REGULATION OF VASCULAR SMOOTH-MUSCLE SOLUBLE GUANYLATE-CYCLASE ACTIVITY, MESSENGER-RNA, AND PROTEIN-LEVELS BY CAMP-ELEVATING AGENTS

Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation o f gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene express ion in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulat ed sGC activity and to correlate activity changes with mRNA and protei n levels in cultured rat aortic smooth muscle cells. Pretreatment of c ells with 50 to 1000 mu mol/L isobutylmethylxanthine or 0.01 to 10 mu mol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-s elective inhibitor (H89 or KT 5720) partially or fully prevented the d ownregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating, agents. Quantification of reverse transcriptase-po lymerase chain reaction products by high-performance liquid chromatogr aphy revealed that mRNA for both alpha(1)- and beta(1)-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and fo rskolin but not with dideoxyforskolin (inactive analogue). Moreover, p rotein levels for the sGC alpha(1)-subunit of cells pretreated with is obutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate t hat cAMP-elevating agents decrease sGC activity, possibly by decreasin g mRNA or protein levels or both.