Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC170
The Endogenous Salivary Peptide Opiorphin Inhibits Ca2+ Signalling in Rat Primary Sensory Neurones
A. Ayar1, M. Ozcan2, O. F. Kalkan1, D. Aydin1
1. Department of Physiology, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey. 2. Department of Biophysics, Firat University, Faculty of Medicine, Elazig, Turkey.
Evidence from recent in vivo behavioural pain studies suggests that the inhibition of enzymes responsible for the inactivation of enkephalins, endogenous opiate receptor agonists with potent analgesic properties, are potential targets for effective endogenous modification to obtain strong, safe and long lasting analgesia. However, the cellular mechanisms by which the inhibitor of multiple enkephalin degrading enzymes are acting, and their possible peripheral antinociceptive effects is not studied yet. The aim of the present study was to investigate the effects of opiorphin, a natural peptide inhibitor of enkephalin degrading-enzyme, on peripheral nociception by investigating its action on intracellular Ca2+ signalling in rat dorsal root ganglion (DRG) neurones. All experimental procedures were approved by the Institutional Ethics Committee. Following enzymatic digestion and mechanical agitation the DRG neurones were cultured on coated coverslips and loaded with 1 umol Fura-2 AM. Intracellular calcium responses were quantified by the changes in 340/380 ratio for individual DRG neurons using fluorescence imaging system consisting of CCD camera coupled to an inverted microscope with a 40x (1.30 NA) objective. Brief exposure to high KCl (30 mM) was used to stimulate intracellular calcium signals, and effects of opiorphin was investigated. All data were analyzed by using unpaired t test, P <.05 defining statistical significance. Acute application of 10, 30 or 100 uM opiorphin failed to produce any significant change in KCl (30 mM)-induced elevation in cytosolic calcium levels (each tested on cells from three different culture dishes). Treatment with opiorphin for 30 minutes caused a complete inhibition of cytosolic calcium responses to KCl (30 mM). The mean 340/380 nm ratio was increased from baseline level of 0.79±0.05 (n=67) to 1.24±0.06 with KCl exposure (30 mM KCl, P<0.05, n= 67) which remained without significant change at 5th minute but abolished after 30 minute treatment with 100 uM opiorphin (30 mM KCl + 100 uM opiorphin: 0.78±0.07, n=55). We report for the first time in these peripheral nociceptive sensory neurones that opiorphin inhibits membrane depolarisation-induced calcium signalling. Although this effect takes time, considering the fact that opiorphin advantages with respect to the abuse liability and drug tolerance, this agent or its proper derivatives are promising candidates for treatment of pain conditions.
Where applicable, experiments conform with Society ethical requirements