Nitric oxide-independent down-regulation of soluble guanylyl cyclase by bacterial endotoxin in astroglial cells

Abstract

Induction of nitric oxide (NO) synthase (NOS) type 2 (NOS-2) in glial cells after exposure to bacterial endotoxin [lipopolysaccharide (LPS)] or inflammatory cytokines has been repeatedly demonstrated both in vitro and in vivo. However, little is known about effects of these agents on NO-dependent cyclic GMP (cGMP) formation. In this work, we show that treatment of rat cerebellar astrocyte-enriched primary cultures with LPS decreases NO donor- stimulated cGMP formation with a similar initial time course (up to 9-12 h) and concentration dependency (0.1-1 ng/ml) as for induction of NOS-2. This effect appears to be due to a down-regulation of soluble guanylyl cyclase (sGC) because LPS treatment decreases sGC activity and sGC β1 subunit levels. In contrast, cGMP phosphodiesterase activity and stimulation of the particulate guanylyl cyclase by atrial natriuretic peptide are not affected. Incubation of astroglial cultures with a transcription inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) for 18-20 h does not decrease sGC activity but totally prevents LPS-induced desensitization of sGC. Inhibition of NOS-2 activity with N(G)-monomethyl-L-arginine or inhibition of NOS-2 induction with the synthetic glucocorticoid dexamethasone failed to prevent the inhibitory effect of LPS on sGC, indicating that NO production is not involved. Moreover, after removal of LPS the time for recovery of sGC responsiveness is much longer than that for NOS-2 return to basal levels. LPS impairment of CGMP formation also occurs in cortical astrocytes but not in cerebellar granule neurons. The decreased responsiveness of sGC to NO stimulation following LPS challenge may prevent inappropriate astroglial cGMP signaling caused by excess production of NO by adjacent activated glial cells.