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OX₂ receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

ABSTRACT: Aims: Orexin-A is known to induce anti-nociceptive effects in animal models of chronic pain. We have found that orexin-A inhibits KCl loading-induced increases in the intracellular calcium ion levels ([Ca²⁺ ]_i ) in C-fiber-like neurons of rats showing inflammatory nociceptive behavior. Here, we examined the effects of orexin-A on the depolarization of C-fiber-like neurons derived from a rat model for another type of chronic pain, namely neuropathic pain. Thus, we analyzed the effects of orexin-A on KCl-induced increases in [Ca²⁺ ]_i in C-fiber-like neurons of rats with sciatic nerve ligation.

Methods: Paw withdrawal and threshold force in response to tactile stimuli were evaluated using von Frey filaments. Sham-operated rats served as controls. [Ca²⁺ ]_i in neurons were visualized by calcium fluorescent probe. Changes in [Ca²⁺ ]_i were assessed using relative fluorescence intensity.

Results: Seven days after sciatic nerve ligation, paw withdrawal and threshold force for tactile stimuli were increased and reduced, respectively. KCl loading to neurons from either sciatic nerve-ligated or control rats increased relative fluorescence intensity. The KCl-induced increase in relative fluorescence intensity in sciatic nerve-ligated, but not that of control, rats was inhibited by orexin-A. The OX₁ and OX₂ receptor antagonist MK-4305 and OX₂ receptor antagonist EMPA, but not the OX₁ receptor antagonist SB 334867, each counteracted orexin-A-induced inhibition of KCl-provoked increases in relative fluorescence intensity.

Conclusion: The present findings constitute neuropharmacological evidence that OX₂ but not OX₁ receptors mediate the inhibitory effects of orexin-A on KCl-induced increases in [Ca²⁺ ]_i in C-fiber-like neurons of rats showing hyperalgesia provoked by sciatic nerve ligation.

SUBMITTER: Yamaguchi M

PROVIDER: S-EPMC7292216 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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OX<sub>2</sub> receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

Yamaguchi Masami M Ishikawa Manabu M Aono Yuri Y Saigusa Tadashi T

Neuropsychopharmacology reports 20191217 1

<h4>Aims</h4>Orexin-A is known to induce anti-nociceptive effects in animal models of chronic pain. We have found that orexin-A inhibits KCl loading-induced increases in the intracellular calcium ion levels ([Ca<sup>2+</sup> ]<sub>i</sub> ) in C-fiber-like neurons of rats showing inflammatory nociceptive behavior. Here, we examined the effects of orexin-A on the depolarization of C-fiber-like neurons derived from a rat model for another type of chronic pain, namely neuropathic pain. Thus, we ana ...[more]

PMID: 31845549

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Project description:Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

Dataset Information

OX₂ receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

Publications

OX<sub>2</sub> receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

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OX2 receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

Publications

OX<sub>2</sub> receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

OX₂ receptors mediate the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from rat dorsal root ganglion in a chronic pain model.