PARENT SESSION
PLATFORM SESSION 20. TESTIS BIOLOGY

Tuesday, August 3, 2004
4:30 PM–6:30 PM
Buchanan A203
Chair: Derek McLean Co-Chair: Rebecca Bott

(596) ACTIVE RESPIRATION, MAINTENANCE OF MITOCHONDRIAL MEMBRANE POTENTIAL (m) AND INNER MITOCHONDRIAL CALCIUM ARE REQUIRED FOR STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) MEDIATED STEROIDOGENESIS.

Shankara, Tristan ¹, Dothager, Christina ¹, Lepore, Fred¹, Hales, Dale¹, ¹ University of Illinios at Chicago, Chicago, IL

ABSTRACT- Steroidogenic Acute Regulatory Protein (StAR) is required for the hormonally controlled cholesterol transfer into the inner mitochondrial membrane, the first and rate-limiting step in steroid hormone biosynthesis. The purpose of the present study was to examine the relationship between the mitochondrial membrane potential (m), mitochondrial respiratory capacity, intra- mitochondrial calcium dynamics, and their relative importance to Leydig cell steroidogenesis. In order to determine what aspect of mitochondrial function is required to support StAR protein function, site-specific mitochondrial inhibitors were tested. MA-10 cells were treated with antimycin A, oligomycin, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), or RU360 and their effects on cAMP-stimulated StAR expression and function were analyzed. Their effects on StAR protein amount was assessed by western blot, and StAR protein synthesis was analyzed by ³⁵[S]-labeling and immunoprecipitation. The effect of inhibitors on membrane potential was determined by ratiometric spectrofluorometry and fluorescence microscopy using the mitochondrial fluorophore tetramethylrhodamine ethyl ester (TMRE). Mitochondrial respiratory capacity was determined by measuring the rate of oxygen consumption using the Strathkelvin Mitocell Oxygen electrode. Results demonstrate that dissipation of the mitochondrial membrane potential with CCCP inhibits StAR protein by preventing its import into the mitochondria, while inhibition of site II/III or site V with antimycin A and oligomycin, respectively, inhibit StAR translation/transcription. Further, depletion of intra mitochondrial calcium levels by blocking reuptake with Ru360, a cell permeable derivative of Ruthenium red which inhibits the mitochondrial calcium uniporter, inhibits StAR protein expression. Moreover, all of these inhibitors block steroid hormone biosynthesis. These results suggest that maintenance of mitochondrial calcium, m, and ongoing respiration are all required for StAR protein mediated steroidogenesis.

KEY WORDS: steroid hormone, respiration, Leydig Cell, mitochondria