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Transport and selective utilization of adenosine as a prosubstrate for luteinizing hormone-sensitive adenylate
Abstract:
The objective of the present studies was to examine adenosine uptake in the rat luteal cell, to characterize the cellular products after uptake, and to assess the role of adenosine transport and conversion to cAMP in amplification of LH-stimulated cAMP accumulation. Adenosine uptake showed an apparent Km of 7.3 +/- 0.6 microM, and a maximum velocity of 2.2 +/- 1.4 pmol/min X 10(5) cells at 24 C; uptake was temperature dependent (Q10 = approximately 3) and inhibited by dipyridamole (IC50 = 7 microM). Radiolabeled adenosine uptake was inhibited by AMP (IC50 = 14 microM), ATP (IC50 = 16 microM), guanosine (IC50 = 20 microM), inosine (IC50 = 22 microM), ADP (IC50 = 26 microM), and theophylline (IC50 = 5 mM); no inhibition by adenine, hypoxanthine, xanthine, prostaglandin F2 alpha (PGF2 alpha), PGE2, or LH was seen. Cellular products of radiolabeled adenosine uptake were found primarily in the trichloroacetic acid-soluble fraction (88%), and 90% of the radioactivity in this fraction comigrated with adenine nucleotides on electrophoresis; time-dependent incorporation of radioactivity into RNA, DNA, and protein was also seen. Adenosine transport did not appear to be related to the functional state of the luteal cell; for example, no change in the characteristics of uptake was seen in cells obtained from hypophysectomized animals or in cells incubated directly with PGF2 alpha or LH. Adenosine increased cell ATP levels in a dose-dependent manner in parallel with amplification of LH-stimulated cAMP accumulation. A substantial proportion of the total cAMP produced by the cells was derived from extracellular adenosine (40-90%). This response was directly related to the concentration of adenosine, and LH increased the magnitude of cAMP derived from adenosine by about 2-fold. Based on the present studies, adenosine uptake in the luteal cell appears to occur by a dipyridamole-sensitive, phosphorylation-dependent transport system which is independent of pituitary hormones or PG regulation. Moreover, amplification of LH-dependent cAMP accumulation by adenosine appears to be primarily a mass effect due chiefly to utilization of extracellular adenosine by the cell as a prosubstrate for conversion into cAMP by adenylate cyclase.