Abstract
Nitric oxide (NO) modulates neurotransmission in the central and peripheral nervous systems. NO acts, in part, by stimulating cGMP production by soluble guanylate cyclase (sGC), an obligate heterodimer composed of alpha and beta subunits. To investigate mechanisms that regulate responsiveness to NO in the nervous system, sGC regulation was examined in a rat pheochromocytoma cell line (PC12) exposed to nerve growth factor (NGF). NGF decreased sGC alpha1 and beta1 subunit mRNA and protein levels as well as NO-stimulated sGC enzyme activity. The NGF-mediated decrease in sGC subunit mRNA levels was blocked by 5'-deoxy-5'-methylthioadenosine (an inhibitor of NGF-induced tyrosine phosphorylation). NGF did not decrease sGC subunit mRNA levels in PC12 cells containing a mutant Ras protein that blocks Ras-dependent intracellular signaling. Incubation of PC12 cells with a transcription inhibitor (actinomycin D) or protein synthesis inhibitors (anisomycin or cycloheximide) attenuated the ability of NGF to decrease sGC subunit mRNA levels. Moreover, sGC subunit mRNA levels decreased more rapidly in NGF-treated cells than in actinomycin D-treated cells, suggesting that NGF decreases sGC subunit mRNA stability. Thus, NGF decreases sGC subunit mRNA levels via mechanisms that are dependent on protein tyrosine phosphorylation and Ras activation. The effect of NGF on sGC subunit mRNA stability appears to be transcription- and translation-dependent. Modulation of sGC subunit levels and enzyme activity in PC12 cells suggests that NO responsiveness may be regulated in the nervous system by NGF.
Highlights
In central and peripheral nervous systems, nitric oxide (NO)1 has an important role as a physiologic messenger molecule [1, 2]
To determine whether the mechanisms involved in the regulation of soluble guanylate cyclase (sGC) subunit gene expression are tyrosine phosphorylation-dependent, sGC subunit mRNA levels were measured in PC12 cells pretreated with 3 mM MeSAdo [21]
Destabilization of sGC Subunit mRNAs by nerve growth factor (NGF) Is Dependent on Gene Transcription—To investigate the role of mRNA stability on the effect of NGF on sGC subunit mRNA levels, we examined the effect of actinomycin D, an RNA polymerase inhibitor, on sGC subunit gene expression in PC12 cells
Summary
Materials—NGF (2.5 S, isolated from mouse submaxillary glands) was purchased from Collaborative Biomedical Products (Bedford, MA). Cell Culture—PC12 rat pheochromocytoma cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 culture medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 112 units/ml penicillin, and 112. Filters were blocked in phosphate-buffered saline containing 5% nonfat milk at room temperature for 1 h and incubated with an antiserum directed against the rat ␣1 sGC subunit The concentration of cGMP in the reaction mixture was measured using a commercial radioimmunoassay kit (Biomedical Technologies Inc., Stoughton, MA). sGC enzyme activity is expressed as pmol of cGMP produced/min/mg of protein in the cell extract supernatant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
