Inhibition of neutral endopeptidase potentiates neutrophil activation during Mg-deficiency in the rat

Abstract

Neutral endopeptidase (NEP), which degrades substance P (SP), may regulate neutrophil activation during Mg-deficiency (MgD). Male Sprague-Dawley rats (180g) were fed MgD (approximately 50 mg Mg/kg) or Mg-sufficient (MgS, 608 mg Mg/kg) diets for 7 days +/- NEP inhibitor phosphoramidon (PR, 5 mg/kg/day, s.c.). MgD alone induced a 9-fold (vs. MgS, p <0.01) elevation in plasma SP; MgD+PR enhanced it further to 18-fold (p <0.001). Neutrophils from MgD+PR rats displayed a 3.9-fold higher (p <0.01) basal .O(2-) generation, but those from MgD or PR alone were not activated. Plasma PGE2-metabolite levels rose 2.67- (p <0.01) and 1.56- (p <0.05) fold, respectively, in MgD+PR and MgD groups; the corresponding red blood cell glutathione levels were decreased 21% (p <0.025) and 7% (NS). MgD+PR significantly reduced neutrophil NEP activity by 48% (p <0.02); PR or MgD alone only reduced this activity 26% and 15%, respectively. We conclude that NEP inhibition potentiates SP-mediated neutrophil .O(2-) production and may promote other inflammatory activities during MgD.

Fig. 1

Effect of the neutral endopeptidase inhibitor, phosphoramidon (PR: 5 mg/kg/day, s. c.) on total plasma SP levels (colorimetric ELISA) during week 1 of MgD treatment. After normalizing to baseline (0.447 ng/ml), time-course data were integrated over the specified time. Values are means ± SEM from 4–5 rats/group; **p <0.01, ***p <0.001 vs MgS; # p <0.025 vs MgD alone.

Fig. 2

Influence of NEP inhibition by phosphoramidon on basal generation of superoxide anions in rat neutrophils. The rats were fed MgS or MgD diets for 7 days with or without receiving PR as s. c. implantation for the entire period. The neutrophils were isolated from whole blood by step gradient centrifugation; basal superoxide anion production was determined as SOD-inhibitable cytochrome c reduction. Results are means ± SEM of 4–6 animal samples; ** p <0.01 vs. MgS and, # p <0.025 vs. MgD alone.

Fig. 3

The relative NEP enzymatic activities in neutrophils isolated from MgS or MgD rats (7 days) with or without PR treatment. NEP activity on intact neutrophils were estimated as thiorphan-inhibitable relative fluorescence (excitation = 320 nm, emission = 405 nm). Values are means ± SEM from 4–6 samples; *p < 0.05 vs. MgS control and MgD alone.

Fig. 4

Effects of NEP inhibition by PR on (A) circulating PGE₂-metabolite (PGEM) level and (B) RBC glutathione content. PGEM content in plasma samples were determined by an EIA kit from Cayman Chemical and RBC glutathione was determined by an enzymatic “recycling” method. Values are means ± SEM from 4-6 samples; * p <0.05, **p <0.01 vs. MgS controls; + p <0.05 vs MgD alone or MgS+PR.