Contribution of phosphodiesterase isoenzymes and cyclic nucleotide efflux to the regulation of cyclic GMP levels in aortic smooth muscle cells

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cycli c monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cell s. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmo dulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monop hosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE comple ment, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in th e presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smoo th muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 mu M zaprinast, a concentra tion that selectively inhibits PDES, did not potentiate the SNP- or ANF-ind uced rise of cGMP, questioning the widespread opinion that only PDE5 accoun ts for cGMP hydrolysis in this tissue. Further evidence came from experimen ts assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at conc entrations inhibiting both type 1 and type 5 isoenzymes, in which this pote ntiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigat ed. Shortly after guanylate cyclase activation, extracellular cGMP levels s urpassed intracellular levels. However, comparison of the amounts of cGMP e xtruded to the extracellular medium with those degraded by PDEs leads to th e conclusion that efflux is of relatively minor importance in regulating in tracellular cGMP levels. In cells made tolerant to SNP, selective PDES inhi bition synergistically increased intra- and extracellular cGMP amounts afte r SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells . (C) 1999 Elsevier Science Inc.