JBC, Vol. 251, Issue 14, 4239-4246, Jul, 1976
Regulation of adenylate cyclase from cultured human cell lines by adenosine
R. B. Clark and M. N. Seney
Broken cell particulate preparations of adenylate cyclase isolated from the
human glioma cell line 132-1N1 were stimulated 2-to 3-fold by 30 muM
adenosine. This concentration of adenosine produced a maximal stimulation
of the cyclase while 3 to 5 muM adenosine produced half-maximal
stimulation. Theophylline, at 40 muM, inhibited the adenosine stimulation
of the adenylate cyclase by about 40% while 200 muM produced near complete
inhibition. The inhibition by theophylline could be overcome by increasing
adenosine to a concentration 10-fold that of theophylline, implying that
the inhibition was competitive. Basal activity was not inhibited by even
1.0 mM theophylline, nor was the epinephrine stimulated activity. In
contrast, 1.0 muM propranolol essentially completely inhibited the 8-fold
stimulation of 1.0 muM epinephrine but had no effect on either basal or
adenosine-stimulated activity. Adenosine and 2-chloroadenosine were
equipotent in stimulating adenylate cyclase from the 132-1N1 line, whereas
neither adenine nor guanosine had any detectable effect. GTP, 10 muM,
produced a small variable stimulation of the adenylate cyclase while the
GTP analogue, 5'-guanylylimidodiphosphate (Gpp(NH)p), produced a marked
stimulation fo the cyclase. Preincubation of the adenylate cyclase
preparation with the analogue greatly increased its potency and maximal
effect. In contrast, both basal and adenosine-stimulated activity decreased
markedly with preincubation. The effects of adenosine or epinephrine in
combination with Gpp(NH)p were at least additive and often synergistic in
comparison to the effects of the compounds alone. The effects of adenosine
on intact and broken cell preparations of the human fibroblast lines WI-38
and VA13-2RA were also examined. In the intact VA13-2RA, adenosine produced
rapid and large increases in intracellular and extracellular cyclic
adenosine 3':5'-monophosphate (cAMP). In the parental fibroblast line, the
WI-38, adenosine slightly elevated basal levels of cAMP, but only produced
marked elevations in the presence of non-methylxanthine phosphodiesterase
inhibitors. The effect of adenosine on the broken cell particulate
preparations of adenylate cyclase from the fibroblasts was similar to its
action on the cyclase from the 132-1N1; 30 muM adenosine produced a maximal
stimulation of the adenylate cyclase, and the stimulation was inhibited by
theophylline.
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