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Molecular Neurobiology

, Volume 55, Issue 5, pp 4297–4310 | Cite as

Endothelin-1 Decreases Excitability of the Dorsal Root Ganglion Neurons via ETB Receptor

  • Nandkishor K. Mule
  • Jitendra N. Singh
  • Kunal U. Shah
  • Anil Gulati
  • Shyam S. Sharma
Article

Abstract

Endothelin-1 (ET-1) has been demonstrated to be a pro-nociceptive as well as an anti-nociceptive agent. However, underlying molecular mechanisms for these pain modulatory actions remain unclear. In the present study, we evaluated the ability of ET-1 to alter the nociceptor excitability using a patch clamp technique in acutely dissociated rat dorsal root ganglion (DRG) neurons. ET-1 produced an increase in threshold current to evoke an action potential (I threshold) and hyperpolarization of resting membrane potential (RMP) indicating decreased excitability of DRG neurons. I threshold increased from 0.25 ± 0.08 to 0.33 ± 0.07 nA and hyperpolarized RMP from −57.51 ± 1.70 to −67.41 ± 2.92 mV by ET-1 (100 nM). The hyperpolarizing effect of ET-1 appears to be orchestrated via modulation of membrane conductances, namely voltage-gated sodium current (I Na) and outward transient potassium current (I KT). ET-1, 30 and 100 nM, decreased the peak I Na by 41.3 ± 6.8 and 74 ± 15.2%, respectively. Additionally, ET-1 (100 nM) significantly potentiated the transient component (I KT) of the potassium currents. ET-1-induced effects were largely attenuated by BQ-788, a selective ETBR blocker. However, a selective ETAR blocker BQ-123 did not alter the effects of ET-1. A selective ETBR agonist, IRL-1620, mimicked the effect of ET-1 on I Na in a concentration-dependent manner (IC50 159.5 ± 92.6 μM). In conclusion, our results demonstrate that ET-1 hyperpolarizes nociceptors by blocking I Na and potentiating I KT through selective activation of ETBR, which may represent one of the underlying mechanisms for reported anti-nociceptive effects of ET-1.

Keywords

Endothelin-1 Neuropathic pain ETBDRG neurons Sodium currents Potassium currents Anti-nociception IRL-1620 

Abbreviations

AP

Action potential

4-AP

4-Amino pyridine

APD

Action potential duration

G

Conductance

τdec

Decay phase time constant

IKD

Delayed rectifier potassium current

DRG

Dorsal root ganglion

ET-1

Endothelin-1

ETAR

Endothelin A receptor

ETBR

Endothelin B receptor

IKT

Fast transient potassium currents

Gmax

Maximum conductance

PBS

Phosphate buffer saline

K+

Potassium

RMP

Resting membrane potential

Rs

Series resistance

INa

Sodium currents

SEM

Standard error of mean

TEA

Tetraethyl ammonium chloride

TTP

Time to peak

VNa

Sodium current reversal potential

Notes

Acknowledgements

The authors thank the Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Government of India, for the financial support.

Authors’ Contributions

SSS and JNS conceptualized the study design. NKM and KS carried out the experiments. NKM, KS, and JNS analyzed the data. NKM, JNS, SSS, and AG wrote the final version of the manuscript.

Compliance with Ethical Standards

All experimental protocols were approved by the Institutional Animal Ethics Committee, National Institute of Pharmaceutical Education and Research, SAS Nagar, India.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2017_640_MOESM1_ESM.pdf (8.8 mb)
ESM 1 (PDF 9014 kb) Typical AP trace recorded from DRG neuron and commonly measured AP parameters are shown (A). The AP was elicited by injection of depolarizing current injections (40 ms pulses) in 20 pA increments (shown in inset); it evoked all-or-none action potential overshoot. The current threshold was defined as the minimum current required to evoke an AP, for instance Ithreshold for this neuron was 0.1 nA. Several AP parameters were calculated for given recording and summarized (B).
12035_2017_640_MOESM2_ESM.pdf (10.4 mb)
ESM 2 (PDF 10692 kb) The sodium current was elicited by stepping voltage from -50 to 50 mV in 5 mV increment with a pre-pulse of -120 mV for 50 ms (A) is shown. Original traces and voltage pulse protocols used to isolate transient (left panel) and delayed (right panel) component of macroscopic potassium currents recorded from the neonatal rat DRG neurons (B) are shown. Owing to its faster inactivation, the transient component of IK can selectively be rendered inactive by giving pre-pulse of -40 mV. The neurons were clamped at -80 mV and IK were elicited by giving a pre-pulse of (-40 mV for IKDR or -120 mV for IKT) followed by steps from -50 to 60 mV in 10 mV increment.
12035_2017_640_Fig6_ESM.gif (578 kb)
ESM 3

Schematic diagram depicting dual actions of ET-1 on pro-nociceptive and anti-nociceptive via activation of ETAR and ETBR, respectively. The pro-nociceptive action of ET-1 is mediated via activation of ETAR and underlying secondary messenger system. The anti-nociceptive actions of ET-1 are reportedly carried out via paracrine activation of ETBR in keratinocytes followed by activation of opioid system (as shown in inset, where MOR: Mu opioid receptors system). Here we present an additional non-opioid mechanism of ET-1-mediated anti-nociception which may be orchestrated through the activation of ETBR and resultant modulation of ion channels expressed on DRG neurons. The modulation of ion channels may be carried out via ETBR downstream-secondary messenger system including phospholipase-C (PLC)-inositol trisphosphate (IP3)/diacylglycerol (DAG) system or guanylyl cyclase (GC)/cGMP further employing PKA-PKC to alter the ionic conductances in DRG neurons. (GIF 577 kb)

12035_2017_640_MOESM3_ESM.tif (2.7 mb)
High Resolution Image (TIFF 2719 kb)

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Electrophysiology Laboratory, Department of Pharmacology and ToxicologyNational Institute of Pharmaceutical Education and Research (NIPER)S.A.S. NagarIndia
  2. 2.Department of Pharmaceutical Sciences, Chicago College of PharmacyMidwestern UniversityDowners GroveUSA

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