REGULATION BY THE NCX OF CALCIUM SIGNALLING AND EXOCYTOSIS AT PHYSIOLOGICAL, BUT NOT AT ROOM TEMPERATURE

JA Arranz-Tagarro1,2, JF Padín2, S Vestring2,3, JC Fernández-Morales2, E Calvo-Gallardo2, M Maroto2, AMG de Diego2,4, AG García2,5. 1Facultad de Farmacia, Universidad de Santiago de Compostela (USC), Departamento de Farmacología, Spain, 2Facultad de Medicina, Universidad Autonoma de Madrid (UAM), Departamento de Farmacología y Terapeutica and Instituto Teofilo Hernando, Spain, 3Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Germany, 4University College London (UCL), Ear Institute, UK, 5Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, UAM, Servicio de Farmacología Clínica, Spain

This study deals with the discovery of a novel physiological function of the plasmalemmal Na+/Ca2+ exchanger (NCX), linked to the regulation of the quantal release of catecholamine triggered by K+ depolarization of bovine adrenal chromaffin cells (BCCs). The NCX uses the energy provided by the Na+ gradient to achieve an electrogenic 3 Na+/1 Ca2+ ion exchange. Under physiological conditions Na+ is transported into the cell and Ca2+ extruded from the cytosol. However, when the electrochemical gradient for Na+ is reversed, such as during membrane depolarization or the opening of gated Na+ channels, the exchanger moves Na+ out of the cell and Ca2+ into the cell. These activities led to the proposition that the NCX contributes to the regulation of the cytosolic Ca2+ concentrations ([Ca2+]c) and the Ca2+-dependent exocytotic release of catecholamines in BBCs. In the present study, aimed at further clarifying the contribution of the plasmalemmal NCX to the regulation of [Ca2+]c transients and catecholamine release responses, we took advantage of the low-rate of Ca2+ extrusion through the NCX at 22 ºC and its much higher rate at 37 ºC. Furthermore, we recoursed to the NCX blocker KB-R7943, that blocks the cardiac NCX at 1-5 µM concentrations. We have discovered that burst spike quantal release responses from BCCs stimulated with K+ depolarising pulses were 3-4-folds higher at 37 ºC compared with those obtained at 22 ºC. Also, the single spike amperometric events were faster and with higher amplitude at 37 ºC although the quantal size was similar. Surprisingly, we unexpectedly found that KB-R7943 drastically inhibited the quantal release responses at 37 ºC, leaving untouched the responses obtained at 22 ºC. This effect is not mediated by blockade of calcium channels because in voltage-clamped cells, KB-R7943 did not block the Ca2+ currents. In addition, the measurement of [Ca2+]c by fluorescence microscopy with fura-2 showed a correlation between the quantal K+-evoked release responses and the [Ca2+]c elevations achieved. On the basis of these results we propose that at the physiological temperature of 37 ºC, the plasmalemmal NCX importantly contributes to maintain low the [Ca2+]c levels at plasmalemmal sites nearby the voltage dependent calcium channels during activation of chromaffin cells with depolarizing stimuli of a few seconds duration. In so doing, the NCX is preventing the Ca2+-dependent inactivation of VDCCs, thus ensuring a healthy catecholamine release response during prolonged stressful stimulation of chromaffin cells.