NORADRENALINE RELEASE IN HUMAN CORPUS CAVERNOSUM AND ITS MODULATION VIA PRESYNAPTIC α2-ADRENOCEPTORS

Strips of human corpus cavernosum were incubated with 3H-noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. The electrically (0.66 Hz) evoked tritium overflow was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid. The alpha 2-adrenoceptor agonist B-HT 920 (6-allyl-2-amino-5, 6, 7, 8-4H-thiazolo-5,4-d-azepine), inhibited the evoked overflow, whereas the alpha 1-adrenoceptor agonist, methoxamine, was ineffective. The alpha 2-adrenoceptor antagonist, rauwolscine, facilitated the evoked tritium overflow. It is concluded that the stimulation-evoked release of noradrenaline from the sympathetic nerve fibres of the human corpus cavernosum is modulated via presynaptic alpha 2-adrenoceptors.     

A search for presynaptic beta3-adrenoceptors in the rat

Presynaptically localized adrenoceptors occur on a variety of neurones. In particular, alpha2-adrenoceptors, occurring on neurones of the peripheral and central nervous system, inhibit the release of the respective transmitters whereas beta2-adrenoceptors on some types of postganglionic sympathetic neurones facilitate noradrenaline release. Since only little information is available whether there are also presynaptic beta3-adrenoceptors, we examined the effect of beta3-adrenoceptor agonists on noradrenaline release from the resistance vessels and the hippocampus of the rat and on serotonin and acetylcholine release from the rat hippocampus. In rat hippocampal slices preincubated with (H-noradrenaline, 3H-serotonin and 3H-choline and superfused in the presence of an inhibitor of the neuronal transporter of the respective neurone, the beta3-adrenoceptor agonist CL 316243 did not affect the electrically evoked tritium overflow. The latter was, however, inhibited by at least 50% by agonists of the respective autoreceptors. CL 316243 and another three beta3-adrenoceptor agonists (BRL 37344, ZD 2079 and CGP 12177) failed to affect the electrically evoked tritium overflow also in slices preincubated with 3H-noradrenaline and superfused in the presence of the alpha2-adrenoceptor antagonist rauwolscine whereas prostaglandin E2 caused a marked inhibition. In pithed and vagotomized rats, the increase in diastolic blood pressure induced by electrical stimulation of the sympathetic outflow was also not affected by CL 316243 but markedly inhibited by the cannabinoid receptor agonist WIN 55212-2. CL 316243 and WIN 55212-2 were devoid of an effect on the rise in diastolic blood pressure induced by exogenous noradrenaline. In conclusion, our data suggest that the noradrenergic neurones innervating the resistance vessels of the rat and the noradrenergic, serotoninergic and cholinergic neurones of the rat hippocampus are not endowed with presynaptic beta3-adrenoceptors.     

Beta-endorphin: a highly selective endogenous opioid agonist for presynaptic mu opioid receptors.

In the presence of physiological cations (in Krebs-4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid buffer) at 37 degrees C the Ki value's of beta-endorphin for mu- and delta-opioid receptor binding sites in rat neocortical membranes, labeled with [3H][D-Ala2,MePhe4,Gly- ol5]enkephalin (DAMGO) and [3H][D-Ala2-D-Leu5]enkephalin (in the presence of unlabeled DAMGO), respectively, amounted to about 9 and 22 nM. Surprisingly, a very different selectivity pattern for the endogenous opioid peptide was found when the affinity of beta-endorphin for functional presynaptic opioid receptors was examined. Thus, beta-endorphin strongly inhibited the electrically evoked release of [3H]NE from rat neocortical slices with an IC50 value of about 0.5 nM, whereas [14C] acetylcholine release from neostriatal slices was inhibited with an IC50 value of about 100 nM. On the other hand, the electrically evoked release of [3H]dopamine from striatal slices was not affected by beta-endorphin. The inhibitory effects of DAMGO and beta-endorphin on [3H]NE release from neocortical slices were equally well antagonized by naloxone. Moreover, 10 nM of the highly selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen- Thr-NH2 antagonized competitively the inhibitory effect of beta-endorphin on [3H]NE release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine and serotonin released from the rat perfused heart by compound 48/80 or by allergen challenge influence noradrenaline or acetylcholine exocytotic release

Summary— Terminal nerve fibres of the autonomic nervous system closely approach mast cells in peripheral organs, and mutual influences between release of neurotransmitters or mast cell mediators may cause neuro-immunological interactions. We have studied the influence of mast cell degranulation on the release of endogenous noradrenaline and newly incorporated acetylcholine (such as ¹⁴C-choline/acetylcholine overflow) evoked by stimulation of extrinsic postganglionic sympathetic or preganglionic vagal nerves in the rat Langendorff heart perfused with Tyrode solution. Compound 48/80 perfused in normal hearts, or ovalbumin infused into hearts from rats sensitized to ovalbumin, enhanced the overflow of endogenous histamine and serotonin. Both stimuli increased the release of mediators to a similar extent and with fast kinetics. Maximum average concentrations in the perfusate of histamine were about 800 nmol/l, and of serotonin 40 nmol/l, in a sample collected within 4 min after mast cell degranulation. Stimulation of autonomic nerves did not affect basal histamine or serotonin overflow. Whereas basal overflows were unaffected, the stimulation-evoked releases of both noradrenaline and acetylcholine, were facilitated when compound 48/80 was perfused before and during nerve stimulation. The facilitation of noradrenaline overflow was more pronounced (by 60%) when compound 48/80-induced mediator overflow started 4 min before nerve stimulation as compared to 30 s (15%), and was reduced by cocaine (by 50%), and, in the presence of cocaine, abolished by cimetidine (but was unaffected by mepyramine and thioperamide) and N^G-nitro-(L)-(-)-arginine. In the presence of cimetidine and cocaine, when the facilitatory components were abolished, the evoked noradrenaline overflow observed 30 s after the start of infusion of compound 48/80 was inhibited, and the inhibition was partly reduced by methiotepin and ketanserin. Ovalbumin infusion in hearts from sensitized animals caused an inhibition of evoked noradrenaline overflow sensitive to methiotepin and also partly to ketanserin, and no facilitation was observed. The facilitation (> 100%) of evoked overflow of acetylcholine observed at 4 min after the start of perfusion with compound 48/80 was partly reduced by thioperamide (but not mepyramine or cimetidine) and to a comparable extent either by tropisetron (3 μmol/l) alone or by tropisetron plus methiotepin. In conclusion, degranulation of immunological cells is followed by histamine and serotonin release in the rat heart and may affect the release of autonomic neurotransmitters in rather unusual ways, by i) an uptake₁-dependent and ii) an H₂-mediated facilitation which probably involves nitric oxide as a permissive mediator, and iii) a serotonergic inhibition, of noradrenaline release, and iv) an H₃- and serotonergic facilitation of acetylcholine release.     

Interaction between serotonin uptake inhibitors and alpha-2 adrenergic heteroreceptors in the rat hypothalamus

The effectiveness of presynaptic receptor agonists to inhibit the electrically evoked release of [3H]monoamines from brain slices is attenuated in the presence of blockade of neuronal uptake for the serotonin (5-HT) and the norepinephrine (NE) systems. There is controversy, however, as to the existence of a functional link between the presynaptic receptors and the neuronal uptake carriers. An alternative hypothesis involves competition for the presynaptic receptor sites between the exogenous agonist and the released neurotransmitter. In order to examine the proposed functional interaction, we studied the alpha-2 adrenoceptor-mediated inhibition of the electrically evoked release of [3H]-5-HT from slices of the rat hypothalamus, a model in which endogenous NE does not activate the alpha-2 heteroreceptors located on 5-HT terminals. The inhibitors of 5-HT uptake, citalopram (0.01-1 microM) and paroxetine (1 microM), which by themselves did not modify [3H]-5-HT release, antagonized the inhibition of [3H]-5-HT overflow produced by UK 14.304, an alpha-2 adrenoceptor agonist. The inhibition of the electrically evoked release of [3H]-5-HT by exogenous NE (0.1-1 microM) was also attenuated in the presence of citalopram. In contrast, citalopram did not modify the electrically evoked release of [3H]-NE or the inhibition of [3H]-NE release mediated by UK 14.304. When the 5-HT autoreceptor was blocked by cyanopindolol, the inhibitory effect of UK 14.304 on [3H]-5-HT release was unaltered in the presence of citalopram.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of brain imidazoline receptors in normotensive and hypertensive rats: differential regulation by chronic imidazoline drug treatment.

The binding of [3H]idazoxan in the presence of l-epinephrine was used to characterize and quantitate imidazoline receptors in the brain of spontaneously hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats before and after chronic imidazoline drug treatment. In the cerebral cortex of WKY and SHR rats, the rank order of potency of imidazoli(di)ne drugs (cirazoline greater than idazoxan greater than naphazoline greater than clonidine much greater than RX821002) competing with [3H]idazoxan showed the specificity for an imidazoline receptor which also appeared heterogeneous in nature. In SHR rats, the density of imidazoline receptors (hypothalamus greater than medulla oblongata greater than cerebral cortex) and proportion of high- and low-affinity sites for the receptor were not different from those in WKY and SD rats, suggesting that the receptor itself is not altered in hypertension. However, chronic treatment with idazoxan and cirazoline (10 and 1 mg/kg, i.p., every 12 h for 7 days) consistently increased (about 35%) the density of imidazoline receptors in the brain of WKY and SD, but not in SHR rats. A similar treatment with RX821002, the 2-methoxy analog of idazoxan, which is a highly selective alpha-2 adrenoceptor antagonist, did not increase the density of brain imidazoline receptors. Moreover, the up-regulation of these receptors induced by cirazoline was still present after alkylation of the alpha-2 adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The lack of regulation by idazoxan and cirazoline of the density of imidazoline receptors in the brain of SHR rats suggests the existence of a relevant abnormality in the adaptive process of these receptors in this genetic model of hypertension.     

The concentration of oxygen,lactate and glucose in the central veins,right heart,and pulmonary artery: a study in patients with pulmonary hypertension

Introduction  

Decreases in oxygen saturation (SO₂) and lactate concentration [Lac] from superior vena cava (SVC) to pulmonary artery have been reported. These gradients (ΔSO₂ and Δ[Lac]) are probably created by diluting SVC blood with blood of lower SO₂ and [Lac]. We tested the hypothesis that ΔSO₂ and Δ[Lac] result from mixing SVC and inferior vena cava (IVC) blood streams.     

Secreted and intracellular phospholipases A2 inhibition by 1-decyl 2-octyl-glycerophosphocholine in rat peritoneal macrophages

Summary— Compounds able to inhibit phospholipases A₂ can be considered as potential anti-inflammatory drugs. In this respect, the inhibitory effect of the phospholipid analogue 1-decyl 2-octyl-rac-glycero-3-phosphocholine (decyloctyl-GPC) added to the culture medium of rat peritoneal macrophages stimulated with ionophore A23187 was determined, (a) The substrate of phospholipase A₂ 1-octadecanoyl 2-[¹⁴C]eicosatetraenoyl-sn-glycero-3-phosphocholine ([¹⁴C]20:4-GPC) was added to the culture medium. In macrophages + extracellular fluids, its hydrolysis at the 2-position, produced [¹⁴C]non-phosphorous lipids which reached 12% of the dose at 0.14 μM, 73% at 0.9 and > 90% at 1.6 μM; in experiments where macrophages and extracellular fluids were analyzed separately, decyloctyl-GPC initially added at 4 μM, significantly inhibited the release of [¹⁴C]fatty acids and the eicosanoid synthesis, demonstrating its ability to inhibit secreted and/or intracellular phospholipases A₂. (b) Extracellular fluids were separated from the macrophages and incubated with [¹⁴C]20:4-GPC: 48% of the dose was hydrolyzed by extracellular fluid-associated secreted phospholipase A₂ and decyloctyl-GPC at 3 μM, reduced this hydrolysis by 50%. (c) [³H]arachidonic acid ([³H]20:4) was added to the culture medium and was esterified in the macrophage membrane phospholipids. Activation of intracellular phospholipase A₂ induced the release of [³H] fatty acids and eicosanoid synthesis. These releases were inhibited by 50% with decyloctyl-GPC added at 4 μM. (d) [³H]20:4 and [¹⁴C]20:4-GPC were added to the culture medium of the macrophages. [³H] and [¹⁴C] fatty acids and eicosanoids were released in macrophages or extracellular fluids. They were significantly reduced by the phospholipid analogue added at 4 μM. It is concluded that secreted and intracellular phospholipases A₂ were both inhibited by decyloctyl-GPC which extensively reduced the 20:4 release from exogenous and membrane phospholipids and therefore eicosanoid synthesis.     

Interaction between tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors and the presynaptic 5-hydroxytryptamine inhibitory autoreceptors in the rat hypothalamus

In slices of the rat hypothalamus prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), exposure to lysergic acid diethylamide or 5-methoxytryptamine decreased, in a concentration-dependent manner, the release of 3H-transmitter elicited by electrical stimulation. These inhibitory effects were antagonized by the 5-HT receptor antagonist methiothepin (1 microM). Exposure to methiothepin on its own increased in a concentration-dependent manner the electrically evoked overflow of [3H]-5-HT. Exposure to tricyclic antidepressants, like imipramine and amitriptyline, and to nontricyclic 5-HT uptake inhibitors, like paroxetine and citalopram, did not modify by themselves the electrically evoked overflow of [3H]-5-HT. Yet, the four inhibitors of neuronal uptake of 5-HT, antagonized the inhibition by lysergic acid diethylamide or 5-methoxytryptamine of the electrically induced release of [3H]-5-HT. After depletion of endogenous stores of 5-HT by pretreatment with para-chlorophenylalanine (300 mg/kg i.p.), the inhibitors of 5-HT uptake increased the electrically evoked release of [3H]-5-HT in a concentration-dependent manner. Their order of potency to enhance 5-HT overflow after pretreatment with parachlorophenylalanine paralleled their potency at inhibiting neuronal uptake of 5-HT (paroxetine = citalopram greater than imipramine greater than amitriptyline). In para-chlorophenylalanine-treated rat hypothalamic slices, these inhibitors of 5-HT uptake antagonized the inhibition by 5-HT autoreceptor agonists of the electrically evoked release of [3H]-5-HT to a similar extent than was observed in control rats. It is concluded that inhibition of 5-HT uptake reduces the effectiveness of 5-HT autoreceptor agonists to inhibit the electrically evoked release of [3H]-5-HT, irrespective of the chemical structure of the uptake inhibitor or of the levels of endogenous 5-HT achieved in the synaptic gap.     

Trace amines inhibit the electrically evoked release of [3H]acetylcholine from slices of rat striatum in the presence of pargyline: similarities between beta-phenylethylamine and amphetamine

Amphetamine (AMPH) inhibits the electrically evoked release of [3H]acetylcholine (ACh) from rat striatal slices through the activation of inhibitory dopamine receptors. Naturally occurring analogs of amphetamine (AMPH) such as beta-phenylethylamine (beta-PEA), tyramine (TYR) and octopamine (OCT) are present in trace amounts in the brain of several species. We have studied in this model, in comparison with AMPH, the effects of beta-PEA, TYR and OCT, in order to explore if their central effects are mediated through an action involving dopaminergic nerve terminals or whether they activate a specific receptor directly. In contrast to the results obtained with AMPH, in the absence of inhibition of monoamine oxidase activity, the three amines beta-PEA (0.1-10 microM), TYR (0.1-10 microM) and OCT (10 microM) did not affect the electrically evoked release of [3H]ACh. On the other hand, in the presence of pargyline (10 microM), the three amines inhibited the electrically evoked release of [3H]ACh and all subsequent experiments were carried out in the presence of pargyline. After pretreatment with reserpine (5 mg/kg s.c., 24 h), which results in a 95% depletion of the endogenous dopamine content, OCT lost its inhibitory effect on [3H]ACh release, whereas beta-PEA and TYR still inhibited the electrically evoked release of [3H]ACh. Reserpine pretreatment (5 mg/kg s.c., 24 h) combined with alpha-methyl-p-tyrosine (300 mg/kg i.p., 2 h) reduced endogenous dopamine levels by 99.9%, but, under these conditions, beta-PEA, TYR and AMPH still retained their inhibitory effect on [3H]ACh, release. These inhibitory effects of beta-PEA and AMPH on [3H] ACh release were antagonized by S-sulpiride (0.1 microM). In striatal slices from untreated rats, the inhibition of [3H]ACh released by beta-PEA (30 microM), TYR (30 microM) or AMPH (10 microM) was abolished completely after a 6-hydroxydopamine lesion of the nigro-striatal dopaminergic system. The present data indicate that in order to inhibit the release of [3H]ACh from rat striatal slices in vitro, OCT requires the integrity of vesicular stores of dopamine. On the other hand, beta-PEA, TYR and AMPH are still active when the dopamine levels are depleted, although they require the integrity of the dopaminergic nerve terminal. Inhibition of monoamine oxidase is essential to demonstrate the inhibitory effects of exogenous beta-PEA, TYR and OCT on cholinergic transmission. Our results indicate that a hypothesis concerning a possible physiopathological role of endogenous beta-PEA or TYR should involve concomitant changes in monoamine oxidase activity.     

Inhibition by 5,6-dihydroxy-2-dimethylaminotetralin (M7) of noradrenergic neurotransmission in the rabbit hypothalamus: role of alpha-2 adrenoceptors and of dopamine receptors

Rabbit hypothalamic slices were prelabeled with [3H]norepinephrine and transmitter release elicited by electrical stimulation. In the presence of 10 microM cocaine and in a low Ca++ medium (0.65 mM), exposure for 8 min to exogenous dopamine (0.01-1 microM) inhibited, in a concentration-dependent manner, the electrically evoked release of [3H]norepinephrine. This inhibitory effect of dopamine on [3H]norepinephrine release was antagonized by the dopamine receptor antagonist S-sulpiride (1 microM), but remained unchanged in the presence of the alpha-2 adrenoceptor antagonists idazoxan (1 microM) or yohimbine (0.1 microM). These results indicate that, in a low Ca++ medium, exposure to dopamine decreased [3H]norepinephrine overflow in rabbit hypothalamic slices through the exclusive activation of presynaptic inhibitory dopamine receptors. M7 (5,6-dihydroxy-2-dimethylaminotetralin) is a potent agonist at central presynaptic dopamine autoreceptors and at peripheral alpha-2 adrenoceptors. Exposure to M7 in a normal Ca++ medium, inhibited in a concentration-dependent manner the electrically evoked release of [3H]norepinephrine without affecting the spontaneous outflow of radioactivity. The slope of the concentration-effect curve for these inhibitory effects of M7 was rather flat and the maximal inhibition obtained was 80%. The selective D2 receptor antagonist S-sulpiride (1 microM) failed to produce a significant shift to the right in the concentration-effect curve for the inhibitory effects of M7 on [3H]norepinephrine release. The preferential alpha-2 adrenoceptor antagonist yohimbine (0.1 microM) significantly antagonized the inhibition of [3H]norepinephrine release elicited by 0.01 microM M7, but not for higher concentrations of this aminotetraline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between density (maximum binding) of alpha adrenoceptor binding sites and contractile response in four canine vascular tissues

In microsomal fractions from dog aorta, saphenous veins, mesenteric arteries and veins, both [3H]prazosin and [3H]rauwolscine displayed monophasic saturation in binding. The Kd for [3H] rauwolscine binding was similar for all these blood vessels, but the maximum number of [3H]rauwolscine binding sites was 3 to 7 times higher in veins compared to arteries. The Kd for [3H] prazosin was higher in saphenous vein than that in the arteries. The maximum number of binding sites for [3H]prazosin was similar, except for that in aorta, which was 3 times greater. Phenylephrine (alpha-1 adrenoceptor selective agonist) or norepinephrine (nonselective adrenoceptor agonist) produced similar maximal responses in all vessels. The alpha-2 adrenoceptor selective agonist, B-HT 920 (2-amino-6-allyl-3,4,7,8-tetrahydro-6H-thiazolo[5,4-d]-azepine)-induced contraction in veins but not in arteries. Prazosin (10(-6) M) inhibited completely the contractions to norepinephrine (3 x 10(-6) M) in mesenteric arteries and to phenylephrine (3 x 10(-6) M) in arteries and veins. Contractile responses of mesenteric artery were unaffected by rauwolscine. Rauwolscine (10(-7) M) caused a greater parallel rightward shift of the concentration-response curve to norepinephrine than did prazosin (10(-7) M) in saphenous veins, and a further rightward shift of responses to norepinephrine after 10(-7) M prazosin in mesenteric vein and saphenous vein and abolished B-HT 920-induced responses at alpha-2 adrenoceptors. The tissues responding to B-HT-920 correspond to those having the highest alpha-2 receptor density as measured by [3H]rauwolscine binding. The density of such sites required for contraction to be initiated in veins was much higher than with alpha-1 adrenoceptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava

In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H2O2 contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H2O2-induced contraction required extracellular Ca2+ influx. Because of this difference in Ca2+ utilization, we hypothesized that signaling pathways mediating H2O2-induced contraction in vena cava under resting tone differed from those mediating H2O2-induced contraction in aorta contracted with KCl. Inhibitors of cyclooxygenase (COX) 1 and 2 (indomethacin, 10 microM), thromboxane A2 (TXA2) receptors [ICI185282 (2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid), 10 microM], p38 mitogen-activated protein kinase (MAPK) [SB203580 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), 10 microM], extracellular signal-regulated kinase (Erk) [PD98059 (2'-amino-3'-methoxyflavone), 10 microM], src [PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, 10 microM], and rho kinase [Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), 10 microM], significantly reduced H2O2-induced contraction in vena cava under resting tone and aorta after KCl (30 mM) contraction. In contrast, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 20 microM] did not reduce aortic or venous H2O2-induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA2 receptor agonist U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF(2alpha), 1 microM), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619, and PI3-K inhibition reduced venous contraction to U46619. Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H2O2 contraction, possibly via rho kinase activation, and that H2O2-induced contraction via p38 and Erk MAPK probably occurs independently of TXA2 receptor activation.     

Inhibitory effects of amiloride on alpha adrenoceptors in canine vascular smooth muscle

Amiloride inhibits vascular smooth muscle contractions from canine aorta and saphenous vein. The mechanisms were studied using radioligand binding and functional techniques. Amiloride inhibited [3H]prazosin and [3H]rauwolscine binding to alpha-1 and alpha-2 adrenoceptors in a concentration-dependent manner. Amiloride increased Kd values for [3H]rauwolscine without affecting the maximum binding of [3H]prazosin. These results suggest that the drug interacts with the alpha-1 adrenoceptor binding sites in a competitive manner and with the alpha-2 adrenoceptor binding sites in a noncompetitive manner. Amiloride reduced maximal contractile responses to agonists selective for both alpha adrenoceptors and to elevated K+, the EC50 values were increased by about 10-fold in the presence of amiloride. In Ca+(+)-free Krebs' solution, contractions induced in saphenous vein after addition of Ca++ in saphenous vein in the presence of adrenoceptor agonists were inhibited by amiloride. Our results suggest that amiloride reduced alpha-1 and alpha-2 adrenoceptor-mediated responses and inhibited Ca++ influx.     

Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. I. Release in the absence of vesicular transmitter stores

The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of reserpine-pretreated rabbits was examined. Although reserpine pretreatment reduced tissue DA levels by greater than 95%, the basal efflux of DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) was slightly greater than that observed in untreated slices. DOPAC constituted the large majority of the basal efflux of endogenous compounds. No overflow of endogenous compounds was evoked by electrical stimulation (3 Hz, 3 min) after reserpine pretreatment. Superfusion with alpha-methyl-p-tyrosine (100 microM) abolished the efflux of endogenous DA and DOPAC. AMPH (0.3-10 microM) produced a concentration-dependent increase in the basal efflux of endogenous DA and a concomitant decrease in endogenous DOPAC efflux. The total efflux of endogenous compounds (DA + DOPAC) tended to be decreased by AMPH. No electrically evoked overflow of endogenous compounds was observed in the presence of AMPH. The increase in synaptic DA produced by AMPH was reflected by a concentration-dependent reduction in the electrically evoked overflow of [3H]acetylcholine (ACh). The ability of AMPH to increase DA efflux and inhibit [3H]ACh release was blocked by inhibition of DA synthesis with alpha-methyl-p-tyrosine (100 microM) or by blockade of the DA neuronal uptake carrier with nomifensine (NOM) (10 microM) and was potentiated by inhibition of monoamine oxidase with pargyline (10 microM). NOM also blocked partially the ability of AMPH to reduce endogenous DOPAC efflux. NOM increased the basal efflux of endogenous DA and inhibited electrically evoked [3H]ACh release but these effects were quantitatively much less than those produced by AMPH. NOM had no effect on DOPAC efflux. Pargyline had little effect on endogenous DA efflux or electrically evoked [3H]ACh release but abolished DOPAC efflux and increased tissue DA levels measured at the end of superfusion. When given in combination, NOM and pargyline produced a similar degree of inhibition of [3H]ACh release as AMPH, although the increase in DA efflux produced by this drug combination was less than that produced by AMPH. These results suggest that in the absence of vesicular transmitter stores (reserpine-pretreatment): synthesis provides a continuous supply of DA which is metabolized rapidly within the neuron and is lost as DOPAC; AMPH facilitates the synthesis-dependent efflux of extravesicular DA probably by an accelerated exchange diffusion mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycine-evoked neurotransmitter release from rat hippocampal brain slices: evidence for the involvement of glutaminergic transmission

Glycine caused a concentration-dependent evoked release of [3H]norepinephrine from rat hippocampal brain slices. Other amino acids evoked [3H]norepinephrine release with a rank order of potency: L-serine greater than or equal to glycine greater than beta-alanine greater than D-serine. Strychnine inhibited [3H]norepinephrine release evoked by both glycine and L-serine, but was less effective in inhibiting the release evoked by N-methyl-D-aspartate (NMDA) and kainic acid. Inhibitors of the NMDA receptor/ionophore complex, MK-801, CPP and Mg++, as well as the strychnine-insensitive glycine receptor antagonist, HA-966, caused an incomplete inhibition (maximum approximately 60%) of glycine-evoked [3H]norepinephrine release. The potencies with which MK-801, CPP and Mg++ inhibited glycine- and NMDA-evoked [3H]norepinephrine release were very similar. The combination of MK-801 plus kynurenic acid, a nonselective glutamate receptor antagonist, caused no greater inhibition of glycine-evoked release than MK-801, alone. omega-Conotoxin GVIA, an inhibitor of neuronal L- and N-type voltage-sensitive calcium channels, inhibited glycine-evoked [3H]norepinephrine release by approximately 50%, whereas the L-channel inhibitor PN 200-110 had no significant effect. The combination of MK-801 plus omega-conotoxin GVIA caused only a slightly greater inhibition (P greater than .05) of glycine-evoked release than MK-801 alone. Tetrodotoxin inhibited glycine-evoked release of [3H]norepinephrine by approximately 75%. The inhibitory effects of tetrodotoxin and omega-conotoxin GVIA suggest that voltage-sensitive sodium channels and N-type voltage-sensitive calcium channels are important mediators of glycine-evoked release of [3H]norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological evidence for alpha-2 adrenoceptor heterogeneity: differential binding properties of [3H]rauwolscine and [3H]idazoxan in rat brain

In the preceding paper, we have reported that the two alpha-2 adrenoceptor antagonists, [3H]rauwolscine and [3H]idazoxan, exhibit markedly different autoradiographic distributions throughout rat brain. Although [3H]idazoxan labeling appears over brain regions receiving noradrenergic innervation, [3H]rauwolscine binding sites are localized most densely in several areas corresponding to dopaminergic terminal fields. We have presently characterized the pharmacological binding properties of high affinity [3H]rauwolscine and [3H]idazoxan labeled sites, using tissue preparation and incubation protocols which are identical to those used in the previous autoradiographic study. Endogenous monoamines inhibited radioligand binding with a rank order of potency of epinephrine = norepinephrine greater than dopamine greater than serotonin. Numerous dopaminergic compounds failed to inhibit either [3H]rauwolscine or [3H]idazoxan binding with high potency, and rauwolscine was a poor inhibitor of [3H]spiroperidol binding. Several adrenergic compounds which selectively label alpha-1 or beta adrenoceptors also exhibited low potency in inhibiting either radioligand. In contrast, alpha-2 adrenoceptor agonists and antagonists possessed high affinity for both [3H]rauwolscine and [3H]idazoxan labeled sites. Their relative potencies at the two sites differed, however. Whereas idazoxan was equipotent in inhibiting either [3H]rauwolscine or [3H]idazoxan binding, rauwolscine exhibited 10-fold higher affinity for its own labeled site. These pharmacological data are consistent with anatomical data presented in the preceding paper, which support the existence of a heterogenous population of alpha-2 adrenoceptors within rat brain, labeled entirely by [3H]idazoxan and only in part by [3H]rauwolscine.     

Release of gamma-[3H]aminobutyric acid (GABA) from electrically stimulated rat cortical slices and its modulation by GABAB autoreceptors

Slices of rat temporo-parietal cortex were prelabeled with gamma-[3H]aminobutyric acid ([3H]GABA), in the presence of the glial GABA uptake inhibitor beta-alanine. The slices were then superfused with a medium containing the GABA transaminase inhibitor aminooxyacetic acid and stimulated electrically (5 min, 2 msec, 36 mA at 5 or 10 Hz), in the presence of the neuronal GABA reuptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid] and of beta-alanine. Representative experiments showed that the tritium released could be accounted for almost entirely by authentic [3H]GABA. The electrically evoked overflow of [3H]GABA was tetrodotoxin sensitive and largely calcium-dependent. Exogenous GABA, added to the superfusion medium at 3 to 30 microM, reduced in a concentration-dependent manner the electrically evoked (5 Hz) release of [3H]GABA. The GABAB receptor agonist (-)-baclofen, but not the GABAA receptor agonist muscimol, mimicked GABA and produced a concentration-inhibition curve almost superimposable to that of the natural transmitter. The effects of GABA and of (-)-baclofen were much more pronounced at 5 than at 10 Hz. The GABA-induced inhibition of [3H]GABA release was sensitive to the novel GABAB receptor antagonist beta-(p-chlorophenyl)-3-amino propyl phosphonic acid which, by itself, increased the [3H]GABA overflow. The inhibitory effect of GABA was not counteracted by the GABAA receptor antagonists bicuculline or SR 95531 [2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide]. The results are compatible with the presence in the rat cerebral cortex of autoreceptors mediating inhibition of GABA release and belonging to the GABAB type. These autoreceptors may be activated tonically under physiological conditions.     

Effects of amiloride and 5-(N,N-hexamethylene)amiloride on dopaminergic and cholinergic neurotransmission

In order to establish the role of the Na+/H+ exchange transport on neurotransmission, we investigated the effects of amiloride and of 5-(N,N-hexamethylene)amiloride (HMA) on dopamine (DA) and acetylcholine (ACh) release and on receptor-mediated modulation of DA and ACh release. Superfused rabbit striatal slices prelabeled with [3H]DA and [14C]choline were stimulated electrically in the presence and absence of several concentrations of these agents. Amiloride (3-10 microM) and HMA (0.3-10 microM) reduced the basal efflux and the stimulation evoked overflow of total 3H and of [3H]-3,4-dihydroxyphenylacetic acid and inhibited monoamine oxidase activity. The inhibition of stimulation evoked overflow of total 3H was blocked by pretreatment with nomifensine but not by sulpiride. Amiloride had no effect on the basal efflux and the stimulation evoked overflow of ACh or it did modify apomorphine-induced inhibition of DA and ACh release. However, at 3 to 10 microM, HMA enhanced the basal efflux of 3H; this effect was not prevented either by uptake inhibition with nomifensine or by low extracellular calcium. These results suggest that amiloride-sensitive Na+ transport and the amiloride and HMA-sensitive Na+/H+ antiporter play no role on the secretion of DA and ACh, or on the mechanisms by which activation of pre- and postsynaptic DA receptors lead to inhibition of neurotransmitter release. Amiloride- and HMA-induced monoamine oxidase inhibition accounts for the effects of amiloride and HMA on DA efflux and overflow. The guanidine moiety present in the amiloride and HMA molecules is most likely responsible for these effects.     

Interaction between neuronal uptake inhibitors and presynaptic serotonin autoreceptors in rat hypothalamic slices: comparison of K+ and electrical depolarization

In rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine ([3H]-5-HT), exposure to the 5-HT receptor agonist lysergic acid diethylamide (0.1-1 microM) or 5-methoxytryptamine (0.1-10 microM) decreased in a concentration-dependent manner the release of 3H-transmitter elicited by high K+ or electrical stimulation. Exposure to the 5-HT autoreceptor antagonist methiothepin (0.1-1 microM) increased in a concentration-dependent manner the K+ stimulation-evoked overflow of [3H]-5-HT and a similar increase was observed under conditions of electrical stimulation. In contrast, exposure to the nontricyclic 5-HT uptake inhibitor citalopram (0.1-1 microM) did not modify by itself the electrically evoked overflow of [3H]-5-HT, but increased in a concentration-dependent manner the release of 3H-transmitter elicited by K+ stimulation. This effect of citalopram on transmitter release was potentiated when the endogenous stores of 5-HT were depleted by pretreatment with para-chlorophenylalanine methyl ester (300 mg/kg i.p.). Citalopram was shown previously to antagonize the inhibition by lysergic acid diethylamide of the electrically evoked release of [3H]-5-HT in rat hypothalamic slices. Yet, this inhibitor of neuronal uptake of 5-HT did not antagonize the effects of lysergic acid diethylamide when the release of [3H]-5-HT was evoked by K+ depolarization. Electrical stimulation represents a more physiological experimental model for transmitter release than exposure to high K+, and therefore the interaction between 5-HT uptake blockade and presynaptic inhibitory 5-HT autoreceptors, observed in the hypothalamus with electrical stimulation but not with K+ depolarization, remains of biological relevance.