,2,3,4 quinoleine (S14001) on adrenergic neurotransmission in isolated canine saphenous veins were investigated in experiments which measured the accumulation, overflow and metabolism of 3H-norepinephrine. 2. 2. S14001 inhibited the accumulation of total tritium ( 3H-norepinephrine and 3H-metabolites of norepinephrine) in a concentration-dependent manner. 3. 3. Under basal conditions, S14001 increased tension and basal effiux of total tritium; the latter consisted predominantly of 3H-DOPEG. The increases in these parameters were not inhibited by desmethylimipramine (DMI). 4. 4. During electrical stimulation, S14001 increased the contraction and overflow of total tritium; the latter consisted predominantly of 3H-DOPEG. The increases in these parameters were inhibited by DMI. 5. 5. These experiments suggest that S14001 has dual effects on adrenergic neurotransmission in the canine saphenous vein: (a) an inhibitory action on the neuronal accumulation; and (b) a pharmacological displacement of the transmitter from adrenergic nerve terminals.

Role of cyclic AMP in the release of noradrenaline from isolated rat atria. Effect of pretreatment with clenbuterol.

The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardio-accelerant nerves was studied in rat atria preincubated with [3H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 mumol/l), or of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 mumol/l), did not affect both basal and evoked overflow. However, in the presence of the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l) both forskolin and IBMX increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 mumol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional alpha 2-adrenoceptors is blocked. Propranolol (0.1 mumol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the beta 2-adrenoceptor agonist clenbuterol (0.3 mg.kg-1, s.c., twice daily, 14 days), a treatment which desensitizes beta-adrenoceptor-mediated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMX in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMX on noradrenaline release are only to be observed after blockade of presynaptic alpha 2-adrenoceptor autoinhibition. beta-adrenoceptor blockade or clenbuterol pretreatment decreases the facilitatory response to forskolin and hence prejunctional beta-adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase.

Stable adenine nucleotides inhibit [3H]-noradrenaline release in rabbit brain cortex slices by direct action at presynaptic adenosine A1-receptors.

Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz. Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADP beta S, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine greater than ATP approximately ATP gamma S approximately beta,gamma-imido-ATP approximately ADP greater than beta,gamma-methylene-ATP. AMP 30 mumol/l and AMPS 30 mumol/l were approximately equieffective with 30 mumol/l of adenosine and ATP gamma S, and ADP beta S 30 mumol/l was approximately equieffective with 30 mumol/l of ADP. alpha,beta-Methylene-ADP, 2-methylthio-ATP, UTP and GTP gamma S did not change the evoked overflow of tritium. alpha,beta-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to alpha,beta-methylene-ATP or when the slices were stimulated by 30 pulses, 10 Hz. Neither adenosine deaminase (100 U/l) nor the blocker of 5'-nucleotidase, alpha,beta-methylene-ADP (10 mumol/l), attenuated the inhibition caused by ATP, ATP gamma S and beta,gamma-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP to the right by very similar degrees. 8-(p-Sulphophenyl)-theophylline (30 and 300 mumol/l) also markedly antagonized the inhibition produced by ATP gamma S. alpha,beta-Methylene-ATP (10 and 30 mumol/l) and suramin (100 mumol/l) did not modify the effects of adenosine, ATP gamma S and beta,gamma-methylene-ATP. It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2x-, P2y- or P3-purinoceptors.

Dopamine efflux from striatum after chronic nicotine: evidence for autoreceptor desensitization.

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H-labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine-treated mice. The overflow of [3H]DA and its 3H-metabolites exhibited similar distribution patterns in [3H]DA-preloaded striatum dissected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (-)-Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation-induced tritium efflux from striatum of untreated mice, whereas (+/-)-sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (-)-quinpirole and (+/-)-sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (+/-)-SKF-38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors.

The human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked 3H overflow was inhibited by histamine and the H3 receptor agonist R-(-)-alpha-methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by alpha 2-adrenoceptor blockade by rauwolscine. S-(+)-alpha-methylhistamine (up to 10 mumol/l) as well as the histamine H1 and H2 receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 mumol/l) and dimaprit (up to 30 mumol/l), respectively, were ineffective. The selective histamine H3 receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H2 and H1 receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H3 class.

Mutual interaction of histamine H3-receptors and alpha 2-adrenoceptors on noradrenergic terminals in mouse and rat brain cortex.

Brain cortex slices were preincubated with 3H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the inhibition of the electrically evoked tritium overflow caused by histamine in the presence of alpha-adrenoceptor ligands (mouse and rat brain cortex), and the inhibition caused by talipexole (the former B-HT 920) in the presence of H3-receptor ligands (mouse brain cortex). In mouse brain cortex slices, the inhibitory effect of histamine on the tritium overflow evoked by 36 pulses, 0.3 Hz was not changed by the alpha 1-adrenoceptor antagonist prazosin, but increased by the alpha 2-adrenoceptor antagonist rauwolscine. When the current strength or the duration of electrical pulses was reduced to compensate for the increase in evoked tritium overflow produced by rauwolscine, the latter still enhanced the effect of histamine. The histamine-induced inhibition of tritium overflow evoked by 360 pulses, 3 Hz was not affected by the alpha 1-adrenoceptor agonist phenylephrine but attenuated by the alpha 2-adrenoceptor agonist talipexole. Finally, the inhibition by histamine of the tritium overflow evoked by 3 pulses, 100 Hz was attenuated by talipexole but not affected by rauwolscine. Conversely, the inhibitory effect of talipexole on tritium overflow elicited by 360 pulses, 3 Hz was slightly attenuated by the H3-receptor agonist R-(-)-alpha-methylhistamine but not affected by the H3-receptor antagonist thioperamide. In rat brain cortex slices, histamine only tended to inhibit tritium overflow evoked by 360 pulses, 3 Hz, both in the absence of alpha-adrenoceptor antagonists and in the presence of prazosin. However, histamine markedly inhibited the evoked overflow in the presence of rauwolscine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of N-methyl-D-aspartate- and kainate-evoked noradrenaline release in human cerebral cortex slices by ethanol.

The effect of ethanol on the release of noradrenaline evoked by various stimuli was investigated in human cerebral cortical slices from patients undergoing neurosurgery. The slices were preincubated with [3H]noradrenaline and then superfused. Tritium overflow was stimulated by exposure to N-methyl-D-aspartate (NMDA; in slices superfused without Mg2+), kainic acid, veratridine or by increasing the K+ concentration. The NMDA-evoked tritium overflow was concentration-dependently inhibited by ethanol; an inhibition by 37% occurred at 48 mmol/l ethanol. This ethanol concentration was not yet effective when kainic acid was used for stimulation, but ethanol 150 mmol/l strongly inhibited the tritium overflow evoked by kainic acid as well. The tritium overflow evoked by veratridine or high K+ was not affected by ethanol in the concentration range investigated. These findings are compatible with the suggestion that the NMDA receptor and, with less susceptibility, the kainate receptor are sites of action underlying the effect of ethanol in the human brain.

Inhibition of noradrenaline release via NEM-sensitive H3 receptors in the mouse brain cortex: more marked effect than in the rat brain

Mouse brain cortex slices preincubated with3H-noradrenaline were superfused and the effect of histamine on the electrically (0.3 or 3 Hz) evoked tritium overflow was examined. The evoked overflow was inhibited by histamine and R-(−)-α-methylhistamine but not by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of histamine was antagonized by thioperamide but not by dimethindene and ranitidine. The degree of inhibition produced by histamine was more marked at 0.3 than at 3 Hz and was attenuated by the α2-adrenoceptor agonist talipexole (the former B-HT 920) but increased by phentolamine and the α2-adrenoceptor antagonist rauwolscine. Pre-exposure of slices toN-ethylmaleimide attenuated the inhibitory effect of histamine. The results suggest that histamine inhibits nordrenaline release in the mouse brain cortex via H3 receptors which interact with presynaptic α2-adrenoceptor and may be coupled to a G (e.g. Gi or Go) protein. The extent of H3 receptor-mediated inhibition of nordrenaline release in the mouse brain is more marked than that in the rat brain.