Investigation of effects of leptin on intracellular calcium signals in rat dorsal root ganglion neurons

Central and peripheral administration of leptin plays an important role in nociceptive behavior, but the cellular mechanism of this action remains unclear. This study investigate the effects of leptin on intracellular calcium, ([Ca2+]i) levels in isolated rat dorsal root ganglion (DRG) neurons, with the aim of exploring possible involvement of leptin-mediated signaling in peripheral nociception.Isolated DRG neurons were loaded with 1 ?mol fura-2 AM and Ca2+ responses were assessed by using the fluorescent ratiometry. Fura-2 AM loaded DRG cells were excited 340 nm and 380 nm, and emission was recorded at 510 nm by using digital microscopic calcium imaging system. Changes in free intracellular calcium concentrations were determined by the change in 340 nm/380 nm ratio in individual DRG neurons. Leptin caused increase in [Ca2+]i in a dose depedent manner. The mean 340 nm/380 nm rations were (baseline 100.0±0.0 % vs leptin): 104.6±0.4 % (0.1 ?M leptin, n=42 cells), 119.5±3.9 % (1 ?M leptin, n=31 cells), 129.4±4.2 % (10 ?M leptin, n=29 cells) and 187.5±7.6 % (100 ?M leptin, n=35 cells) following addition of respective concentrations of leptin. The PKC inhibitor chelerythrine chloride significantly decreased the leptin (1 ?M)-induced calcium responses (from 100.0±0.0 % to 56.7±6.4 %, n=27 cells). After depletion of thapsigargin-sensitive calcium stores by treatment of cells with thapsigargin (TG) attenuated but not completely blocked the subsequent calcium responses to 1 ?M leptin (TG + 1?M leptin = 106.1±2.4 % (n=18 cells) vs. 1 ?M leptin= 119.5±3.9 (n=23 cells).We first demonstrated that leptin mediate peripheral nociceptive signalling through mechanism that involves PKC-dependent changes in intracellular calcium levels in rat primary sensory neurons. Results from this study indicates that in addition to mediating the sensing of energy reserves in the body, leptin involved in the sensing process of pain.Key Words: Leptin, Calcium Imaging, Dorsal Root Ganglion, Pain, Protein Kinase C.