Elsevier

Brain Research

Volume 739, Issues 1–2, 11 November 1996, Pages 163-168
Brain Research

Research report
Substance P potentiates ATP-activated currents in rat primary sensory neurons

https://doi.org/10.1016/S0006-8993(96)00821-9Get rights and content

Abstract

The aim of this study was to explore whether substance P could modulate the response mediated by ATP receptor. Experiments were carried out on rat dorsal root ganglion (DRG) neurons isolated acutely with enzymatic and mechanical treatment. The ATP-activated inward currents were recorded using the whole-cell patch-clamp technique. The majority of the neurons examined (8285, 96.5%) were sensitive to ATP (1–1000 μM). Application of substance P (0.01–100 μM) also caused an inward current. Several differences between these two kinds of currents were observed. 0.01, 0.1 and 1 μM substance P increased the ATP (10 μM)-activated current to 113.7 ± 3.1%, (n = 8); 127.2 ± 6.7%, (n = 12) and 154.7 ± 14.4% (n = 6) (means ± S.E.M.), respectively. This potentiating effect can be blocked by spantide, an NK1 receptor antagonist, and intracellular application of H7 (which is a potent inhibitor of PKC) could also block this kind of potentiation of SP on ATP-activated current. Since the substance P receptor and ATP receptor can coexist in rat DRG neurons and activation of substance P receptor can modulate the response mediated by ATP receptor, it suggests that they may cooperate with each other in activating peripheral nociceptive endings of sensory neurons, especially during tissue damage and/or inflammation.

Introduction

It is generally agreed that adenosine-5′-triphosphate (ATP) plays a role of extracellular chemical messenger, either as a neurotransmitter or a co-transmitter acting on peripheral neurons including primary sensory, sympathetic and parasympathetic neurons and on central neurons in a variety of brain areas 1, 10. The receptor for ATP is referred to as P2 purinoceptor which is further classified into P2x and P2y subtypes pharmacologically according to the potency order of the agonists [5]. The activation of ATP receptor which belongs predominantly to the super-family of ligand-gated ion channel receptors results in the opening of nonselective cationic channels, thereby depolarizing its target cells. Growing evidence indicates that ATP exerts an excitatory effect on effector cells innervated by purinergic nerves such as the cardiac and smooth muscle and exocrinic glands while it may also mediate the fast and slow transmission in neuro-neuronal synapses both in the peripheral and central nervous systems 2, 3, 4.

Since the evidence for the existence of ATP receptor in the membrane of sensory neurons including dorsal root ganglion (DRG) neurons first proved by Krishtal et al. [11], a number of works have been carried out in order to elucidate the characteristics, pharmacology and kinetics for ATP receptor. It is well known that many other ligand-gated ion channel receptors such as nAChR, GABAAR, NM-DAR, 5-HT3R, etc., could be modulated by a number of agents [20]and it has been identified in recent years that ATP-activated inward current could be potentiated by micromolar concentration of Zn2+ and inhibited by ETOH 12, 13, 14. Recently, we found that some neurotransmitters can also exert modulatory effects on ATP-activated currents [6]. In the present work the effect of peptide neuro-transmitter substance P (SP) on the ATP-mediated current was studied. A preliminary report has been published elsewhere [8].

Section snippets

Isolation of DRG neurons

2–3-week-old Sprague-Dawley rats, irrespective of sex, were decapitated, and the thoracic and lumbar segments of vertebrate column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The DRGs, together with dorsal and ventral roots and attached spinal nerves, were taken out from the inner side of each half of the dissected vertebrate and transferred immediately into Dulbecco's Modified Eagle's Medium (DMEM, Sigma) at pH = 7.4, 340

ATP- and SP-activated currents in the membrane of DRG neurons

In the present study, the majority of the cells examined responded to externally applied ATP (1–1000 μM) in a concentration-dependent manner (8285, 96.5%); many of them were also sensitive to substance P. In other words, there was a coexistence of ATP and SP receptor in the membrane of these neurons. The neurons chosen for this experiment were requested to be sensitive both to ATP and SP, the size distribution of them modified from Scroggs et al. [19]were as follows: large cells (> 45 μm) 4;

Discussion

For investigating the modulatory effect of SP on ATP-activated currents it was required that the experiments were carried out on neurons endowed with these two (SP and ATP) receptors. It is practicable because there are a large number of DRG cells in response to SP and ATP according to the data obtained from our previous work, in which the cells sensitive to SP and ATP are 90.6% [18]and 94.5% [6]respectively.

It is evident from Fig. 3 that the enhancement of amplitude of ATP-activated currents

Acknowledgements

We are grateful to Mrs. Y.Z. Fan for preparing isolated neurons, Dr. W.Z. Wei for technical help, and Prof. X.R. Jin for reading the manuscript. This work was supported by Grant 39270244 from the National Natural Science Foundation of China and a fund from the Ministry of Health of China.

References (21)

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