Tritium Overflow Research Articles

Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors.

1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2ń

Estimation of the biophase concentration of noradrenaline at presynaptic alpha 2-adrenoceptors in brain slices.

The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic alpha 2-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the alpha 2-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [3H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC50 value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the alpha 2-adrenoceptor antagonist yohimbine (1 or 10 mumol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine H3A receptor-mediated inhibition of noradrenaline release in the mouse brain cortex.

Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with alpha 2-adrenoceptor antagonist properties, and the effects of histamine receptor ligands on the electrically (0.3 Hz) evoked tritium overflow were studied. The evoked overflow (from slices superfused with phentolamine) was inhibited by histamine (pIC35 6.53), the H3 receptor agonist R-(-)-alpha-methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H1 receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mumol/l and the H2 receptor agonist dimaprit 10 mumol/l. The inhibitory effect of histamine was not affected by the H1 receptor antagonist dimetindene 1 mumol/l and the H2 receptor antagonist ranitidine 10 mumol/l. The concentration-response curve of histamine (determined in the presence of rauwolscine) was shifted to the right by the H3 receptor antagonists thioperamide (apparent pA2 8.67), impromidine (7.30) and burimamide (6.82) as well as by dimaprit (6.16). The pA2 values of the four drugs were compared with their affinities for H3A and H3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H3A, but not for the H3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H3A receptors and that dimaprit is an H3 receptor antagonist of moderate potency.

Effects of thromboxane agonists on cardiac adrenergic neurotransmission

The effects of thromboxane B 2 (TxB 2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA 2 (ONO 11006) and 9,11-dideoxy-11,9-epoxymethano prostaglandin F 2α (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [ 3H]noradrenaline. In addition to this influence on adrenergic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle.

Anpirtoline, a novel, highly potent 5-HT1B receptor agonist with antinociceptive/antidepressant-like actions in rodents.

1. The purpose of the present study was to relate the effects of the novel drug, anpirtoline, on 5-hydroxytryptamine (5-HT) receptor subtypes to its antinociceptive and antidepressant-like actions in rodents. 2. Binding assays with rat brain membranes have shown that anpirtoline bound with a much higher affinity to 5-HT1B receptor (Ki = 28 nM) than to 5-HT1A (Ki = 150 nM) and 5-HT2 (Ki = 1.49 microM) receptors. 3. Like 5-HT, anpirtoline concentration-dependently inhibited forskolin-stimulated adenylate cyclase activity in homogenates from the rat substantia nigra. Both effects were not additive, and could be prevented by 5-HT1B receptor antagonists such as propranolol and penbutolol. 4. In superfused rat and pig brain cortex slices preincubated with [3H]-5-HT, the electrically evoked tritium overflow was inhibited by anpirtoline and 5-HT. Whereas 5-HT was equipotent in both tissues (EC50 = 69 nM), anpirtoline was markedly less potent in pig brain cortex slices (EC50 = 1190 nM) than in rat brain cortex slices (EC50 = 55 nM). The concentration-response curve for anpirtoline was shifted to the right by metitepine in both preparations. 5. In the social behaviour deficit test, anpirtoline and trifluoromethylphenyl-piperazine were effective in reversing the isolation-induced impairments in mice, an effect shown only by compounds with agonist properties at the 5-HT1B receptor. 6. In the electrostimulated pain test using mice, anpirtoline dose-dependently increased the pain threshold with an ED50 of 0.52 mg kg-1, i.p. The antinociceptive activity of anpirtoline was abolished by pretreatment with cyproheptadine or propranolol.7. In the forced swimming test in rats, anpirtoline induced a dose-related increase in swimming activity. With an ED50 value of 4.6mgkg-1, i.p., anpirtoline was 4 times more potent than the two standard compounds imipramine and desipramine. The decrease of immobility time or the increase of active periods in this model of behavioural despair is suggested to be characteristic of antidepressant drugs.8. Anpirtoline exhibits both antinociceptive and antidepressant-like activities in animals. It is probable that anpirtoline elicits these pharmacological effects via its agonist effect on 5-HT1B and 5-HT1A receptors.

Presynaptic site of action underlying the ethanol-induced inhibition of norepinephrine release evoked by stimulation of N-methyl- d-aspartate (NMDA) receptors in rat cerebral cortex

Rat brain cortex synaptosomes pre-incubated with [ 3H]norepinephrine were used (1) to provide evidence that part of the NMDA receptors mediating stimulation of norepinephrine (NE) release are located on the noradrenergic varicosities themselves, (2) to characterize these receptors and (3) to examine whether ethanol specifically inhibits the NMDA-evoked NE release via a presynaptic site of action. In synaptosomes superfused with Mg 2+-free Krebs-Henseleit solution, NMDA (2-min exposur) stimulated tritium overflow in a concentration- and glycine-dependent manner. The stimulatory effect of NMDA was not altered by tetrodotoxin but was abolished by omission of Ca 2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg 2+. dl-(E)-2-Amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; a competitive NMDA receptor antagonist) produced a parallel shift of the concentration-response curve for NMDA to the right, whereas dizocilpine (MK-801; an antagonist at the phenyclidine, PCP, recognition site of the NMDA-gated ion channel) reduced the maximum effect of NMDA. Ethanol inhibited the NMDA-evoked tritium overflow in a concentration-dependent manner. In contrast, in synaptosomes superfused with Ca 2+-free Krebs-Henseleit solution containing 15 mM K + throughout, ethanol did not affect the tritium overflow evoked by 2 min introduction of 75 μM Ca 2+ into the superfusion fluid. This Ca 2+ -evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca 2+ channel antagonist Cd 2+. Three main conclusions can be drawn: (1) NMDA receptors mediating stimulation of NE release are also located on the cortical noradrenergic varicosities (and not only on so far unknown excitatory interneurones within the cortex); (2) these receptors exhibit the characteristic pharmacological features of the NMDA receptor system; (3) ethanol selectively inhibits the NE release evoked by stimulation of the presynaptic NMDA receptors, leaving the Ca 2+-evoked release promoted by high K + unaffected. This finding is compatible with the suggestions that the NMDA receptor system itself is a site of action of ethanol.

Prejunctional opioid mu-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors.

Experiments were designed to study the interaction between prejunctional alpha 2-adrenoceptors and both adenosine and opioid receptors at the postganglionic sympathetic nerve endings innervating the tail artery of the rat. Segments of this vessel were preincubated with [3H]-noradrenaline and then perfused/superfused with [3H]-noradrenaline-free medium. Their perivascular nerves were field stimulated with standard stimulation parameters: 24 pulses at 0.4 Hz, 0.3 ms, 200 mA. In some experiments, the stimulation parameters were adjusted in order to obtain similar reference release values despite the presence of a first release-modulating drug. The adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.3-10 mumol/l) and [D-Ala2,MePhe4,Glyol5]enkephalin (DAGO; 0.3-10 mumol/l) depressed the stimulation-evoked overflow of tritium in a concentration dependent manner. The release-inhibiting effect of both NECA and DAGO was enhanced in the presence of the alpha 2-adrenoceptor antagonist rauwolscine (3 mumol/1) while it was attenuated in the presence of the alpha 2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK-14,304; 0.1 mumol/l). These changes occurred both at standard and adjusted stimulation parameters. These results demonstrate that the prejunctional adenosine A1- and opioid mu-receptors interact with the prejunctional alpha 2-adrenoceptors. The level at which these interactions take place (receptors themselves or transduction mechanisms) as well as the physiological significance of the phenomenon remain to be determined.

Quantitative evaluation of the autoinhibitory feedback of release of 5-HT in the caudate nucleus of the rabbit where an endogenous tone on α 2-adrenoceptors does not exist

Slices of caudate nucleus of the rabbit were preincubated with [ 3H]serotonin ([ 3H]5-HT) in the presence of nomifensine, then superfused and twice stimulated electrically. The stimulation-evoked overflow of tritium, representing action potential-induced, exocytotic release of 5-HT, was inhibited concentration-dependency by 5-HT receptor ligands, the potencies of which were compatible with the assumption of a 5-HT 1D-like autoreceptor. The inhibitor of the uptake of 5-HT, 6-nitroquipazine, markedly changed the shape of the concentration-response curve of the 5-HT autoreceptor agonist, 5-carboxamido-tryptamine (5-COHT). The maximum effects of the concentration-response curves of 5-COHT and of exogenous 5-HT became more pronounced in the additional presence of the 5-HT autoreceptor antagonists, metitepin or metergoline. Nonlinear regression analysis of these curves was used to estimate the p K d-value of endogenous 5-HT and the 5-HT biophase concentration at the autoreceptor, in the absence and in the presence of 6-nitroquipazine and in the additional presence of metitepin or metergoline. This revealed a highly operative autoinhibitory feedback, via a 5-HT 1D type autoreceptor of release of 5-HT in tissue from the caudate nucleus. Also the inhibition by the α 2-adrenoceptor agonists, clonidine and UK-14,304, of release of 5-HT was concentration-dependent. There was neither an enhancement of release by rauwolscine, being a 5-HT autoreceptor agonist and α 2-adrenoceptor antagonist, in the presence of metitepin, or by the α-adrenoceptor antagonist, phentolamine (10 −6M). At variance with the noradrenergic innervation of α 2-adrenoceptors on 5-HT terminals in the hippocampus, it was confirmed functionally that α 2-adrenoceptors on 5-HT nerve endings in the caudate nucleus were not activated by endogenous noradrenaline, as to be expected in this tissue which hardly contains any noradrenaline.

Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens.

Effects of electrical stimulation and nicotine on ATP and tritium outflow and smooth muscle tension were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured using the luciferase technique. Electrical stimulation caused biphasic contractions and an acceleration of ATP and tritium outflow. The contraction amplitude and the overflow of ATP increased markedly, whereas the overflow of tritium increased only slightly with the frequency of stimulation (1-10 Hz; constant number of 60 pulses). The contraction amplitude did not increase with an increase in pulse number (20-540 pulses; constant frequency of 5 Hz), whereas the overflow of ATP increased slightly, and that of tritium markedly. Nicotine caused monophasic, transient contractions and, again, an acceleration of ATP and tritium outflow. Contractions, ATP and tritium overflow increased with the concentration of nicotine (56-320 mumol/l) in an approximately parallel manner. The influence of some drugs on responses to electrical stimulation (60 pulses, 5 Hz) and nicotine (180 mumol/l) was investigated. Tetrodotoxin blocked all effects of electrical stimulation but did not change those of nicotine. The reverse was true for hexamethonium. Neither electrical stimulation nor nicotine caused contraction or an increase in ATP outflow after pretreatment with 6-hydroxydopamine. The main effects of prazosin 0.3 mumol/l were to reduce electrically evoked contractions (above all second phase) as well as nicotine-evoked contractions and the nicotine-evoked overflow of ATP (the latter by about 81%). Prazosin also tended to diminish the electrically evoked overflow of ATP. alpha,beta-Methylene-ATP 10 mumol/l elicited a transient contraction and ATP overflow on its own. The main change in the subsequent state of desensitization was a decrease of the first phase of electrically evoked contractions. The main effects of prazosin combined with desensitization by alpha,beta-methylene-ATP were marked decreases of electrically evoked contractions (by 94%), the electrically evoked overflow ATP (by 66%), nicotine-evoked contractions (by 97%) and the nicotine-evoked overflow of ATP (by 70%). It is concluded that both electrical stimulation and nicotine release noradrenaline and ATP in guinea-pig vas deferens. Only part of the evoked overflow of ATP (about 32%) is neural in origin. Another part probably originates from smooth muscle cells where it is released by neurogenic noradrenaline acting at alpha 1-adrenoceptors. Corelease leads to cotransmission: electrically as well as nicotine-evoked contractions consist of adrenergic and purinergic components. Varying types of stimulation release cotransmitter mixtures of varying composition.(ABSTRACT TRUNCATED AT 400 WORDS)

Ethanol inhibits the N-methyl-D-aspartate (NMDA)-induced attenuation of the NMDA-evoked noradrenaline release in the rat brain cortex: interaction with NMDA-induced desensitization

The influence of ethanol, AP5 (DL-2-amino-5-phosphonopentanoic acid) and dizocilpine (MK-801) ((+)-5-methyl-10,11-dihydro-5H-dibenzo (a,b)-cyclo-hept-5,10-imine hydrogen maleate) on the NMDA-induced attenuation of the NMDA-evoked noradrenaline release was examined in rat brain cortex slices preincubated with 3H-noradrenaline. The slices were superfused with Mg(2+)-free Krebs-Henseleit solution. Tritium overflow (corresponding to 3H-noradrenaline release) was stimulated by 300 mumol/l NMDA for 2 min. Presence of 10-100 mumol/l NMDA from 20 to 2 min before stimulation concentration-dependently inhibited the NMDA (300 mumol/l)-evoked 3H overflow, suggesting an agonist-induced desensitization attributable to the modification of events at the NMDA receptor itself and/or distal to this receptor system. The desensitizing effect of 100 mumol/l NMDA was almost complete and was not diminished when the time of preexposure was decreased to 10 min, and when NMDA was removed from the superfusion fluid for up to 5 min before the stimulus; however, the desensitizing effect was reduced after further decrease in the duration of preexposure to NMDA or further prolongation of the interval between preexposure and stimulation. Ethanol inhibited the NMDA-induced 3H overflow (IC50 45 mmol/l); this effect was almost abolished when ethanol was omitted from the superfusion fluid from 2 min before stimulation onward. Ethanol, when simultaneously present with 100 mumol/l NMDA in the superfusion fluid from 20 to 2 min before the NMDA stimulus, concentration-dependently (IC50 112 mmol/l) decreased the inhibitory effect of NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of 8-hydroxy-2-(N-dipropylamino) tetralin (8-OH-DPAT) at alpha 2-adrenoceptors.

1 We have examined the actions of the 5-HT1A-receptor ligand 8-OH-DPAT at alpha 2-adrenoceptor ligand binding sites in human platelet and rat kidney membranes and at functional pre- and postjunctional alpha 2-adrenoceptors in rat atrium and human saphenous vein, respectively. 2 8-OH-DPAT had low affinity for the alpha 2A ligand binding site of human platelet but showed 10 times higher affinity for the alpha 2B ligand binding site of rat kidney (K1 of 6.41, -log M). 3 In functional studies, 8-OH-DPAT had low potency as an antagonist of noradrenaline-induced contractions in human saphenous vein, with a KB of 5.40 (-log M). 4 In rat atria preincubated with [3H]-noradrenaline, 8-OH-DPAT potentiated stimulation-evoked overflow of tritium with an EC30 (concentration producing 30% increase in the stimulation-evoked overflow) of 6.37 (-log M). 5 It is concluded that 8-OH-DPAT shows selectively for the alpha 4B ligand binding site of rat kidney and for the functional prejunctional alpha 2-adrenoceptor of rat atrium, which resembles the alpha 2B ligand binding site.

Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic ?2-adrenoceptors and lack of autoinhibition

Sympathetic neurons from twelve day old chick embryos were plated on polystyrol discs and kept in culture for five days. After incubation with 3H-noradrenaline the discs were transferred to small chambers and superfused. Electrical field stimulation (36 pulses at 3 Hz) increased the outflow of tritium. The evoked overflow of tritium was abolished in the absence of extracellular calcium and was diminished by about 90% in the presence of tetrodotoxin (1 mumol/l). The alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the alpha 1-adrenoceptor agonist methoxamine was ineffective at up to 1 mumol/l. The concentration-response curve of UK-14,304 was shifted to the right by the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 mumol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating alpha 2-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition.

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

The effect of Bay K 8644 (a dihydropyridine Ca(2+)-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [3H]-noradrenaline. Bay K 8644 (3 mumol/l) significantly increased atrial rate from 206 +/- 7 to 259 +/- 9 beats.min-1 (P less than 0.05) and also tritium outflow (expressed as fractional rate of loss in min-1 x 10(3)) from 6.49 +/- 0.35 to 8.61 +/- 0.74 (P less than 0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([3H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.

Protein Kinase C and α2-Adrenoceptor-Mediated Inhibition of Noradrenaline Release from the Rat Tail Artery

In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release.

Estradiol effect on indomethacin and prostaglandin E 2-modulation of adrenergic transmission in rabbit oviduct

The effect of prostaglandin E 2/PGE 2/ and indomethacin on 3H-noradrenaline (3H-NA) release- and on contractions-evoked by field electrical stimulation (FES) was studied in vitro in oviductal isthmus of mature rabbits (untreated and treated with estradiol). FES evoked guanethidine-sensitive contractions and calcium-dependent tritium overflow, which reflected 3H-NA overflow. Markeed and concentration-dependent decrease of FES-evoked contractions by PGE 2 (0.1–100 nM) was observed in both groups of animals. The inhibitory effect of PGE 2 was more pronounced in estradiol treated animals (RC 50 1.5 nM, n=9) than in untreated animals (IC 50 18 nM, n=6). Indomethacin, 1 μM, induced a remarkably pronounced increase of FES-evoked contractions in estradiol treated (by 57.3 ± 6.3%, n=8), in comparison with untreated rabbits (21.4 ± 3.8%, n=7). The amount of FES-evoked release of tritium was significantly higher in untreated than in estradiol treated rabbits. PGE 2 decreased and indomethacin increased tritium-evoked release. The effects of PGE 2 and indoemethacin on tritium-evoked release showed no estradiol dependence. The competitive results of PGE 2 and indomethacin on both evoked contraction and 3H-NA release suggest that endogenous prostaglandin E 2 takes part in modulation of adrenergic mediated contraction and that estradiol enhanced the prostaglandin effect.

Subclassification of the presynaptic α2‐autoreceptors in rabbit brain cortex

1. alpha 2-Adrenoceptor binding sites have been subclassified into alpha 2A sites of which a main characteristic is very low affinity for prazosin, and alpha 2B sites with relatively high affinity for prazosin. The presynaptic alpha 2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of alpha 2A and alpha 2B. Release of [3H]-noradrenaline in cortical slices was elicited by trains of 4 pulses delivered at 100 Hz. 2. Clonidine caused concentration-dependent inhibition of the stimulation-evoked overflow of tritium, with an EC50 of 7.5 nM and a maximal inhibition by 96%. 3. The following alpha-adrenoceptor antagonists shifted the concentration-response curve of clonidine to the right (antagonist-receptor dissociation constants KD in brackets): yohimbine (14 nM), 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408; 15 nM) and 1,2-dimethyl-2,3,9,13betetrahydro-1H-dibenzo[c,f]imidazo[1,5-a]aze pine (BRL 41992; 630 nM). Prazosin 1 microM and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolinedione (AR-C 239) 1 microM failed to antagonize the effect of clonidine. Higher concentrations of prazosin and AR-C 239 greatly accelerated the basal efflux of tritium. 4. The method used permits the functional determination of antagonist affinities undistorted by endogenous alpha 2-autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic alpha 2-autoreceptors in rabbit brain cortex are markedly different from the alpha 2B-subtype and probably belong to the prazosin-insensitive alpha 2A-subtype.

A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins

1. Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]-noradrenaline. 2. Activation of PKC by stepwise increasing concentrations (0.01 nM-1 microM) of 12-O-tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) or mezerein caused a significant and concentration-dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3. PKC inhibitors, polymyxin B (1 and 10 microM) and the isoquinolinesulphonamide, H-7 (1 microM), inhibited significantly the phorbol ester-potentiated tritium overflow evoked by TNS with no effects on the contraction. H-7 and the related inhibitor H-8 at 10 microM reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS-induced contraction in the absence of TPA. 4. None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5. PDBu (0.01 and 0.1 microM) elevated resting tension of the veins more effectively than TPA and mezerein. 6. These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.

Species differences in presynaptic serotonin autoreceptors: mainly 5-HT1B but possibly in addition 5-HT1D in the rat, 5-HT1D in the rabbit and guinea-pig brain cortex

The pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with 3H-serotonin and then superfused with medium containing fluvoxamine 3 mumol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC50 values and maximal effects and antagonist KB values. Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 greater than serotonin greater than 8-OH-DPAT in the rat and 5-CT greater than serotonin greater than RU 24969 greater than 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the KB values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 mumol/l did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (-)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species. The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT1D. The serotonin axons of rat brain cortex may possess 5-HT1D in addition to 5-HT1B autoreceptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Prejunctional prostanoid receptors on cardiac adrenergic terminals belong to the EP3 subtype.

1. The effects of prostaglandin E2 (PGE2) and of several synthetic prostanoids on the cardiac response to sympathetic nerve stimulation in guinea-pig atria have been evaluated. 2. PGE2 (0.01-100 nM), sulprostone (0.01-100 nM) and misoprostol (0.1-100 nM), but not butaprost (0.1-100 nM), dose-dependently reduced the increase in cardiac contractility induced by electrical field stimulation of sympathetic terminals. 3. The EP1 antagonist AH6809 (1 microM) did not modify the inhibition of cardiac response induced by PGE2, sulprostone and misoprostol. 4. In preparations preloaded with [3H]-noradrenaline, tritium overflow induced by electrical field stimulation was greatly and significantly reduced by 100 nM PGE2 and by 100 nM sulprostone. 5. These results indicate that PGE2 and other synthetic prostanoids reduce noradrenaline release from cardiac adrenergic nerve terminals acting on prejunctional inhibitory receptors belonging to the EP3 subtype.

Adenosine receptors mediate a pertussis toxin-insensitive prejunctional inhibition of noradrenaline release on a papillary muscle model

The effects of adenosine receptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea-pig papillary muscles preincubated with [3H] noradrenaline were studied. N6-cyclopentyladenosine (CPA), N6-(R-phenylisopropyl)-adenosine, and 5'-N-ethylcarboxamidoadenosine caused concentration-dependent inhibition of evoked overflow with a rank order of potency typical for interaction of the compounds with the A1-subtype of adenosine receptors. Maximum inhibition was 80%. The A1-selective antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) induced a rightward shift of the concentration-response curve for CPA with a pA2 of 8.35. However, DPCPX per se had no effect on stimulation-evoked tritium overflow. On the other hand, in the presence of 4-nitrobenzylthioinosine (2 mumol/l) and deoxycoformycin (1 mumol/l), inhibitors of adenosine uptake and deamination, respectively, DPCPX produced a concentration-dependent increase in overflow with a pD2 of 8.1. Pretreatment of the animals with pertussis toxin caused a substantial reduction in the activity of toxin-sensitive G proteins, as indicated by a lack of [32P]ADP ribosylation in a ventricular membrane preparation. Nevertheless, the inhibitory effect of the adenosine receptor agonists on stimulus-evoked overflow remained unaffected. These results are compatible with the existence of inhibitory prejunctional adenosine receptors in guinea-pig papillary muscle, which appear to be coupled to a pertussis toxin-insensitive G protein. The role of endogenous adenosine in occupying these receptors seems minimal under basal conditions.