Neutral endopeptidase inhibition enhances substance P mediated inflammation due to hypomagnesemia

Abstract

During dietary deficiency of magnesium neurogenic inflammation is mediated, primarily, by elevated levels of substance P (SP). The enzyme most specific for degrading this neuropeptide is neutral endopeptidase (NEP). In recent studies we found that pharmacological inhibition of NEP by phosphoramidon resulted in elevated plasma levels of SP and greater oxidative stress. We also observed that hypomagnesemia reduced cardiac and intestinal expression of NEP. In these magnesium-deficient rats increased intestinal permeability and impaired cardiac contractility occurred. In our colony of genetically-engineered NEP knockout mice that have reduced ability to degrade SP, we found increased oxidative stress that was prevented by SP (neurokinin-1) receptor blockade. Thus, we submit that inhibition of NEP by pharmacological, genetic and dietary approaches (magnesium restriction), causes greater neurogenic inflammation that may result in increased intestinal and cardiac dysfunction.

Figure 1

Three weeks of hypomagnesemia decreases circulating neutrophil NEP activity (assayed using a fluorogenic peptide substrate) and increases basal superoxide anion generation (assayed by SOD-inhibitable cytochrome c reduction) in rats. Assay methods for can be found in reference . Values are means ± SE of 4–6 rats. *p<0.05, **p<0.01 vs MgS control.

Figure 2

(A) Immunohistochemical staining for NEP in heart (upper panels) and small intestine (lower) of 3 week MgS (left) & MgD (right) rats. Rabbit anti-NEP & FITC were used. (Mag. 20×). (B) Quantification (pixel number) of diminished NEP staining in the small intestines (A: lower panels) of 3 week MgD rats is evident (49 % decreased vs MgS, p<0.05). Values are means ± SE of 5.

Figure 3

NEP protein content in 3 and 5 week MgS and MgD rat cardiac ventricles. Homogenized tissue protein (70 μg) was separated by SDS-page gel electrophoresis; transferred to PVDF membranes; probed with rabbit anti NEP antibody; incubated with donkey anti-rabbit; developed with ECL-plus. Values are means ± SE of 3–5. **: p<0.01 vs MgS; †: p=0.0002 for 5 week MgD vs 3 week MgD.

Figure 4

Circulating substance P (SP) (A) and RBC glutathione (B) levels in hypomagnesemic and control mice at dietary week 2. SP measurements were made by chem.-ELISA and data is modified from reference . The procedure for RBC glutathione measurements is described in reference . Values are means ± SE of 3–5 mice. ** p<0.01, *** p<0.001 vs MgS.

Figure 5

Effect of NK-1 blockade on RBC glutathione levels in B6 [NEP(+/+) or wildtype] or NEP knockout [NEP(−/−)] mice on 1 week control (MgS) or low Mg (MgD) diets. Mice received L-703,606 (1.5 mg / kg / day, s.c. pellet) or placebo. The procedure for RBC glutathione measurements is described in reference . Values are means ± SE of 4–5 mice. * p<0.05 vs untreated MgS; + p<0.05 vs untreated MgD.