AUGMENTATION OF [Ca2+]i RESPONSES TO G-PROTEIN COUPLING AGONISTS IN TRP 5-EXPRESSED NEUROSECRETORY PC 12 CELLS

1. To determine the role of TRP 5 proteins in neuronal cells, we expressed TRP 5 in non-differentiated rat pheochromocytoma cells (PC12). Increases in cytosolic Ca2+ concentration ([Ca2+]i), Na+ concentration ([Na+]i) and current in response to receptor agonists were examined in cells transfected with TRP 5 (TRP 5-cells) and vector (control-cell). [Ca2+]i and [Na+]i were monitored with ratiometric imaging using fura-2 and SBFI, respectively. Membrane currents were measured with conventional whole cell patch-clamp technique. 2. RT-PCR revealed that TRP 1, 3, 4, 5, 6, and 7 were expressed in rat brain and adrenal medulla, and that TRP 1 and 3 in PC 12. Expression of TRP 5 protein in TRP 5-cells was confirmed with anti-TRP 5-immunohistochemistry. 3. Bradykinin (BK), UTP and caffeine induced dose-dependent increases of [Ca2+]i in PC 12. Fifty percent effective concentrations for BK, UTP and caffeine were 0.22 μM, 28 μM and 20 mM, respectively. 4. The resting [Ca2+]i (51±3.2nM) was significantly higher in TRP 5-cells than that in control-cells (40±0.5nM). Pretreatment of TRP 5-cells with dibutyryl cAMP and 8-bromo cGMP did not affect the resting [Ca2+]i. 5. [Ca2+]i increases induced by BK and UTP were 3-times and 1.5-times larger in TRP 5-cells than those in control-cells, respectively. [Ca2+]i responses to caffeine and high-KCl were almost the same magnitude as those in control-cells. In TRP 5-cells, Mn2+ influx in TRP 5-cells 1 min after agonist stimulation was significantly higher than that in control cells. 6. In a Ca2+-free solution, BK, UTP and caffeine caused a transient increase in [Ca2+]i due to Ca2+ release from internal Ca2+ stores. Subsequent administration of Ca2+ evoked a [Ca2+]i increase due to capacitative Ca2+ entry. Both increases in [Ca2+]i were not different in magnitude between TRP 5 and control cells. Similar results were obtained with a SERCA inhibitor, thapsigargin. 7. In control and TRP 5 cells, SK & F96365, a blocker of receptor-activated Ca2+ channel, inhibited [Ca2+]i responses to BK, but not those to UTP. 8. In TRP 5 cells, the resting [Na+]i and BK-induced [Na+]i increase were significantly higher than those in control cells. At a holding potential of-60 mV, an inward current evoked by BK became prominent by TRP 5 transfection. 9. The expression of TRP 5 in PC 12 led to an increase of the resting [Ca2+]i and Ca2+ entry induced by BK and UTP. It is unlikely that TRP 5 channels are associated with Ca2+ release from internal stores an