Biochemical and 31P-NMR spectroscopic evaluation of immobilized perfused rat Sertoli cells

Cell immobilization and perfusion are used for physiologic studies of Sertoli cells with phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy and biochemical methods. In this study the 31P NMR spectra of Sertoli cells isolated from 18-to 21-day-old rats and immobilized in agarose threads continuously perfused with oxygenated Dulbecco's modifed Eagle medium were obtained at 81 MHz on an NMR system. Cytosolic Ca2+, intracellular Mg2+, lactate and pyruvate, and oxygen consumption were measured with standard biochemical methods. Perfused Sertoli cells maintain a stable intracellular adenosine triphosphate concentration for more than 10 hours. Sertoli cells placed in cold storage overnight and then subjected to perfusion partially regenerate cellular adenosine triphosphate levels. Sertoli cells consume an average of 4.8 +/- 0.4 nmol O2/min/10(6) cells and maintain average ambient lactate and pyruvate levels of 7.1 +/- 0.8 mg/dl and 0.65 +/- 0.05 mg/dl, respectively, with a lactate/pyruvate ratio in the range 8 to 12. The basal Ca2+(i) of Sertoli cells is 98 +/- 0.7 nmol/L (n = 58), which declines to a level less than 10 nmol/L when the Sertoli cells are perfused with a calcium-free medium. Perfusion of Sertoli cells with a sodium-free medium, with 10(-6) mol/L carbonyl cyanide P-trifluoromethoxy-thenylhydrozone, or with Ca2+ ionophore A23187 at a concentration of 10(-6) mol/L increases the Ca2+(i) to a level of 426 +/- 107 nmol/L, 274 +/- 29 nmol/L, or 282 +/- 57 nmol/L, respectively. A bioreactor for physiologic studies of Sertoli cells in real time with NMR spectroscopy has been developed. These data demonstrate that isolated, immobilized, and perfused Sertoli cells are stable for prolonged periods. In addition, these data suggest that Sertoli cells possess a functional Na+-Ca2+ antiporter and that they sequester extracellular Ca2+ in one or more intracellular compartments.