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

Abstract

The measurement of intracellular Ca(2+), cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca(2+) levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca(2+). In this study, we describe a Ca(2+) 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. 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 Ca(2+) levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca(2+) 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 Ca(2+) levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated. After SOC entry activation, cytosolic Ca(2+) levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca(2+) 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 Ca(2+) levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca(2+) levels were similar in aorta from young and old (3 and 22 months, respectively) rats. We confirmed that the system provides valuable data about intracellular Ca(2+) dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca(2+) 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 Ca(2+) levels and promotes understanding vascular physiology and disease.