Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle.

The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanosine 3':5'-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilation, and septic shock. In the pulmonary bed the opposite response may occur, pulmonary hypertension. We hypothesized that sGC activity becomes downregulated in the face of Escherichia coli lipopolysaccharide (LPS). We tested the effects of LPS on alpha1-subunit sGC mRNA abundance, Western analysis, and enzyme activity in cultured rat pulmonary artery smooth muscle cells. LPS increased extracellular cGMP production by pulmonary artery smooth muscle cells, with increased levels being first detectable at 3-6 h (10 microg/ml LPS) and exceeding 140 pmol/ml by 24 h (P < 0.05). The response was inhibited by 0.05 mM l-NG-monomethyl-l-arginine (l-NMA) and, in turn, restored by 1 mM l-arginine, indicating a NO synthase-dependent response. Pretreating cells with LPS for >/= 3 h inhibited subsequent cGMP synthesis in response to 10(-4) M SNAP for 60 min. Coincubating cells with 0.05 mM l-NMA also reversed this effect. Soluble GC enzyme activity in cells exposed to basal medium alone measured 0.74 pmol cGMP/ml per minute; activity in cells exposed to 10 microg/ml LPS for 24 h decreased to 0.04 pmol cGMP/ml per minute (P < 0.05). LPS pretreatment decreased sGC mRNA abundance and protein mass, but did not totally eliminate them. It is concluded that LPS affects cGMP synthesis at the level of enzyme activity, enzyme mass, and mRNA abundance. Over the short term (<24 h) LPS causes the synthesis of large amounts of cGMP. As the duration of exposure progresses (>/=3 h), mechanisms come into play that decrease cGMP production significantly and include decreases in mRNA abundance, enzyme mass, and enzyme activity.