Kisspeptin leads to calcium signaling in cultured rat dorsal root ganglion neurons

Kisspeptin, a hypothalamic neuropeptide, is a member of the RF-amide family known to alter pain sensitivity in rodents. It is known that kisspeptin and G protein-coupled receptor (GPR54) is expressed in dorsal root ganglion (DRG) neurons, the cellular model of pain, kisspeptin is associated with pain modulation in rodents. Despite this, there are no studies yet on the effects and mechanisms of kisspeptin on sensory neurons. Therefore, the purpose of this study is to investigate the nociceptive/antinociceptive effects and mechanism of kisspeptin on intracellular free calcium ([Ca2+]i) levels in rat DRG neuron culture. In this thesis study, DRG neurons were prepared using primary cell culture, Spraque-Dawley 1-2 day old rats (n = 20). DRG neurons were loaded with 1 µM Fura-2 acetoxy methyl ester (AM) and intracellular free calcium responses were measured with excitation at 340 nm and 380 nm and emission at 510 nm by digital imaging microscopy with changes of the ratio 340 nm/ 380 nm. The effect of kisspeptin-10 administration at different doses (100 nM, 300 nM and 1000 nM, at least 3 replicates) on [Ca2+]i levels was observed. In the absence of extracellular calcium, the effects of kisspeptin were also observed with the Protein Kinase C (PKC) inhibitor chelerythrine chloride (ChCl) pretreatment to determine whether it exhibited its possible effects on [Ca2+]i signal via the PKC pathway. Bath application of kisspeptin-10 to DRG neurons increased [Ca2+]i levels in a dose-dependent and significant way. 340 nm / 380 nm ratios respectively (baseline 100.0 ± 0.0% vs kisspeptin): 118.4 ± 4.6% (100 nM kisspeptin, n = 41, p <0.01), 126.6 ± 4.1% ( 300 nM kisspeptin, n = 36, p <0.001) and 140.2 ± 4.9% (1000 nM kisspeptin, n = 44, p <0.001). In the absence of extracellular calcium, the kisspeptin induced attenuated but still significant increase in [Ca2+]i levels. [Ca2+]i responses persisted in the presence of protein kinase C (PKC) inhibitor. Results, it has been revealed that kisspeptin-10 activates the [Ca2+]i signal in cultured rat sensory neurons independent of PKC; and this suggests that the peripheral site is also involved in the pain modulating effect of kisspeptin.