Simultaneous measurement of cytosolic and mitochondrial calcium levels: Observations in TRPC1-silenced hepatocellular carcinoma cells

Introduction: The measurement of intracellular Ca2+, cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca2+ levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca2+. In this study, we describe a Ca2+ monitoring system that allows studying both adherent cells and tissues and discuss data obtained from hepatocellular carcinoma cells and rat thoracic aorta by using this system. Methods: For this purpose, two apparatus, one for adherent cells and the other for intact rat aorta, were designed and produced. With this system, changes in cytosolic Ca2+ levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca2+ levels were simultaneously measured in TRPC1-silenced Huh7 hepatocellular carcinoma cells. In addition, the effects of trifluoromethylphenylimidazole (TRIM) on cyclopiazonic acid (CPA)-, serotonin (5-HT)-, and phenylephrine (PE)-induced changes in isometric force and cytosolic Ca2+ levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated. Results: After SOC entry activation, cytosolic Ca2+ levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca2+ levels following CPA-induced ER depletion were significantly (p < .05) diminished in TRPC1-silenced Huh7 cells. In addition, TRIM partially inhibited both 5-HT-induced contractions and cytosolic Ca2+ levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca2+ levels were similar in aorta from young and old (3 and 22 months, respectively) rats. Discussion: We confirmed that the system provides valuable data about intracellular Ca2+ dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca2+ loading following CPA-induced ER depletion in TRPC1-silenced Huh7 cells suggests a possible role of TRPC1 in hepatocellular carcinoma cell apoptosis. The system also enables the simultaneous measurement of isometric force and cytosolic Ca2+ levels and promotes understanding vascular physiology and disease. (C) 2015 Elsevier Inc. All rights reserved.