Nitric Oxide down regulates the activity of soluble guanylyl cyclase activity

and its biologic significance

Introduction

Guanylyl cyclase (GC) is a family of enzyme that catalyze the formation of the 2nd messenger system cGMP from GTP. GC are subdivided into 2 forms, soluble guanylyl cyclase (sGC) and guanylyl cyclase that are membrane bound and linked to a receptor. Soluble guanylyl cyclase is activated by NO and  membrane bound  GC is activated by peptide hormones

Mammalian sGC is a heterodimer consisting of two subunits, a and b, of which there are four types (a1, a2, b1, b2). Each subunit contains a N-terminal portion, C-terminal cyclase catalytic domain and a central dimerization region. The N-terminal portion constitutes the heme-binding domain and is the least conserved re4gion of the enzyme. Both the a and b subunits are required for the biologic activity of sGC.

It has been noted that cGMP plays a role in the smooth muscle relaxation, platelet aggregation inhibition, and in retinal phosphotransduction. It was also found to participate in signal transduction within the nervous system. cGMP is also involved in regulating water and electrolyte balance as well as bone metabolism. cGMP- signalling is maediated by three different groups of cGMP effector molecules. The cGMP activated protein kinase, the cGMP- regulated phosphodiesterases, and the cGMP- gated ion channel.

Nitric oxide can be synthesized endogenously by NO synthases in a Ca2+ dependent mechanism. Stimulated sGC contains a prosthetic heme group which provides the acceptor cell for NO. NO- heme complex formation leads to a conformational change that can produce an up to 200-fold increase in the activity of the enzyme.

Two isoforms of the NO- sensitive heterodimeric enzyme have already been identified, the ubiquitous  a1b1 and the less broadly distributed a2b1 isoform. Both isoforms show the same regulatory properties but differ in their subcellular distribution. It is the N- terminal of the subunit which is responsible for heme binding and heme coordination. Whereas the cyclase catalytic domains are located in the C- terminal regions. The cyclase catalytic domain is conserved in the membrane bound guanilyl cyclase as well as in the adenylyl cyclase.

Aside from NO, only a few other substances can activate sGC. Carbon monoxide has been reported to bind heme groups wit high affinity but it can only induce enzyme activity marginally, about 3 to 5- folds. An about 10- fold increase of activity has been reported for the NO sensitive GC when using YC- 1 as an activator. YC-1 also induces NO and CO sensitivity of the enzyme. It can also inhibit phosphodiesterases, thus preventing cGMP degradation.

NO is produced indogenously within the vascular endothelium and functions as a natural vasodilator. Discoverers of NO identified it as a signal molecule in the nervous system, at a weapon against infection, and as modulator of blood pressure. This discovery led to the investigation of the role of inhaled NO as a means of treating pulmonary hypertension.

The NO/ cGMP pathway controls several physiologic functions of the nervous system. the effect of NO/cGMP on the survival and differentiation of neurons and synaptic plasticity suggests that this pathway can regulate gene expression.

Discussion

      Prolonged exposure of bovine chromaffin cell with NO led to the desentization of sGC to subsequent NO stimulation and a time dependent decrease on cGMP production. Decrease sGC catalytic activity occurred together with a decrease in sGC protein levels. This brought about a diminished level of the  b1 subunit and the mRNA’s that code for a1 and b1 subunit.

      Studies conducted before had recorded prolonged exposure of cell to NO cause a decrease in the intracellular level of reduced thiols. Reduced glutathione (GSH) is the major intracellular redox buffer in most cells and decrease level of this can lead to inactivation of several proteins.

      To determine if the decreased level of reduced thiol is responsible for the desentization of sGC, a permeable reduced glutathione was added to the set-up. However, the effect of NO on sGC activity was not prevented. Thisa indicated that the intracellular level of GSH  was not affected by the low concentration of NO employed in the experiment. However, results of the studies shows that intracellular level of GSH clearly affects the activity of sGC. This was proven by depleting the cell with GSH by exposing it to an inhibitor of synthesis (BSO) of GSH. The NO- stimulated cGMP increase was significantly diminished both in cells treated with BSO alone and in cells treated with NO plus BSO. Thiol groups participation in sGC activity has been reported previously. The amino acid residue cysteine, contains a thiol group which is important for the structural and functional properties of proteins. The catalytic site of guanylyl cyclase was also found to contain critical thiol groups involved in its activation by NO that are sensitive to oxidative inactivation. This demonstrates that NO activation of sGC leads to disulfide formation which reversibly inactivates the enzyme.

      It was also found that prolonged treatment of chromaffin cells with NO reduce the amount of sGC subunit to b1. process of decreasing  b1 subunit is through a translation- independent mechanism. This was based on the observation that  b1 subunit levels decreased more faster in NO exposed chromaffin cell than those exposed with a drug called cycloheximide. Pretreatment of chromaffin cell with cycloheximide did not prevent the effect of NO on altering the sGC  b1 subunit levels, a greater decrease was noted.

      The half- life of sGC in physiologic conditions is long. It was established to be 55 hours. b1 subunit levels and the a1 subunit level was decreased after the cell is treated for 48- hrs with cycloheximide. This result reveals that the estimated half-life of 55 hrs. reflects that of the  b1 subunit. However, whereas NO treatment of chromaffin cell decreased sGC a1 and b1 subunits mRNA levels, the decrease in protein level noted was only on the  b1 subunit. This may show that a1 subunit has a higher stability than b1 subunit during exposure to NO.

      The decrease in b1 subunit level triggered by long exposure to NO may be explained by the two actions of NO. First, NO decreases the level of mRNA that code for  b1 subunit and second, it destabilizes the  b1 subunit protein, thus making it more susceptible to proteolytic enzymes. Destabilization of the subunit protein can occur in three ways; nitrosylation of cysteine or tyrosine residues, oxidation of the ferrous component of the heme group, and oxidation of different residues but mostly cysteine. Among the three, oxidation may be the most effective method. Destabilization could induce conformational changes in the structure of sGC making it unstable and susceptible to digestion.

The effect  of NO on mRNA protein levels was mediated by cGMP and cGMP- dependent protein kinase (PKG) mechanism. It was suggests that cGMP regulates gene expression of the sGC subunit. The presence of cGMP- dependent protein kinase inhibitors effectively prevented the NO induced down- regulation of the sGC subunit mRNA and partially inhibited the reduction in b1 subunits.

  Conclusion

    In conclusion, prolonged exposure of chromaffin cell leads to a decrease production of cGMP by regulating the activity of soluble guanylyl cyclase and by decreasing subunit mRNA levels through a cGMP- dependent mechanism and by destabilizing b1 subunit. The result also suggests that activation of cGMP- dependent protein kinase mediates the drop in sGC subunit mRNA levels.      

 References

Koesling D, Friebe A (1999) Structure and regulation of soluble guanylyl cyclase. Rev Physiol. Biochem. Pharmacol. 135, 35-41

Wedel, BJ, Garbers DL (2001) The guanylyl cyclase family at Y2K. Annu. Rev. Physiol. 63, 215-233

Moncada S, Higgs EA (1995) Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J. 13: 1319-30

Harrison DG, Bates JN (1993) The nitrovasodilators. New ideas about old drugs. Circulation 87: 1461-1467

 

 

   

 

 

 

   

 

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