Differential regulation of the kinetics of cytosolic ca2+ in chromaffin cells from normotensive and hypertensive rats

JF Padín1, E Dantas da Silva Júnior2, A Caricati-Neto2, M Maroto1, AMG de Diego1,3, AG García1,4. 1Facultad de Medicina, Universidad Autónoma de Madrid (UAM), Instituto Teofilo Hernando and Departamento de Farmacología y Terapéutica, Spain, 2Escola Paulista de Medicina, Universidade Federal de São Paulo, Departamento de Farmacología, Brazil, 3University College London (UCL), Ear Institute, UK, 4Instituto de Investigación Sanitaria, Hospital Universitario la Princesa, Universidad Autónoma de Madrid (UAM), Servicio de Farmacología Clínica, Spain

Enhanced quantal release of catecholamines in spontaneously hypertensive rats (SHR) in comparison with normotensive rats, has recently been demonstrated in our laboratory (Miranda-Ferreira et al., 2008 JPET 324:685-93; 2009 JPET 329:231-40). Whether this alteration is secondary to differences in the regulation of calcium homeostatic mechanisms, was investigated here in chromaffin cells from control Wistar Kyoto/NCrl rats (WKY) and in SHR. Cultured adrenal chromaffin cells loaded with fura-2 AM from 6-10-month-old WKY and SHR rats were used; this allowed the monitoring of the kinetics of the Ca2+ changes in the cytosol ([Ca2+]c) upon challenging the cells with 5-30 s pulses of acetylcholine (ACh). We found that the amplitudes and areas of the [Ca2+]c transients elicited by ACh pulses of increasing duration were 2-3-fold higher in SHR cells, compared with WKY cells. This difference was paralleled by the ability of 2 µM protonophore FCCP to enhance more than 3-fold the basal [Ca2+]c in SHR, with respect to WKY, suggesting that chromaffin cells from hypertensive animals are overloaded with Ca2+. We finally observed that SHR cells exhibited greater frequency of spontaneous [Ca2+]c oscillations, compared with WKY cells. We conclude that SHRs exhibit a poorer regulation of the mechanism controlling the role of mitochondria in buffering the [Ca2+]c transients triggered by ACh, compared with WKY. Mitochondrial Ca2+ overload could explain the lower level of ATP found in SHR chromaffin cells, that are more vulnerable than WKY chromaffin cells. These disturbances in Ca2+ homeostasis could underlie the greater catecholamine release responses that could contribute to the development and/or maintenance of a hypertensive state. Therefore, targeting mitochondria could become an efficient strategy to develop new anti-hypertensive drugs with potential cardiovascular protective effects.