Abstract
Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.
Highlights
Efflux of CAMP occurs in many different cell types
Several properties of efflux as it occurs in avian erythrocytes were presented in the original report by Davoren et al [3], namely it continued in the presence of sufficient antagonist to cause a sharp drop in cellular CAMP, it was inhibited by probenecid and by caffeine, and it did not occur at 0 “C. Adherent cell lines as well as avian erythrocytes have been used to characterize further the properties of efflux
Cellular nucleotides were labeled with [3H]adenosine, and the cells were stimulated with 100 PM adenosine; in the first experiment, 1.6%, and, in the second, less than l%, of total labeled nucleotide plus adenosine appeared in the medium over a period of 15 min
Summary
Fig. lA shows a time course of change in intracellular and extracellular CAMP in response to 20 pM adenosine. A plot of extracellular CAMP uersm the time integral of intracellular CAMP for the time course shown in Fig. 1A is linear, with an apparent first-order rate constant of O.O25/min. The ‘; rate constant for efflux for the time course shown in Fig. 2 was O.O57/min in the absence of probenecid and too low to. The points fit a rectangular hyperbola, with an EC& for adenosine of 14 pM These data suggest that the efflux process itself is regulated by the occupancy of an adenosine receptor independent of intracellular CAMP concentration. Sine is consistent with the interpretation of efflux as an A, receptor-mediated event [31]
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