Inhibition of ferrochelatase impairs vascular eNOS/NO and sGC/cGMP signaling.

Bin Zhang,Michael S Wolin,An Huang,Dong Sun,Wensheng Deng,Sharath Kandhi,Norah Alruwaili

doi:10.1371/journal.pone.0200307

Abstract

Ferrochelatase (FECH) is an enzyme necessary for heme synthesis, which is essential for maintaining normal functions of endothelial nitric oxide synthase (eNOS) and soluble guanylyl cyclase (sGC). We tested the hypothesis that inhibition of vascular FECH to attenuate heme synthesis downregulates eNOS and sGC expression, resulting in impaired NO/cGMP-dependent relaxation. To this end, isolated bovine coronary arteries (BCAs) were in vitro incubated without (as controls) or with N-methyl protoporphyrin (NMPP; 10−5–10-7M; a selective FECH antagonist) for 24 and 72 hours respectively. Tissue FECH activity, heme, nitrite/NO and superoxide levels were sequentially measured. Protein expression of FECH, eNOS and sGC was detected by western blot analysis. Vascular responses to various vasoactive agents were evaluated via isometric tension studies. Treatment of BCAs with NMPP initiated a time- and dose-dependent attenuation of FECH activity without changes in its protein expression, followed by significant reduction in the heme level. Moreover, ACh-induced relaxation and ACh-stimulated release of NO were significant reduced, associated with suppression of eNOS protein expression in NMPP-treated groups. Decreased relaxation to NO donor spermine-NONOate reached the statistical significance in BCAs incubated with NMPP for 72 hours, concomitantly with downregulation of sGCβ1 expression that was independent of heat shock protein 90 (HSP90), nor did it significantly affect BCA relaxation caused by BAY 58–2667 that activates sGC in the heme-deficiency. Neither vascular responses to non-NO/sGC-mediators nor production of superoxide was affected by NMPP-treatment. In conclusion, deletion of vascular heme production via inhibiting FECH elicits downregulation of eNOS and sGC expression, leading to an impaired NO-mediated relaxation in an oxidative stress-independent manner.

Highlights

We provided direct evidence indicating that endothelial nitric oxide synthase (eNOS) and soluble guanylyl cyclase (sGC) are two independent targets for N-methyl protoporphyrin (NMPP) that inhibits FECH-dependent heme synthesis to downregulate eNOS and sGC expression, leading to impaired NO/sGC/cGMP-mediated vasodilator responses, which are independent of vascular oxidative stress
Accumulating evidence has indicated an oxidationdependent alteration of FECH/heme/sGC signaling in vasculatures, characterized as impaired NO-mediated responses [12,16,18,24,28,29,30], two important points have not been unaddressed yet, as what is direct effect of deleting FECH/heme on eNOS expression, and 2) whether an alternative mechanism independent of oxidative stress is involved
The present study was focused on direct correlation between the FECH/heme and two heme-dependent enzymes, via assessing eNOS and sGC protein expression and activity, as a function of NMPP-inhibition of FECH/heme without interruptions from oxidative stress

Summary

Introduction

While an in vivo inhibition of heme synthesis initiated significant reductions in systemic nitrite/nitrate excretion and renal NOS activity of rats [19], it is worth noting that the in vivo intervention of heme synthesis can initiate systemic changes in multiple signal pathways and/or molecules involved, such as oxidations and antioxidative defense or iron-sulfur cluster scaffold proteins and components of the electron transport chain, etc., all of which may not necessarily be direct consequences of FECH inhibition or heme loss, but rather a biological complexity that links multiple events operating in concert to elicit responses observed. Most previous studies were focused on oxidative stress that disrupts heme/NO/sGC/cGMP signaling involving both FECH/heme-dependent and -independent mechanisms To this end, the present study aimed to test the hypothesis that the inhibition of FECH, followed by heme deficiency [14] directly downregulates eNOS and sGC expression to alter NO/cGMP-mediated responses

Methods

Results

Conclusion