Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by α2-adrenoceptors

doi:10.1016/S0306-4522(97)00306-0

Neuroscience

Volume 82, Issue 2, 17 October 1997, Pages 511-520

https://doi.org/10.1016/S0306-4522(97)00306-0 Get rights and content

Abstract

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 μg/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 μM) 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 α₂-antagonist and BRL44408, the subtype-selective α_2AD antagonist augmented the evoked release of [³H]noradrenaline, ARC239, a selective α_2BC antagonist was without effect. In contrast, neither of the α₂-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 α₁-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 α_2AD 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.

Section snippets

ATP and [³H]noradrenaline release experiments

Male Sprague–Dawley rats (140–160 g, obtained from Charles River, Hungary) were decapitated, the brain was quickly removed, and the whole hypothalamus together with the median eminence was rapidly dissected in ice-cold Krebs' solution saturated with 95% O₂ and 5% CO₂. 400 μm-thick slices were cut in rostrocaudal direction using a McIlwain tissue chopper which yielded eight slices of each hypothalamus. Four-four slices were incubated separately for 60 min in 1 ml of Krebs' solution (mM: NaCl 113,

Resting and stimulation-evoked release of ATP and [³H]noradrenaline

ATP and [³H]NA were released simultaneously from the superfused hypothalamic slices during the resting condition and in response to electrical depolarization (Fig. 1). After 60 min preperfusion the basal release of ATP was 4.1±0.2 pmol/g/min, while the basal tritium efflux was 0.102±0.005%/min (n = 13). Low frequency electrical field stimulation (20 V, 2 Hz, 240 shock) increased several-fold the outflow of both ATP and [³H]NA: the stimulation-evoked release was 45.2±7.7 pmol/g and 1.68±0.21%,

Discussion

Despite the fact that P₂-purinoceptors mediating the action of ATP in the intercellular communication are abundantly expressed in the central nervous system,3, 14the number of identified pathways which utilize ATP as a transmitter or co-transmitter in the mammalian brain is still relatively low.[29]Apart from medial habenula, where the first electrophysiological[18]and neurochemical[51]evidence for the fast neurotransmitter role of ATP was provided, research interest has been recently

Conclusions

In summary we showed that ATP and [³H]NA are co-released from central noradrenergic nerve terminals, and the majority of ATP release evoked by electrical stimulation is derived from the ventral noradrenergic bundle, originating in the A1 cell group in the caudal medulla oblongata. Parallel release of ATP and [³H]NA can also be induced by nicotinic receptor activation, but this release is derived from a different pool. The α₂-autoreceptors of the hypothalamus belong to the α_2AD subtype and the

Acknowledgements

This work was supported by the grants of Hungarian Research Foundation (OTKA F013019) Council for Tobacco Research Inc. and the Hungarian Medical Research Council (ETT144). The authors are grateful to Dr Gábor Makara for helpful suggestions, and to Mrs Audrey Hashim for performing the HPLC assay. The excellent technical assistance of Mrs Zsuzsanna Körössy is gratefully acknowledged.

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Co-release of endogenous ATP and [3H]noradrenaline from rat hypothalamic slices: origin and modulation by α2-adrenoceptors

Abstract

Section snippets

ATP and [3H]noradrenaline release experiments

Resting and stimulation-evoked release of ATP and [3H]noradrenaline

Discussion

Conclusions

Acknowledgements

Brain Res.

Neuroscience

Neurochem. Int.

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Biochem. Pharmac.

Neurosci. Lett.

Neuropharmacology

Brain Res.

Front. Neuroendocrinol.

Neuroscience

Eur. J. Pharmac.

Molec. Brain Res.

Brain Res.

Neuroscience

Neuroscience

Semin. Neurosci.

Eur. J. Pharmac.

Neuroscience

Neuroscience

Trends pharmac. Sci.

Semin. Neurosci.

Eur. J. Pharmac.

Trends Neurosci.

Autoradiography of P2x ATP receptors in rat brain

Br. J. Pharmac.

Rat astroglial P2z (P2x7) receptors regulate intracellular calcium and purine release

NeuroReport

Distribution of [3H]α,β-methylene ATP binding sites in rat brain and spinal cord

NeuroReport

α2-Adrenoceptor-mediated autoinhibition of sympathetic transmitter release in guinea-pig vas deferens studied by intracellular and focal extracellular recording of junction potentials and currents

Naunyn-Schmiedeberg's Arch. Pharmac.

α-2A and α2B-adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype

J. Pharmac. exp. Ther.

Co-release of endogenous ATP and [³H]noradrenaline from rat hypothalamic slices: origin and modulation by α₂-adrenoceptors

ATP and [³H]noradrenaline release experiments

Resting and stimulation-evoked release of ATP and [³H]noradrenaline

Autoradiography of P_2x ATP receptors in rat brain

Rat astroglial P_2z (P_2x7) receptors regulate intracellular calcium and purine release

Distribution of [³H]α,β-methylene ATP binding sites in rat brain and spinal cord

α₂-Adrenoceptor-mediated autoinhibition of sympathetic transmitter release in guinea-pig vas deferens studied by intracellular and focal extracellular recording of junction potentials and currents

α-_2A and α_2B-adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype