Imaging of Calcium Oscillations and Activity of Cytochrome P-450_scc in Adrenocortical Cells

Summary

We have investigated the molecular mechanisms of signal transduction in steroidogenesis of adrenal fasciculata cells using noninvasive methods. With fluorescence video-microscopy, we discovered that the Ca²⁺ oscillations superimposed on step Ca²⁺ increase occurred in individual Calcium Green-l-loaded cells on stimulation with adrenocorticotropic hormone (ACTH) at 1 pM while the cAMP level was not increased. The Ca²⁺ oscillation lasted for several minutes with a frequency around 0.04 Hz. When the Ca²⁺ signaling was inhibited by the addition of ethyleneglycotetraacetic acid (EGTA), the corticoid production was also considerably suppressed. The results suggest that Ca²⁺ signaling is a second messenger for ACTH-induced steroid hormone synthesis in fasciculata cells. Imaging of cholesterol side-chain cleavage by cytochrome P-450_scc in intact cells was investigated with 3β-hydroxy22,23-bisnor-5-cholenyl ether (cholesterol-resorufin) incorporated into adrenocortical cells using a confocal microscope. On conversion of cholesterol-resorufin by P-450_scc to pregnenolone and resorufin, the P-450_scc activity was localized by observing the large increase in fluorescence (strong fluorescence spots and weak fluorescence patches) of the free resorufin. A good signal-to-noise ratio for the image was obtained by stimulation of cells with ACTH and NADPH when low density lipoprotein (LDL) was used for delivery of cholesterol-resorufin. Cholesterol traffic was investigated by measuring the movement of LDL in individual cells with video and confocal microscopy. We found a rapid directed motion and very slow motion of endosomes containing fluorescent-labeled LDL molecules in the cytoplasm of the cell, implying that LDL-containing endosomes are actively transported toward lysosomes.