Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

Abstract
Activation of store-operated Ca2+entry inhibits type 6 adenylyl cyclase (EC 4.6.1.1 ; AC6; Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712–6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC6and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca2+entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca2+inhibition of AC6is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca2+inhibition of AC6. Enzyme activity was assessed using PMVEC membranes, where Ca2+and cAMP concentrations were independently controlled. Endogenous AC6activity exhibited high- and low-affinity Ca2+inhibition, similar to that observed in C6-2B cells, which predominantly express AC6. Ca2+inhibition of AC6in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca2+inhibition of AC6is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC6to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.