Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy.

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

Marijuana smoke induces severe pulmonary hyperresponsiveness, inflammation, and emphysema in a predictive mouse model not via CB1 receptor activation.

Sporadic clinical reports suggested that marijuana smoking induces spontaneous pneumothorax, but no animal models were available to validate these observations and to study the underlying mechanisms. Therefore, we performed a systematic study in CD1 mice as a predictive animal model and assessed the pathophysiological alterations in response to 4-mo-long whole body marijuana smoke with integrative methodologies in comparison with tobacco smoke. Bronchial responsiveness was measured with unrestrained whole body plethysmography, cell profile in the bronchoalveolar lavage fluid with flow cytometry, myeloperoxidase activity with spectrophotometry, inflammatory cytokines with ELISA, and histopathological alterations with light microscopy. Daily marijuana inhalation evoked severe bronchial hyperreactivity after a week. Characteristic perivascular/peribronchial edema, atelectasis, apical emphysema, and neutrophil and macrophage infiltration developed after 1 mo of marijuana smoking; lymphocyte accumulation after 2 mo; macrophage-like giant cells, irregular or destroyed bronchial mucosa, goblet cell hyperplasia after 3 mo; and severe atelectasis, emphysema, obstructed or damaged bronchioles, and endothelial proliferation at 4 mo. Myeloperoxidase activity, inflammatory cell, and cytokine profile correlated with these changes. Airway hyperresponsiveness and inflammation were not altered in mice lacking the CB1 cannabinoid receptor. In comparison, tobacco smoke induced hyperresponsiveness after 2 mo and significantly later caused inflammatory cell infiltration/activation with only mild emphysema. We provide the first systematic and comparative experimental evidence that marijuana causes severe airway hyperresponsiveness, inflammation, tissue destruction, and emphysema, which are not mediated by the CB1 receptor.

ATP-Evoked Intracellular Ca²âº Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein 1: recent developments concerning substrates and inhibitors of a promising therapeutic target.

SSAO/VAP-1 is not only involved in the metabolism of biogenic and xenobiotic primary amines and in the production of metabolites with cytotoxic effects or certain physiological actions, but also plays a role, for example, as an adhesion molecule, in leukocyte trafficking, in regulating glucose uptake and in adipocyte homeostasis. Interest in the enzyme has been stimulated by the findings that the activities of the SSAOs are altered (mostly increased) in various human disorders, including diabetes, congestive heart failure, liver cirrhosis, Alzheimer's disease and several inflammatory diseases, although the underlying causes are often unknown. On the basis of their insulin-mimicking effect, SSAO substrates are possibly capable of ameliorating metabolic changes in diabetes, while SSAO inhibitors (somewhat of a contradiction) are of potential benefit in preventing diabetes complications, atherosclerosis and oxidative stress contributing to several disorders or modulating inflammation, and hence may be of substantial therapeutic value. Great efforts have been made to develop novel compounds which may lead to future drugs useful in therapy, based on their effects on SSAO/VAP-1, and some of the results relating to novel substrates and inhibitors are surveyed in the present review.

Purinergic modulation of glutamate release under ischemic-like conditions in the hippocampus.

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Contribution of platelet P2Y12 receptors to chronic Complete Freund's adjuvant-induced inflammatory pain.

Essentials The role of platelet P2Y12 receptors in the regulation of chronic inflammatory pain is unknown. Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain model was used in mice. Gene deficiency and antagonists of P2Y12 receptors attenuate hyperalgesia and local inflammation. Platelet P2Y12 receptors contribute to these effects in the chronic phase of inflammation. SUMMARY: Background P2Y12 receptor antagonists are widely used in clinical practice to inhibit platelet aggregation. P2Y12 receptors are also known to regulate different forms of pain as well as local and systemic inflammation. However, it is not known whether platelet P2Y12 receptors contribute to these effects. Objectives To explore the contribution of platelet P2Y12 receptors to chronic inflammatory pain in mice. Methods Complete Freund's adjuvant (CFA)-induced chronic inflammatory pain was induced in wild-type and P2ry12 gene-deficient (P2ry12-/- ) mice, and the potent, direct-acting and reversible P2Y12 receptor antagonists PSB-0739 and cangrelor were used. Results CFA-induced mechanical hyperalgesia was significantly decreased in P2ry12-/- mice for up to 14 days, and increased neutrophil myeloperoxidase activity and tumor necrosis factor (TNF)-α and CXCL1 (KC) levels in the hind paws were also attenuated in the acute inflammation phase. At day 14, increased interleukin (IL)-1ß, IL-6, TNF-α and KC levels were attenuated in P2ry12-/- mice. PSB-0739 and cangrelor reversed hyperalgesia in wild-type mice but had no effect in P2ry12-/- mice, and PSB-0739 was also effective when applied locally. The effects of both local and systemic PSB-0739 were prevented by A-803467, a selective NaV1.8 channel antagonist, suggesting the involvement of NaV1.8 channels in the antihyperalgesic effect. Platelet depletion by anti-mouse CD41 antibody decreased hyperalgesia and attenuated the proinflammatory cytokine response in wild-type but not in P2ry12-/- mice on day 14. Conclusions In conclusion, P2Y12 receptors regulate CFA-induced hyperalgesia and the local inflammatory response, and platelet P2Y12 receptors contribute to these effects in the chronic inflammation phase.

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons.

To understand the functional significance and mechanisms of action in the CNS of endogenous and exogenous cannabinoids, it is crucial to identify the neural elements that serve as the structural substrate of these actions. We used a recently developed antibody against the CB1 cannabinoid receptor to study this question in hippocampal networks. Interneurons with features typical of basket cells showed a selective, intense staining for CB1 in all hippocampal subfields and layers. Most of them (85.6%) contained cholecystokinin (CCK), which corresponded to 96.9% of all CCK-positive interneurons, whereas only 4.6% of the parvalbumin (PV)-containing basket cells expressed CB1. Accordingly, electron microscopy revealed that CB1-immunoreactive axon terminals of CCK-containing basket cells surrounded the somata and proximal dendrites of pyramidal neurons, whereas PV-positive basket cell terminals in similar locations were negative for CB1. The synthetic cannabinoid agonist WIN 55,212-2 (0.01-3 microM) reduced dose-dependently the electrical field stimulation-induced [3H]GABA release from superfused hippocampal slices, with an EC50 value of 0. 041 microM. Inhibition of GABA release by WIN 55,212-2 was not mediated by inhibition of glutamatergic transmission because the WIN 55,212-2 effect was not reduced by the glutamate blockers AP5 and CNQX. In contrast, the CB1 cannabinoid receptor antagonist SR 141716A (1 microM) prevented this effect, whereas by itself it did not change the outflow of [3H]GABA. These results suggest that cannabinoid-mediated modulation of hippocampal interneuron networks operate largely via presynaptic receptors on CCK-immunoreactive basket cell terminals. Reduction of GABA release from these terminals is the likely mechanism by which both endogenous and exogenous CB1 ligands interfere with hippocampal network oscillations and associated cognitive functions.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

OBJECTIVE: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. METHODS: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. RESULTS: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. CONCLUSIONS: Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.

Ischemic-like condition releases norepinephrine and purines from different sources in superfused rat spleen strips.

Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.

Is the neuronal ATP release from guinea-pig vas deferens subject to alpha 2-adrenoceptor-mediated modulation?

The effects of a variety of alpha 2-adrenoceptor agonists and antagonists were studied on stimulation-evoked release of endogenous ATP, measured by the luciferin-luciferase assay, and on the release of [3H]noradrenaline from the guinea-pig vas deferens. The biphasic mechanical contraction of the guinea-pig smooth muscle was recorded concomitantly. The alpha 2-adrenoceptor agonist, xylazine (1 microM) inhibited the field stimulation-evoked (8 Hz, 0.1 ms, 480 shocks) release of ATP and [3H]noradrenaline, and both phases of the contraction. The inhibitory effect of xylazine on the release of ATP, noradrenaline and muscle contraction was prevented by the selective alpha 2-adrenoceptor antagonist, CH 38083 [7,8-(methylenedioxi)-14 alpha-alloberbanol, 1 microM]. In the presence of prazosin (0.1-1 microM) or WB 4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl- 1,4-benzodioxane hydrochloride, 0.1-1 microM], i.e. under the condition when the effect of noradrenaline on postjunctional alpha 1-adrenoceptors was excluded, the stimulation-evoked release of [3H]noradrenaline was significantly enhanced, however, the release of endogenous ATP and also both phases of contraction were reduced. In the presence of prazosin, xylazine was able to inhibit the stimulation-evoked release of ATP. In vas deferens dissected from reserpine pretreated (2 x 5 mg/kg, i.p.) guinea-pigs, the content of noradrenaline was 0.5% of control and there was no detectable evoked release of noradrenaline. Under this condition, the release of ATP evoked by electrical stimulation was still detectable, but the amount of ATP was much smaller than that measured from control animals. Xylazine did not reduce the release of ATP. Oxymetazoline, a relatively selective alpha 2-adrenoceptor agonist failed to inhibit the release of [3H]noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that ATP released from the postsynaptic site by noradrenaline, is involved in mechanical responses of guinea-pig vas deferens: cascade transmission.

The release of endogenous ATP and [3H]noradrenaline, and the mechanical response of the guinea-pig vas deferens to field stimulation of its motor nerves were examined using a perfusion system. The release of ATP at rest was 0.83 +/- 0.13 pmol/g per min, and ATP released by field stimulation (8 Hz, 480 shocks) was 5.47 +/- 1.23 pmol/g. The evoked release was completely inhibited when Ca2+ was removed and 1 mM EGTA was added, or by 1 microM tetrodotoxin. The release of ATP and [3H]noradrenaline in response to field stimulation was constant with an S2/S1 ratio of 1.10 +/- 0.11 for ATP and 0.92 +/- 0.03 for [3H]noradrenaline, respectively (where S1 and S2 are stimulation periods). Prazosin (1 microM), a potent alpha 1-adrenoceptor antagonist, significantly reduced the stimulation-evoked release of ATP by 75% and significantly reduced both mechanical twitch and tonic responses, but enhanced the release of [3H]noradrenaline. This finding indicates that there is an alpha 1-adrenoceptor-mediated release of endogenous ATP. However, the prazosin-insensitive portion of ATP release (25%) is considered to be of presynaptic origin. The stimulation of alpha 1-adrenoceptors by 1-noradrenaline or methoxamine in concentrations ranging from 10 to 100 microM resulted in a concentration-dependent release of ATP and a biphasic contraction of the vas deferens: a twitch response was followed by a tonic contraction. Prazosin (1 microM) completely prevented the effect of 1-noradrenaline or methoxamine on both ATP release and mechanical response. When Ca2+ was omitted and EGTA (1 mM) was added, 1-noradrenaline was still able to release ATP but failed to produce contraction. Nifedipine, a Ca-channel and ATP receptor antagonist, reduced the twitch contraction and enhanced the release of ATP from muscle in response to noradrenaline administration. This finding indicates that the release of ATP from the muscle is not linked to mechanical contraction. When the vas deferens was made deficient in noradrenaline by 6-hydroxydopamine pretreatment (100 + 250 mg/kg, i.p.), electrical field stimulation failed to release [3H]noradrenaline and ATP. Under these conditions, exogenous 1-noradrenaline was much more effective in releasing ATP from the smooth muscle, and producing twitch responses, followed by a tonic contraction. After reserpine pretreatment (2 x 5 mg/kg, i.p.), the field stimulation-evoked release of ATP and both phases of contraction were markedly reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP acts as fast neurotransmitter in rat habenula: neurochemical and enzymecytochemical evidence.

The release of ATP and ADP, the putative central neurotransmitters, from the isolated habenula preparation was investigated in the rat, at rest and during electrical stimulation, using the luciferin-luciferase assay and the creatine phosphokinase assay. Electrical field stimulation (2 Hz, 360 pulses) released a considerable amount of ATP (2450 +/- 280 pmol/g wet tissue) from the tissue; inhibition of the voltage Na+ entry by tetrodotoxin (1 microM) reduced significantly the evoked release (by 66.25 +/- 6.65%), but not the resting release of ATP. Endogenous ADP also appeared in the effluent, but its amount differed during resting condition and after stimulation from that of ATP, suggesting that the majority of the released compound is ATP in response to stimulation. When ATP was added to the tissue, it readily decomposed to ADP and AMP (Km = 811.6 +/- 68.88 microM, vmax = 23.1 +/- 2.75 nmol/min per prep., measured by high-performance liquid chromatography combined with ultraviolet detection), indicating that the habenula contains ectoATPases. In addition, the inactivation of extracellular ATP by the ectoATPase enzyme was also visualized by electron microscopic enzyme cytochemistry. The ectoATPase enzyme was present on the membranes of the dendrites and nerve terminals and in the synapses of the habenula. Taking into account the fact that ATP is ubiquitous in excitable cells (storage) and the findings published by Edwards et al. in 1992 ("ATP receptor-mediated synaptic currents in the central nervous system", Nature, Vol. 359, pp. 144-147), our data provides evidence for the release by axonal stimulation and extracellular decomposition of ATP, all needed for an endogenous substance qualified as a transmitter.

Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by alpha2-adrenoceptors.

The release of endogenous ATP, measured by the luciferin-luciferase assay, and of [3H]noradrenaline from the in vitro superfused rat hypothalamic slices were studied. ATP and [3H]noradrenaline were released simultaneously during resting conditions and in response to low and high frequency field electrical stimulation; the release of both substances were frequency dependent between 2 Hz and 16 Hz. The stimulation-induced release of ATP and [3H]noradrenaline was diminished by more than 80% under Ca2+-free conditions. Tetrodotoxin inhibited the majority of the evoked release of both ATP and [3H]noradrenaline, however, it was less effective in reducing the release of [3H]noradrenaline, than that of ATP. Bilateral stereotaxic injection of 6-hydroxydopamine (4 microg/side) to the ventral part of the ventral noradrenergic bundle, originating from the A1 cell group in the brainstem, resulted in a 55% reduction of endogenous noradrenaline content of the hypothalamic slices, and the tritium uptake and the stimulation-evoked release of [3H]noradrenaline was also markedly reduced. While the basal release of ATP was not affected, the evoked release was diminished by 72% by this treatment. Perfusion of the slices with noradrenaline (100 microM) initiated rapid and continuous tritium release; on the other hand, it did not release any ATP. In contrast, 6 min perfusion of (-)nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide evoked parallel release of ATP and [3H]noradrenaline which was inhibited by the nicotinic receptor antagonist mecamylamine; 6-hydroxydopamine lesion of the ventral part of the ventral noradrenergic bundle did not affect the nicotine-evoked ATP and [3H]noradrenaline release. While CH 38083, a non subtype-selective alpha2-antagonist and BRL44408, the subtype-selective alpha2AD antagonist augmented the evoked release of [3H]noradrenaline, ARC239, a selective alpha2BC antagonist was without effect. In contrast, neither of the alpha2-antagonists significantly affected the evoked-release of ATP. In summary, we report here that endogenous ATP and [3H]noradrenaline are co-released stimulation-dependently from superfused rat hypothalamic slices. A significant part of the release of both compounds is derived from the nerve terminals, originating from the A1 catecholaminergic cell group of brainstem nuclei. Unlike that from the peripheral sympathetic transmission, noradrenaline and alpha1-adrenoceptor agonists were unable to promote the release of ATP. Conversely, parallel ATP and noradrenaline release could be induced by nicotine receptor activation, but this release does not originate from the same nerve endings. The evoked-release of [3H]noradrenaline is inhibited by endogenous noradrenaline via alpha2AD subtype of adrenoreceptors, while the release of ATP is not subject to this autoinhibitory modulation. In conclusion, our results support the view that ATP is involved in the neurotransmission in the hypothalamus, but the sources of the released ATP and noradrenaline seem to be not identical under different stimulatory and modulatory conditions.

The triangular septal nucleus as the major source of ATP release in the rat habenula: a combined neurochemical and morphological study.

The role of ATP as a fast neurotransmitter is emerging from several lines of physiological and pharmacological studies. The bulk of experimental data on release properties and purinergic receptor-mediated postsynaptic potentials derives from studies in the habenula, but the source of the stimulation-evoked ATP release in this region is still unknown. In the present study, retrograde and anterograde tracing techniques were used to establish that both calretinin-containing and calretinin-negative neurons in the triangular septal and septofimbrial nuclei send a massive projection to the medial habenula, where they form asymmetrical synapses with their target neurons. The cells of origin, their axon terminals, as well as their synaptic targets remained unstained in sections immunostained for GABA. Electrolytic lesions of this anatomically circumscribed pathway resulted in an over 80% decrease in ATP release from habenula slices evoked by electric field stimulation. The possibility of transneuronal effects and release from local collaterals of habenular projection neurons accounting for the decreased ATP release has been excluded, since (i) there were no signs of neuronal degeneration, chromatolysis or atrophy in the habenula, (ii) the projection neurons have extremely sparse local collaterals and (iii) there are apparently no interneurons in the habenula. We conclude that the projection from the triangular septal and septofimbrial nucleus to the habenula uses ATP as a fast neurotransmitter, and its co-transmitter, if any, is likely to be glutamate.

Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP.

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca2+]o-dependent manner. The Na+ channel blocker, tetrodotoxin (1 microM) inhibited the stimulation-evoked release of endogenous ATP and of [3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz. when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 microM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A1-adenosine receptors. When the temperature was reduced to 7 degrees C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9 +/- 5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the Km (apparent Michaelis constant) and Vmax (apparent maximal velocity) were 475 +/- 24 microM and 3.50 +/- 0.18 nmol/min per mg for ectoATPase and 1550 +/- 120 microM and 14.5 +/- 0.9 nmol/min per mg tissue for 5'-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca2+]o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue. The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.

Non-synaptic release of [3H]noradrenaline in response to oxidative stress combined with mitochondrial dysfunction in rat hippocampal slices.

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.

Local regulation of [(3)H]-noradrenaline release from the isolated guinea-pig right atrium by P(2X)-receptors located on axon terminals.

In this study the regulation of cardiac sympathetic outflow by presynaptic P(2X) receptor-gated ion channels was examined. ATP (30 microM - 1 mM) and other P2-receptor agonists elicited [(3)H]-noradrenaline ([(3)H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP>2-methyl-thioATP>alpha,beta-methylene-ATP=ADP, whereas ss, gamma-methylene-L-ATP was inactive. Ca(2+)-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [(3)H]-NA outflow was not reduced by omega-Conotoxin-GVIA (100 nM), Cd(2+) (100 microM) and tetrodotoxin (1 microM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [(3)H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3, 7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 microM), antagonists of A(1)-, A(2)- and inhibitory P(2) receptors. Zn(2+) (50 microM), the P(2X)-receptor modulator potentiated, and P(2X) receptor antagonists, i.e. suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and 2'-o-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP, 30 microM) antagonized the ATP (1 mM)-evoked response. RT - PCR study revealed the expression of P(2X2) and P(2X3) receptor mRNAs in guinea-pig superior cervical ganglion. PPADS (30 microM) significantly reduced the EFS-induced [(3)H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 microM). In summary a P(2X)-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P(2X3) or hetero-oligomeric P(2X2)/P(2X3) complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis.

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.

ATP and [3H]noradrenaline release and the presence of ecto-Ca(2+)-ATPases in the capsule-glomerulosa fraction of the rat adrenal gland.

Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective alpha 2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and alpha, beta-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca(2+)-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue.