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Progesterone Maintains Basal Intracellular Adenosine Triphosphate Levels and Viability of Spontaneously Immortalized Granulosa Cells by Promoting an Interaction between 14-3-3 and ATP Synthaseß/Precursor through a Protein Kinase G-Dependent Mechanism

Departments of Cell Biology (J.J.P., X.L., J.R.) and Obstetrics and Gynecology (J.J.P.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: John J. Peluso, University of Connecticut Health Center, Department of Physiology (MC3505), 263 Farmington Avenue, Farmington, Connecticut 06030-1230. E-mail: peluso{at}nso2.uchc.edu.

The present studies were designed to 1) describe changes in both the mitochondrial membrane potential and ATP content of spontaneously immortalized granulosa cells as they undergo apoptosis, 2) identify some of the downstream events that are activated by progesterone (P4), and 3) relate these downstream events to changes in mitochondrial function and apoptotic cell death. These studies revealed that in response to serum deprivation, the mitochondrial membrane potential initially hyperpolarizes and ATP content increases. That this increase in ATP is required for apoptosis was demonstrated by the finding that oligomycin inhibited the increase in ATP and apoptosis. Piridoxalphosphate-6-azopeyl-2'-4'-disulfonic acid, an inhibitor of purinergic receptors, which are activated by ATP, also inhibited apoptosis due to serum withdrawal. This study provides additional support for ATP’s causative role in apoptosis. Moreover, 8-Br-cGMP, a protein kinase G (PKG) activator, mimicked P4’s action, whereas a PKG antagonist, DT-3, attenuated P4’s suppressive effect on ATP and apoptosis. Finally, DT-3 treatment was shown to attenuate P4-regulated phosphorylation of 14-3-3 and its binding partner, ATP synthaseß/precursor and the amount of ATP synthaseß/precursor that bound to 14-3-3. Based on these data, it is proposed that P4 prevents apoptosis in part by activating PKG, which in turn maintains the interaction between ATP synthaseß/precursor and 14-3-3. In the absence of P4-induced PKG activity, we further propose that some ATP synthaseß precursor dissociates from 14-3-3, resulting in its activation and incorporation into the ATP synthase complex, which ultimately results in an increase in ATP and apoptosis.