Stimulation of soluble guanylyl cyclase (sGC) activity by NO produces different degrees of relaxation in pulmonary veins (PV) and arteries (PA) isolated from control lambs (C) and from lambs with PPHN following surgical ligation of the ductus arteriosus (L). We previously reported a rank order for relaxations to NO and NO donors of CPV = LPV >> CPA >> LPA. The current study examined whether relaxations to NO corresponded to levels of specific subunits which make up the sGC heterodimer. Western blot analysis was used to determine protein content for the α1, α2, and β1 subunits of sGC in third generation PA and PV isolated from lambs delivered 9 days following ductal ligation (n=4) and their twin controls (n=4). 50 μg of the total protein from the cytosolic fraction of each vessel homogenate was separated by gel electrophoresis and transferred to a polyvinlyidene difluroide membrane. Immunodetection was performed using a monoclonal antibody for the α1 subunit (H6 clone from Dr. F Murad) and a polyclonal antibody for the β1 subunit which cross reacts with the α2 subunit (Alexis, CA). A single band corresponding to the α1 subunit (73 kDa) was detected by the H6 antibody; and 2 major bands corresponding to the α2 (79 KDa) and β1 (70 kDa) subunits were detected by the polyclonal antibody. Bands were analyzed by densitometry and mean OD values are shown in the table. (* P <0.05 CPV vs CPA; ** CPA vs LPA by ANOVA). We conclude that content of the α1 subunit of sGC corresponds well with responsivity to NO. An inverse relationship appears to exist for theα2 subunit. We speculate that increased expression of the α2 subunit in LPA may produce a less catalytically active sGC heterodimer.

Table 1 No caption available.