Proceedings of The Physiological Society
University College Dublin (2009) Proc Physiol Soc 15, PC68
Spinorphin Inhibits Membrane Depolarisation-Induced Intracellular Calcium Signals in Cultured Rat Dorsal Root Ganglion Neurons
A. Ayar1, M. Ozcan2, T. Kuzgun3, E. Alcin2
1. Physiology, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey. 2. Biophysics, Faculty of Medicine, Firat University, Elazig, Turkey. 3. Physiology, Faculty of Medicine, Firat University, Elazig, Turkey.
Spinorphin, an endogenous peptide with antagonist actions on enkephalin-degrading enzymes and P2X3 receptors, presents potential antinociceptive effects. It is known that the primary afferent sensory neurons are functionally heterogeneous and small sized subpopulation of cultured DRG neurons may serve as a cellular model for studying the peripheral nociception. The aim of the present study was to determine the effects of spinorphin on Ca2+ transients, evoked by high-K+ (30 mM), and whether there were differences in spinorphin effect among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Following enzymatic digestion and mechanical agitation the DRG neurons were cultured on coated coverslips and loaded with 5 uM Fura-2 AM. Standard fura-2 ratiometric technique was utilised for quantifying [Ca2+]i responses in individual DRG neurons using fluorescence imaging system consisting of CCD camera coupled to an inverted microscope with a 40x (1.30 NA) objective. All data were analyzed by using unpaired t test, P <.05 defining statistical significance. Spinorphin, inhibited the Ca2+ transients evoked with 30 mM K+ in concentration dependant manner in a subpopulation of sensory neurons. Spinorphin dose dependently inhibited the HiK+-induced [Ca2+]i responses (1.40±0.09 vs. 1.42±0.08, n=15, NS for 10 uM;1.45±0.09 vs. 0.91±0.08 for 100 uM spinorphin n=16, P<0.05; and 1.39 ±0.09 vs. 0.84 ±0.08 for 300 uM spinorphin n=20, P<0.05, respectively) only in small-diameter DRG neurones. Additionally, after application of spinorphin a significant percent of small-diameter nociceptive DRG neurones did not respond to stimulation by HiK+ (response rate after application of 10, 100 and 300 uM spinorphin: 95%, 55%, and 54%, respectively) while the percentage of the response was not significantly changed in large-diameter non-nociceptive DRG neurones. Results from this study indicates that spinorphin significantly inhibits calcium signalling, transient changes in free intracellular Ca2+ concentration which are key for the modulation of cell membrane excitability and neurotransmitter release, in only small-diameter nociceptive subpopulation of DRG neurons and this endogenous agent may be effective analgesic with potential combination with opioids.
Where applicable, experiments conform with Society ethical requirements