Tritium Overflow Research Articles

Antibodies Against the NH2-Terminus of the GluA Subunits Affect the AMPA-Evoked Releasing Activity: The Role of Complement.

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.

N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain.

Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6-8.8) in an atropine-sensitive manner (apparent pA2 8.6-8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5-6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32μM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1μM, yielding a biphasic curve with a pIC50 of 8.6 for the falling part and a pEC50 of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10μM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory Gs protein comes into play at high concentrations of a muscarinic receptor agonist.

A search for presynaptic inhibitory histamine receptors in guinea-pig tissues: Further H3 receptors but no evidence for H4 receptors

The histamine H4 receptor is coupled to Gi/o proteins and expressed on inflammatory cells and lymphoid tissues; it was suggested that this receptor also occurs in the brain or on peripheral neurones. Since many Gi/o protein-coupled receptors, including the H3 receptor, serve as presynaptic inhibitory receptors, we studied whether the sympathetic neurones supplying four peripheral tissues and the cholinergic neurones in the hippocampus from the guinea-pig are equipped with release-modulating H4 and H3 receptors. For this purpose, we preincubated tissue pieces from the aorta, atrium, renal cortex and vas deferens with 3H-noradrenaline and hippocampal slices with 3H-choline and determined the electrically evoked tritium overflow. The stimulation-evoked overflow in the five superfused tissues was inhibited by the muscarinic receptor agonist oxotremorine, which served as a positive control, but not affected by the H4 receptor agonist 4-methylhistamine. The H3 receptor agonist R-α-methylhistamine inhibited noradrenaline release in the peripheral tissues without affecting acetylcholine release in the hippocampal slices. Thioperamide shifted the concentration–response curve of histamine in the aorta and the renal cortex to the right, yielding apparent pA2 values of 8.0 and 8.1, respectively, which are close to its affinity at other H3 receptors but higher by one log unit than its pKi at the H4 receptor of the guinea-pig. In conclusion, histamine H4 receptors could not be identified in five experimental models of the guinea-pig that are suited for the detection of presynaptic inhibitory receptors whereas H3 receptors could be shown in the peripheral tissues but not in the hippocampus.This article is part of the Special Issue entitled ‘Histamine Receptors’.

Neuropeptide Y as a presynaptic modulator of norepinephrine release from the sympathetic nerve fibers in the pig pineal gland.

Norepinephrine (NE) released from the sympathetic nerve endings is the main neurotransmitter controlling melatonin synthesis in the mammalian pineal gland. Although neuropeptide Y (NPY) co-exists with NE in the pineal sympathetic nerve fibers it also occurs in a population of non-adrenergic nerve fibers located in this gland. The role of NPY in pineal physiology is still enigmatic. The present study characterizes the effect of NPY on the depolarization-evoked 3H-NE release from the pig pineal explants. The explants of the pig pineal gland were loaded with 3H-NE in the presence of pargyline and superfused with Tyrode medium. They were exposed twice to the modified Tyrode medium containing 60 mM of K+ to evoke the 3H-NE release via depolarization. NPY, specific agonists of Y1- and Y2- receptors and pharmacologically active ligands of α2-adrenoceptors were added to the medium before and during the second depolarization. The radioactivity was measured in medium fractions collected every 2 minutes during the superfusion. NPY (0.1-10 μM) significantly decreased the depolarization-induced 3H-NE release. Similar effect was observed after the treatment with Y2-agonist: NPY13-36, but not with Y1-agonist: [Leu31,Pro34]-NPY. The tritium overflow was lower in the explants exposed to the 5 μM NPY and 1 μM rauwolscine than to rauwolscine only. The effects of 5 μM NPY and 0.05 μM UK 14,304 on the depolarization-evoked 3H-NE release were additive. The results show that NPY is involved in the regulation of NE release from the sympathetic terminals in the pig pineal gland, inhibiting this process via Y2-receptors.

Endothelial dysfunction impairs vascular neurotransmission in tail arteries

The present study intends to clarify if endothelium dysfunction impairs vascular sympathetic neurotransmission. Electrically-evoked tritium overflow (100 pulses/5 Hz) was evaluated in arteries (intact and denuded) or exhibiting some degree of endothelium dysfunction (spontaneously hypertensive arteries), pre-incubated with [3H]-noradrenaline in the presence of enzymes (nitric oxide synthase (NOS); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; xanthine oxidase; cyclooxygenase; adenosine kinase) inhibitors and a nucleoside transporter inhibitor. Inhibition of endothelial nitric oxide synthase with L-NIO dihydrochloride reduced tritium overflow in intact arteries whereas inhibition of neuronal nitric oxide synthase with Nω-Propyl-L-arginine hydrochloride was devoid of effect showing that only endothelial nitric oxide synthase is involved in vascular sympathetic neuromodulation. Inhibition of enzymes involved in reactive oxygen species or prostaglandins production with apocynin and allopurinol or indomethacin, respectively, failed to alter tritium overflow. A facilitation or reduction of tritium overflow was observed in the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or of 5-iodotubericidin, respectively, but only in intact arteries. These effects can be ascribed to a tonic inhibitory effect mediated by A1 receptors. In denuded and hypertensive arteries, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) reduced tritium overflow, suggesting the occurrence of a tonic activation of A2A receptors. When endogenous adenosine bioavailability was increased by the nucleoside transporter inhibitor, S-(4-Nitrobenzyl)-6-thioinosine, tritium overflow increased in intact, denuded and hypertensive arteries. Among the endothelium-derived substances studied that could alter vascular sympathetic transmission only adenosine/adenosine receptor mediated mechanisms were clearly impaired by endothelium injury/dysfunction.

Attenuated aortic vasodilation and sympathetic prejunctional facilitation in epinephrine-deficient mice: selective impairment of β2-adrenoceptor responses.

It has been suggested that there is a link between epinephrine synthesis and the development of β2-adrenoceptor-mediated effects, but it remains to be determined whether this development is triggered by epinephrine. The aim of this study was to characterize β-adrenoceptor-mediated relaxation and facilitation of norepinephrine release in the aorta of phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice. Catecholamines were quantified by reverse-phase high-performance liquid chromatography-electrochemical detection. Aortic rings were mounted in a myograph to determine concentration-response curves to selective β1- or β2-adrenoceptor agonists in the absence or presence of selective β1- or β2-adrenoceptor antagonists. Aortic rings were also preincubated with [(3)H]norepinephrine to measure tritium overflow elicited by electrical stimulation in the presence of increasing concentrations of nonselective β- or selective β2-adrenoceptor agonists. β2-Adrenoceptor protein density was evaluated by Western blotting and β2-adrenoceptor localization by immunohistochemistry. Epinephrine is absent in Pnmt-KO mice. The potency and the maximal effect of the β2-adrenoceptor agonist terbutaline were lower in Pnmt-KO than in wild-type (WT) mice. The selective β2-adrenoceptor antagonist ICI 118,551 [(±)-erythro-(S*,S*)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride] antagonized the relaxation caused by terbutaline in WT but not in Pnmt-KO mice. Isoproterenol and terbutaline induced concentration-dependent increases in tritium overflow in WT mice only. β2-Adrenoceptor protein density was decreased in membrane aorta homogenates of Pnmt-KO mice, and this finding was supported by immunofluorescence confocal microscopy. In conclusion, epinephrine is crucial for β2-adrenoceptor-mediated vasodilation and facilitation of norepinephrine release. In the absence of epinephrine, β2-adrenoceptor protein density was decreased in aorta cell membranes, thus potentially hindering its functional activity.

Differential inhibition of noradrenaline release mediated by inhibitory A1-adenosine receptors in the mesenteric vein and artery from normotensive and hypertensive rats

Mesenteric arteries and veins are densely innervated by sympathetic nerves and are crucial in the regulation of peripheral resistance and capacitance, respectively, thus, in the control of blood pressure. Presynaptic adenosine receptors are involved in vascular tonus regulation, by modulating noradrenaline release from vascular postganglionic sympathetic nerve endings. Some studies also suggest that adenosine receptors (AR) may have a role in hypertension. We aim at investigating the role of presynaptic adenosine receptors in mesenteric vessels and establish a relationship between their effects (in mesenteric vessels) and hypertension, using the spontaneously hypertensive rats (SHR) as a model of hypertension. Adenosine receptor-mediated modulation of noradrenaline release was investigated through the effects of selective agonists and antagonists on electrically-evoked [3H]-noradrenaline overflow. CPA (A1AR selective agonist: 1–100nM) inhibited tritium overflow, but the inhibition was lower in SHR mesenteric vessels. IB-MECA (A3AR selective agonist: 1–100nM) also inhibited tritium overflow but only in WKY mesenteric veins. CGS 21680 (A2AAR selective agonist: up to 100nM) failed to facilitate noradrenaline release in mesenteric veins, from both strains, but induced a similar facilitation in the mesenteric arteries. NECA (non-selective AR agonist: 1, 3 and 10μM), in the presence of A1 (DPCPX, 20nM) and A3 (MRS 1523, 1μM) AR selective antagonists, failed to change tritium overflow. In summary, the modulatory effects mediated by presynaptic adenosine receptors were characterized, for the first time, in mesenteric vessels: a major inhibition exerted by the A1 subtype in both vessels; a slight inhibition mediated by A3 receptors in mesenteric vein; a facilitation mediated by A2A receptors only in mesenteric artery (from both strains). The less efficient prejunctional adenosine receptor mediated inhibitory effects can contribute to an increase of noradrenaline in the synaptic cleft (both in arteries and veins), which might conduce to increased vascular reactivity.

Regulation of K+‐induced [3H]D‐Aspartate Release by Synthetic Neuroprostanes in Isolated Bovine Retina

Neuroprostanes (nPs) are a series of isoprostane‐like compounds that are spontaneously derived by free‐radical catalyzed peroxidation of the polyunsaturated fatty acid, docosahexanoic acid. Although nPs are elevated in neurodegenerative conditions (Musiek et al., Brain Pathol. 15:149,2005), their biological effects remain largely unknown. We investigated the role of synthetic nPs, CO5‐667, CO5‐668 and CO5‐738 on K+‐induced glutamate release (using [3H]D‐aspartate as a marker) in isolated bovine retina. Isolated neural retina were incubated in oxygenated Krebs solution containing 200nM of [3H] D‐aspartame for 60 mins and then prepared for studies of neurotransmitter release using the superfusion method. Release of [3H]D‐aspartate was evoked by iso‐osmotic concentration of K+ (50mM)‐stimuli applied at 80–88 mins (S1) and 116–124 mins (S2) after the onset of superfusion. In the concentration range, 1 nM to 10 μM, the nPs enhanced K+‐induced [3H]D‐aspartate release from retina without affecting basal tritium overflow. For instance, C05‐668 achieved a maximal excitatory response of about 80% (p<0.01, n=4) at the 10 nM concentration of the nP. At an equimolar concentration of 10 nM, the rank order of activity was as follows: CO5‐668> CO5‐738> CO5‐667. In conclusion, the novel synthetic nPs exert an excitatory effect on K+‐evoked [3H]D‐aspartate release in isolated bovine retina.

A pre-junctional action of 5-hydroxytryptamine and methysergide on noradrenergic nerves in dog isolated saphenous vein.

Electrical stimulation (2 Hz for 2 min) of dog isolated saphenous vein strips pre-incubated with tritiated noradrenaline increased the overflow of tritium of which about 80% was noradrenaline. 5-Hydroxytryptamine (5-HT; 1.0 x 10-9-1.0 x 10-7 mol litre-1) and methysergide (3.0 x 10-8-3.0 x 10-6 mol litre-1) inhibited the induced overflow of total tritium by a maximum of 78 +/- 4% and 47 +/- 7% respectively (mean +/- s.e. mean, n=6 for each). Methysergide was about 30 times less potent than 5-HT and the maximum inhibition obtained was less than with 5-HT. Both compounds inhibited electrically-induced contractions and overflow of tritiated noradrenaline. Their inhibitory actions on tritium overflow were little affected by phentolamine (1.0 x 10-6 mol litre-1) or cyproheptadine (1.0 x 10-6 mol litre-1), nor was the inhibitory effect of methysergide on electrically induced contractions antagonized by atropine, mepyramine, cimetidine or propranolol. The findings suggest that the prejunctional inhibitory effect of methysergide may be mediated via stimulation of a 5-HT receptor which, unlike the D-receptor, is not blocked by cyproheptadine. The possibility that the pre-junctional 5-HT receptor in the dog saphenous vein is the same as the post-junctional receptor in this preparation is discussed.

Effect of cocaine on tritium overflow evoked from vasa deferentia previously loaded with [3H]noradrenaline by stimulation using different types of electrode.

In mouse and guinea-pig vasa deferentia previously incubated with [3H]noradrenaline, electrical stimulation applied through parallel electrodes (transmurally) increased overflow of tritium 2- to 5-fold above the resting value. Electrical stimulation applied using methods involving more substantial conduction of nerve impulses in neuronal elements in the tissues evoked a tritium overflow which was smaller (70%) than that evoked by transmural stimulation. Cinchocaine (25 microM), tetrodotoxin (0.5 microM) or the absence of calcium effectively abolished evoked overflow in both tissues whichever method of stimulation was used. In mouse vas deferens, cocaine (10 microM) did not alter overflow evoked by either transmural or axonal stimulation while 100 microM produced a reduction. In guinea-pig vas deferens, cocaine (10 microM) produced a statistically significant increase in evoked overflow of about 50% or more with both transmural and axonal stimulation. As in mouse vas deferens, 100 microM cocaine produced a reduction. It is concluded that the action of cocaine is independent of these methods of stimulation and that some difference in the arrangement of the noradrenergic nerves in the two species may account for the differential effect of cocaine observed.

A contribution, from a possible local anaesthetic action, to the effects of yohimbine on evoked noradrenaline overflow.

Both yohimbine (0.1 to 10 microM) and phentolamine (10 microM) increased the tritium overflow evoked by electrical stimulation (2.5 Hz, 1 ms, 15 V, for 90 s every 20 min) of mouse isolated vas deferens previously incubated with (-)-[3H]noradrenaline. At their maximally effective concentrations, phentolamine (10 microM) produced an effect that was sustained over the 2 h of the experiment while the effect of yohimbine (6 microM) decreased by about 60% over the first 40 min and was then sustained at a lower level. At higher concentrations of yohimbine, the increase in evoked tritium overflow was less marked and at the highest concentration tested (30 microM) evoked overflow was reduced below the levels seen before exposure to the drug. It is concluded that at concentrations maximally effective in inhibiting the presynaptic alpha-adrenoceptor-mediated mechanism controlling transmitter release, the known local anaesthetic effect of yohimbine may contribute to the overall effect on evoked transmitter overflow.

The effects of bromocriptine on pre-synaptic and post-synaptic alpha-adrenoceptors in the mouse vas deferens.

Noradrenaline (NA) and dopamine (DA) contracted the mouse vas deferens and reduced the responses to low frequency nerve stimulation (0.1 Hz). The relative potencies of antagonists suggested that these effects were due to stimulation of post-synaptic and pre-synaptic alpha-adrenoceptors respectively. Bromocriptine produced a non-competitive antagonism of contractile responses to NA (pD2' = 7.6) and DA (pD2' = 8.0) but had no effect on responses to carbachol. Bromocriptine also reduced single twitch responses of the vas to low frequency field stimulation (0.1 Hz), but did not affect stimulation at higher frequencies (1--20 Hz). Yohimbine selectively and rapidly reversed the inhibiting effects of bromocriptine on single twitches, although they could not easily be reversed by washing. Bromocriptine produced a yohimbine-reversible reduction in the stimulated overflow of tritium from vasa previously loaded with 3H--NA. Thus the mouse vas deferens does not appear to contain specific DA receptors and the results suggest that bromocriptine acts as a pre-synaptic alpha-adrenoceptor agonist and post-synaptic alpha-adrenoceptor antagonist in this tissue.

Effects of Urapidil on Catecholamine Turnover and Release in the Central Nervous System of the Rat

The actions of urapidil, prazosin, idazoxan, and haloperidol on the turnover of noradrenaline in the hypothalamus and dopamine in the nucleus accumbens of the rat were investigated using changes in the ratios of 3-methoxy-4-hydroxyphenyl-glycol/noradrenaline (MHPG/NA) and 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA), respectively, as measures for drug-induced effects. Urapidil (2.5-30 mg kg-1 i.v.) increased the ratios of MHPG/NA and DOPAC/DA. Its effects on NA turnover were maximal at 60 min (160% of control at 30 mg kg-1), and on DA turnover at 30 min (138% of control at 30 mg kg-1). Prazosin (0.5-2.5 mg kg-1 i.v.) had no effect, but the high dose of 5 mg kg-1 i.v. significantly increased the ratio of MHPG/NA in the hypothalamus. Idazoxan (2-50 mg kg-1 i.v.) and haloperidol (0.02-0.5 mg kg-1 i.v.) selectively enhanced turnover of NA and DA, respectively. In experiments on field-stimulated overflow of tritium from slices of hypothalamus and nucleus accumbens labelled with [3H]NA or [3H]DA, respectively, urapidil (1 mumol L-1) facilitated the evoked responses in both regions. Prazosin (0.1 mumol L-1) had no effect in either of the two areas. Idazoxan (0.1 mumol L-1) increased stimulated overflow of [3H]NA from the hypothalamus but not of [3H]DA from the nucleus accumbens. Conversely, haloperidol (0.1 mumol L-1) greatly enhanced evoked overflow of [3H]DA but not of [3H]NA. From the present results it is concluded that urapidil has an antagonistic effect at central alpha 2-adrenoceptors and also a weak antagonistic action at central dopamine D2-receptors.

Yohimbine affects the evoked overflow of neurotransmitters from rat brain slices by more than one mechanism.

Both yohimbine (0.1-10 microM) and phentolamine (10 microM) increased the tritium overflow evoked by electrical stimulation (3 Hz, 2 ms, 18 mA for 120 s every 20 min) of rat brain cortex slices previously incubated with [3H](-)-noradrenaline. At their maximally effective concentrations, neither of these compounds produced an effect which was fully maintained over the 1 h of the experiment, but the decline in effect of yohimbine (1.25 microM) was more marked, falling sharply to reach, after 1 h, 25% of the effect observed after 20 min, whereas phentolamine only declined to 60% of the effect after 20 min. In rat brain cortex slices previously incubated with [3H]5-hydroxytryptamine ([3H]5-HT), the tritium overflow evoked by electrical stimulation (3 Hz, 2 ms, 40 mA for 120 s) was increased by yohimbine (0.1-1 microM) and phentolamine (0.1-10 microM), but a higher concentration of yohimbine (10 microM) decreased evoked overflow below the levels seen in the absence of either drug. It is concluded that, at concentrations effective in inhibiting the presynaptic alpha-adrenoceptor-mediated mechanisms controlling transmitter release in rat brain slices, a non-alpha-adrenoceptor-mediated, possible local anaesthetic, action of yohimbine contributes to the overall effect of this drug on transmitter overflow.

Noradrenaline release in rodent tissues is inhibited by interleukin-1β but is not affected by urotensin II, MCH, NPW and NPFF

We studied whether noradrenaline release is affected by interleukin-1β and the neuropeptides urotensin II, melanin-concentrating hormone (MCH), neuropeptide W (NPW) and neuropeptide FF (NPFF). Rodent tissues preincubated with [3H]noradrenaline were superfused, and the effect of peptides on the electrically-evoked tritium overflow (“noradrenaline release”) was studied. In mouse brain cortex, interleukin-1β at 0.3nM and the prostaglandin E2 analogue sulprostone at 3nM inhibited noradrenaline release by about 40%; the effect of interleukin-1β developed gradually, whereas the effect of sulprostone occurred promptly. Urotensin II at 0.001–1µM did not affect noradrenaline release in rat kidney cortex, whereas 0.01µM angiotensin II increased it (positive control). MCH at 0.01–1µM did not alter noradrenaline release in the rat brain cortex, and NPW 1µM did not affect noradrenaline release in the mouse hypothalamus or hippocampus. In each model, 0.1µM sulprostone inhibited noradrenaline release (positive control). NPFF and the NPFF2 receptor agonist dNPA (1µM) did not affect noradrenaline release in the mouse atria; the inhibitory effect of the δ opioid receptor agonist 1µM DPDPE on noradrenaline release in this tissue was not altered by NPFF or dNPA at 0.32µM but was counteracted by the δ opioid antagonist naltrindole at 0.001µM. In conclusion, interleukin-1β inhibits noradrenaline release in the mouse cortex; the effect develops gradually, suggesting that it affects protein biosynthesis. Noradrenergic neurons in various tissues from rodents are devoid of presynaptic receptors for urotensin II, MCH, NPW and NPFF. Finally, an interaction between a δ opioid agonist and NPFF could not be detected.

Functional crosstalk of prejunctional receptors on the modulation of noradrenaline release in mesenteric vessels: A differential study of artery and vein

The role of angiotensin II receptors, bradykinin receptors and β-adrenoceptors in the modulation of noradrenaline release and the influence of α 2-autoinhibition in these effects was investigated in the mesenteric artery and vein. Rings of mesenteric vessels of male Wistar rats were labelled with [ 3H]-noradrenaline and the effects of modulators on tritium overflow evoked by 100 pulses at 2 Hz (marked α 2-autoinhibition) and by 20 pulses at 50 Hz or 100 pulses at 2 Hz plus yohimbine (1 μM; reduced α 2-autoinhibition) were evaluated. Angiotensin II and bradykinin enhanced noradrenaline release evoked by 100 pulses at 2 Hz, in a concentration-dependent manner, in both vessels. These effects were attenuated under conditions of reduced α 2-autoinhibition. The attenuation was partially reversed by activation of adenosine A 1 receptors in both vessels and by activation of P2Y receptors in the vein. Isoprenaline and the selective β 2-adrenoceptor agonist formoterol enhanced tritium overflow independently of α 2-autoinhibition in the vein. In the artery, the enhancement by formoterol was only observed under reduced α 2-autoinhibition. Pharmacological characterization of the β-adrenoceptors indicated that in the artery the effect of isoprenaline was mediated by the β 1-subtype under marked α 2-autoinhibition and by the β 2-subtype under reduced α 2-autoinhibition whereas in the vein the effect was independent of α 2-autoinhibition. The results indicate that α 2-autoinhibition is a key determinant of the magnitude of facilitation caused by angiotensin II and bradykinin in both types of mesenteric vessels and regulates the effects mediated by β 1-and β 2-adrenoceptors which co-exist in the artery.

Prostaglandins of the E series inhibit monoamine release via EP3 receptors: proof with the competitive EP3 receptor antagonist L-826,266

Prostaglandin E(2) (PGE(2)) and its analogue sulprostone inhibit noradrenaline and serotonin release in rodent tissues. We examined whether the receptor involved is blocked by the EP(3) antagonist L-826,266, whether such receptors also occur on central cholinergic neurones and retinal dopaminergic cells, whether PGE(2) is produced by the degradation of the endocannabinoid virodhamine and whether EP(3) receptor activation stimulates (35)S-GTPgammaS binding. Transmitter release was studied as electrically evoked tritium overflow in superfused tissues preincubated with (3)H-noradrenaline (which in the guinea pig retina labels dopaminergic cells), (3)H-serotonin or (3)H-choline. (35)S-GTPgammaS binding, a measure of G protein activation, was studied in mouse and guinea pig hippocampal membranes. L-826,266 antagonised the effect of sulprostone on noradrenaline release in the rat cortex, yielding a Schild plot-based pA(2) value of 7.56. Apparent pA(2) values in mouse cortex and rat vas deferens (noradrenaline release) and rat cortex (serotonin release) were 7.55, 7.87 and 7.67, respectively. PGE(2) did not affect acetylcholine release in rat brain and dopamine release in guinea pig retina. In seven mice tissues, noradrenaline release was inhibited by sulprostone but not affected by virodhamine. (35)S-GTPgammaS binding was not altered by sulprostone but stimulated by the cannabinoid agonist WIN 55,212-2. Prostaglandins of the E series inhibit monoamine release via EP(3) receptors at which L-826,266 is a competitive antagonist. EP(3) receptors that inhibit transmitter release are not present on central cholinergic neurones and retinal dopaminergic cells. Virodhamine is not converted to PGE(2). An EP(3) receptor model based on (35)S-GTPgammaS binding could not be identified.

Heterogeneity of release-regulating muscarinic receptors in rat sympathetic neurons: evidence for inhibitory presynaptic M1 receptors

Of the 5 known subtypes of mAChRs, M2, M3, and M4 have been reported to act as inhibitory presynaptic receptors in the nervous system, in general, and in sympathetic neurons, in particular. M1 receptors, in contrast, have rather been viewed as facilitatory presynaptic receptors. In superior cervical ganglion (SCG) neurons, M1 receptors are well known to inhibit KCNQ channels. Previously, we were able to show that non-presynaptic M1 receptors in SCG neurons enhance noradrenaline release through an inhibition of KCNQ channels. However, M1 receptors also mediate an inhibition of voltage-activated Ca2+ channels, which represents the predominant mechanism of presynaptic inhibition. Hence, presynaptic M1 receptors may exert inibitory presynaptic modulation. To test this possibility, we performed experiments on rat superior cervical ganglion neurons. In primary cultures tritium overflow was assayed to investigate the release of [3H]noradrenaline, and the perforated patch-clamp technique was employed to record Ca2+ currents. The muscarinic agonist oxotremorine M transiently enhanced 3H outflow and reduced electrically evoked release, once the stimulatory effect had faded. The stimulatory effect was enhanced by pertussis toxin and was abolished by blocking M1 receptors, by opening KCNQ channels, and by preventing action potential propagation. The inhibitory effect, in contrast, was not altered by preventing action potentials or by opening KCNQ channels, but was reduced by pertussis toxin. The inhibition remaining after pertussis toxin treatment was abolished by blockage of M1 receptors or inhibition of phospholipase C. When [3H]noradrenaline release was triggered independently of voltage-activated Ca2+ channels, oxotremorine M failed to cause any inhibition. The inhibition of Ca2+ currents by oxotremorine M was reduced by pertussis toxin and then abolished by the blockage of M1 receptors. This demonstrates that M1, in addition to M2, M3, and M4, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close voltage-activated Ca2+ channels. In addition, our results contradict the widely accepted concept that all inhibitory presynaptic receptors restrict transmitter release through a direct inhibition of Ca2+ channels via G protein βγ subunits and offer an alternative mechanism.

A 2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and βγ subunits formed after α 2-adrenoceptor activation

This work aimed to investigate the molecular mechanisms involved in the interaction of α 2-adrenoceptors and adenosine A 2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by α 2-adrenoceptors and A 2A-receptors interact. The selective adenosine A 2A-receptor agonist 2- p-(2-carboxy ethyl) phenethylamino-5′- N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A 2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 μM). CGS 21680-mediated effects were also abolished by drugs that disrupted G i/o-protein coupling with receptors, PTX (2 μg/ml) or NEM (40 μM), by the anti-G sα peptide (2 μg/ml) anti-G βγ peptide (10 μg/ml) indicating coupling of A 2A-receptors to G sα and suggesting a crucial role for G βγ subunits in the A 2A-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 μM) or forskolin (1 μM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A 2A-receptors couple to G sα subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G i/o-protein coupling (it disappears after disruption of G i/o-protein coupling, PTX or NEM) and/or G βγ subunits (anti-G βγ). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G sα subunits (released after adenosine A 2A-receptor activation; prime-activation), G βγ subunits (released after activation of G i/o-protein coupled receptors) which can directly synergistically stimulate the prime-activated AC or indirectly via G βγ activation of the PLC-PKC pathway.

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.