³¹P NMR investigation of energy metabolism in perifused MMQ cells

Abstract

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. 31P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the 31P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.

In-cell NMR: Why and how?

“…A number of bioreactors inside spectrometers were designed at that time (see Section 3.7.). These permitted continuous monitoring of cell metabolism, notably to characterize the impact of drug-treatment and irradiation on mammalian cancer cell metabolism by 13 C-and 31 P-NMR [768,[777][778][779][780][781][782][783][784][785][786][787][788][789][790][791][792][793][794], or to study the effects of ethanol and various chemicals on hepatocytes [795,796], as well as metabolism in hybridoma [771,797]. However, in-cell NMR studies of metabolic activities appeared to be limited to cases involving the study of high concentration metabolites.…”

“…Bioreactors have been used to characterize cell metabolism in real-time with 13 C-and 31 P-NMR. A broad variety of studies using NMR-bioreactors have been concerned with the effects of drugexposure and irradiation on mammalian cancer cell metabolism [768,[777][778][779][780][781][782][783][784][785][786][787][788][789][790][791][792][793][794]1093,1094,1099,1100,1103,1858,1866,1890], the impact of ethanol and chemicals on hepatocytes [795,796], metabolism in hybridoma [771,797] and bacteria [1819,1883,[1891][1892][1893][1894], comparisons of the metabolism of cells in suspension or embedded in gels, which has industrial applications for yeast metabolism [1804][1805][1806]1811,1831,1832,1859], and antibody secretion of hybridoma …”

“…As discussed above (Section 2.3.2. ), in combination with 13 C-NMR, 31 P-NMR and bioreactors were used to study the effects of various drugs on mammalian cancer cell metabolism [768,[777][778][779][780][781][782][783][784][785][786][787][788][789][790][791][792][793][794], of ethanol and various chemicals on hepatocytes [795,796], and the metabolism of hybridoma [771,797], and yeast immobilized in gel matrices [866]. More recently, 31 P-NMR also provided interesting checks for cellular health during in-cell structural biology studies [867][868][869].…”