Loss of neutral endopeptidase activity contributes to neutrophil activation and cardiac dysfunction during chronic hypomagnesemia: Protection by substance P receptor blockade

Prolonged hypomagnesemia (Hypo-Mg) increases cardiovascular risk of morbidity and mortality after an episode of myocardial ischemia, and is common in hospitalized patients. In rodents, Hypo-Mg leads to neurogenic inflammation associated with substance P elevations. Neutral endopeptidase is a proteolytic substance P-degrading enzyme, which is expressed on mature neutrophils. This study investigated the effects of Hypo-Mg on neutral endopeptidase activity and cell activation in male Sprague-Dawley rats that were either fed a modified Mg-deficient diet or a Mg-sufficient diet for up to five weeks.

Abstract

BACKGROUND/OBJECTIVE:

Hypomagnesemia (Hypo-Mg) in rodents leads to neurogenic inflammation associated with substance P (SP) elevations; neutral endopeptidase (NEP) is a principle cell surface proteolytic enzyme, which degrades SP. The effects of chronic Hypo-Mg on neutrophil NEP activity, cell activation and the associated cardiac dysfunction were examined.

METHODS/RESULTS:

Male Sprague-Dawley rats (180 g) were fed Mg-sufficient or Mg-deficient (Hypo-Mg) diets for five weeks. Enriched blood neutrophils were isolated at the end of one, three and five weeks by step gradient centrifugation. NEP enzymatic activity decreased by 20% (P value was nonsignificant), 50% (P<0.025) and 57% (P<0.01), respectively, for week 1, 3 and 5 Hypo-Mg rats. In association, neutrophil basal superoxide (•O₂⁻)-generating activities were elevated: 30% at week 1 (P value was nonsignificant), and fourfold to sevenfold for weeks 3 to 5 (P<0.01). Maximal phorbol myristate acetate-stimulated •O₂⁻ production by Hypo-Mg neutrophils increased twofold at week 5. Also, plasma 8-isoprostane levels were elevated twofold to threefold, and red blood cell glutathione decreased by 50% (P<0.01) after three to five weeks of chronic Hypo-Mg. When Hypo-Mg rats were treated with the SP receptor blocker (L-703,606), neutrophil NEP activities were retained at 75% (week 3) and 77% (week 5) (P<0.05); activation of neutrophil •O₂⁻ and other oxidative indexes were also significantly (P<0.05) attenuated. After five weeks, histochemical (hematoxylin and eosin) staining of Hypo-Mg-treated rat ventricles revealed significant white blood cell infiltration, which was substantially reduced by L-703,606. Echocardiography after three weeks of Hypo-Mg only showed modest diastolic impairment, but five weeks resulted in significant (P<0.05) depression in both left ventricular systolic and diastolic functions; changes in these functional parameters were attenuated by L-703,606.

CONCLUSION:

NEP activity regulates neutrophil •O₂⁻ formation by controlling SP bioavailability. When oxidative inactivation of NEP is prevented by SP receptor blockade, partial protection is afforded against cardiac contractile dysfunction.

Hypomagnesemia (Hypo-Mg), resulting from low Mg intake or from administration of Mg-wasting drugs, is common in hospitalized patients and in patients with acute myocardial infarction (1,2). Prolonged Hypo-Mg is known to increase cardiovascular risk of morbidity and mortality after an episode of myocardial ischemia (3). In experimental animal models (4,5), increased oxidative stress promotes cardiovascular injury during Hypo-Mg. Recent studies (6–8) indicate that neutrophils are activated to produce reactive oxygen species, which may play a key role in contributing to oxidative stress and cardiac inflammation during Hypo-Mg. We have documented that Hypo-Mg results in early and sustained elevations of plasma substance P (SP), which precedes the activation of neutrophils and subsequent cardiomyopathy (4,8–10). SP plays a direct role in the activation of neutrophils (7,8). Neutral endopeptidase (NEP), the principal proteolytic SP-degrading enzyme, is prominently expressed on mature neutrophils (11). In chronic obstructive pulmonary disease, maintaining tissue NEP activity has been reported to protect against pulmonary vascular remodelling in response to chronic smoke exposure and hypoxia (12). Therefore, we hypothesize that NEP inactivation during the course of Hypo-Mg may potentiate SP-mediated systemic inflammation, neutrophil activation and cardiac contractile dysfunction. The relationship between neutrophil NEP status and SP bioactivity remains unclear; we believe that SP-mediated oxidative activity contributes to the loss of neutrophil (and other tissue) NEP activity.

METHODS

Animal dietary models and drug treatment

All animal experiments were performed by adhering to the principles for the care and use of laboratory animals as recommended by the United States Department of Health and Human Services and approved by The George Washington University (Washington, DC, USA) Animal Care and Use Committee. Male Sprague-Dawley rats (180 g) were either fed a modified Mg-deficient diet containing approximately 2 mmol of Mg/kg food, or a Mg-sufficient diet containing 25 mmol of MgO/kg, for up to five weeks (4–9). The SP receptor (neurokinin 1 [NK-1]) blocker – L-703,606 (1 mg/kg/day) – was administered as subcutaneous continuous-release pellet implants at the onset and throughout the dietary period as described (5,7). Plasma aliquots were obtained from blood samples collected by tail bleeding or on the day of sacrifice, and treated with aprotinin (0.016 U/mL) and heparin (358 U/mL) to prevent SP degradation; the plasma aliquots were stored at −80°C.

Neutrophil NEP and superoxide anion activities, and blood oxidative indexes

Neutrophils were isolated from whole blood using a modified ficoll-hypaque gradient method (8,13). Superoxide anion generation by neutrophils with or without 0.125 μg/mL of phorbol myristate acetate (PMA) was measured in Hank’s buffered salt solution (pH 7.8) containing 5 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, and 75 μM cytochrome c with or without 50 μg of superoxide dismutase. Neutrophil NEP enzymatic activity was determined according to the fluorimetric procedure of Miners et al (14), using fluorigenic peptide substrate (R&D Systems, USA) in HEPES buffer (pH 7.4) with or without the specific inhibitor thiorphan. Relative fluorescence intensity was read at excitation of 320 nm and emission of 405 nm. Plasma 8-isoprostane levels were determined according to the instructions provided in the enzyme immunoassay kit from Cayman Chemical (USA) as described previously (13). Red blood cell glutathione was determined enzymatically using the DTNB-glutathione disulfide reductase method described (6–8,13).

Histochemistry and echocardiography

Histochemistry analysis:

Cardiac tissue was rapidly excised, rinsed, quickly embedded in an optimal cutting temperature compound and frozen at −70°C until used (13). Hematoxylin and eosin staining was used to assess the presence of inflammatory infiltrates and lesions.

Echocardiography:

The VingMed System Five (GE Healthcare Canada) was used with a 10 MHz probe (5) on anesthetized animals (2% inhaled isoflurane mixed with 100% oxygen). Aortic and pulmonary artery diameters were measured to calculate stroke volumes. The left ventricular wall thickness and internal diameter were measured to detect dilated or hypertrophic cardiomyopathy. Left ventricular internal diameters in systole and diastole were used to calculate the shortening fraction. Spectral Doppler velocities of the pulmonic and aortic outflows were measured to calculate cardiac output, and the tricuspid and mitral inflows were measured to assess ventricular diastolic function.

Statistics

Data were reported as mean ± SEM of four to six animals per group. Statistical comparisons were evaluated by Student’s t test when only two groups were compared; P<0.05 was considered to be significant. Selected data were analyzed using one-way ANOVA followed by Tukey’s test.

RESULTS

The impact of prolonged Hypo-Mg on neutrophil NEP activity was examined up to five weeks. At the end of week 1, neutrophils obtained from the Hypo-Mg rats displayed only a modest (20%; P value was non-significant) loss of NEP enzymatic activity. However, neutrophils obtained from three- and five-week Hypo-Mg rats exhibited 50% and 57% significantly (P<0.01) lower NEP activity, respectively (Figure 1). Concurrently, the effect of SP (NK-1) receptor blockade on neutrophil NEP activity was assessed; treatment with the specific NK-1 receptor blocker (L-703,606) significantly (P<0.05) attenuated the loss of neutrophil NEP activity due to three or five weeks of Hypo-Mg.

Figure 1).

Time-dependent loss of neutrophil neutral endopeptidase (NEP) activity during hypomagnesemia (Hypo-Mg) and attenuation by substance P receptor blockade (L-703,606 [L]) in Hypo-Mg rats after week (Wk) 1, 3 and 5. Data presented as mean ± SE of four to five rats. *P<0.01 versus MgS control; ^†P<0.05 versus Hypo-Mg alone

The authors previously showed that neutrophils isolated from Hypo-Mg rats were activated after two weeks (6,7). Figure 2A shows that neutrophils isolated from three-week Hypo-Mg rats exhibited higher basal superoxide-generating activity (more than sixfold). At five weeks, the basal activity of the Hypo-Mg neutrophils still remained approximately fourfold to fivefold higher than the control group (Figure 2A); importantly, treatment with L-703,606 at three and five weeks substantially suppressed (approximately 70%) this elevated basal superoxide-generating activity. When PMA (0.125 μg/mL) was included in the assay, all samples were stimulated to produce higher levels of superoxide anion (Figure 2B). However, the neutrophils from the five-week Hypo-Mg rats exhibited a twofold higher activity compared with the stimulated MgS controls. Interestingly, when treated with L-703-606, the PMA-stimulated activity of the five-week Hypo-Mg samples was reduced to control levels (Figure 2B).

Figure 2).

A Comparative basal superoxide-generating activities in neutrophils obtained from MgS and hypomagnesemia (Hypo-Mg) rats after week (Wk) 1, 3 and 5 with or without L-703,606 (L) treatment. *P<0.001 versus MgS control; ^†P<0.01 versus Hypo-Mg alone. B L prevents upregulation of phorbol myristate acetate-stimulated superoxide activity in neutrophils from chronic Hypo-Mg (five weeks). Data presented as mean ± SE of four to five rats. *P<0.01 versus MgS control; ^†P<0.05 versus Hypo-Mg alone

As a key index of systemic oxidative stress, plasma levels of F2-like isoprostanes, which are derived from nonenzymatic peroxidation of poly-unsaturated fatty acids, were determined (13); in Figure 3A, Hypo-Mg alone resulted in a moderate elevation (90% higher) at week 3, which was substantially increased (150% higher) at week 5. Treatment with L-703,606 almost completely suppressed the elevation of 8-isoprostane over the entire duration of Hypo-Mg. Red blood cell glutathione, a key component of systemic antioxidant defense, was depleted by approximately 50% (P<0.01) after three or five weeks of Hypo-Mg (Figure 3B); not surprisingly, administration of the NK-1 receptor blocker significantly attenuated the losses of glutathione by approximately 65%.

Figure 3).

Hypomagnesemia (Hypo-Mg)-induced elevations in plasma 8-isoprostane (A) and loss of red blood cell (RBC) glutathione (GSH) (B); and effects of substance P receptor blockade by L-703,606 (L). A *P<0.01; ^†P<0.05; ^‡P<0.05 versus MgS control; ^§P<0.01 versus Hypo-Mg alone. B *P<0.001 versus MgS control; ^†P<0.01; ^‡P<0.05 versus Hypo-Mg alone. HgB Hemoglobin B

Previously, it was observed that Hypo-Mg for three weeks caused a modest amount of white blood cell (WBC) infiltration in the rat heart (15). In the current study, hematoxylin and eosin staining of the ventricular sections from five-week Hypo-Mg rats (Figure 4) revealed substantial WBC infiltration ranging in pattern from a mainly localized collection of WBCs (Figure 4B), to lesion (necrotic) foci enriched with WBC infiltrates (Figure 4C). In sections from L-703,606-treated Hypo-Mg rats, there was a noticeable decrease of WBC infiltrates in comparison with the five-week Hypo-Mg alone group (Figure 4D).

Figure 4).

Hematoxylin and eosin staining of five-week MgS (A) and hypomagnesemia (Hypo-Mg) (B and C) ventricles indicating that chronic Hypo-Mg promoted white blood cell infiltration and lesion formation, which were partially diminished by L-703,606 (L) (D). Original magnification ×20

The impact of chronic Hypo-Mg on in situ cardiac function in rats was assessed by echocardiography. Three weeks of Hypo-Mg only caused a 20% decline (P<0.05) in the mitral valve early/late ventricular filling velocity ratio – a sign of early diastolic dysfunction (data not shown). However, five weeks of Hypo-Mg led to significant (P<0.05) depressions in left ventricular systolic function (Figure 5 [right]: percentage of fractional shortening decreased by 16.9%) and left ventricular diastolic function (Figure 5 [left]: mitral valve early/late (atrial [A]) ventricular filling velocity ratio decreased by 27.7%) (5). Treatment with the NK-1 receptor blocker during five weeks of Hypo-Mg provided a partial, but significant (P<0.05) functional improvement (44% to 54%) in these parameters.

Figure 5).

Effect of five weeks of hypomagnesemia (Hypo-Mg) on echocardio-graphic parameters of Hypo-Mg rats with or without neurokinin 1 (NK-1) receptor blockade (L-703,606 [L]). % FS Percentage of left ventricular (LV) fractional shortening; E/A Mitral valve early/late (atrial [A]) ventricular filling velocity ratio. Data presented as mean ± SE of four to five rats, and expressed as percentage changes versus MgS control; *P<0.05 versus MgS; ^†P<0.02 versus Hypo-Mg alone

DISCUSSION

Chronic Hypo-Mg can potentially be regarded as a neurogenic inflammatory disorder due to the involvement of SP. Using experimental rodent models, we previously documented that significant elevations of circulating SP occurred after one week of diet-induced Hypo-Mg, which likely reflected its release from neuronal C-fibres (4,5,9,10,16). More recently, we observed that chronic Hypo-Mg (up to five weeks) led to sustained elevations (approximately 6.5-fold higher versus control) of circulating SP (5), which were likely derived from new SP synthesis (16), and as the consequence of progressive loss of NEP activity (Figure 1). In response to heightened SP levels, upregulation of SP receptors was observed in circulating T lymphocytes (17) and neutrophils (18), as well as in the heart (19). Sustained elevations in neutrophil-free radical generation (Figure 2), systemic lipid peroxidation (Figure 3) and cardiac inflammation (Figure 4) occurred; this oxidative/inflammatory stress ultimately progressed to cardiac contractile dysfunction (Figure 5). The protective effects afforded by the NK-1 receptor blocker (L-703,606) observed in the present study and in our previous studies (4,9,20) indicate that SP plays an important role not only in the initiation of the inflammatory cascade, but also in the amplification of the systemic neurogenic inflammation during chronic Hypo-Mg (up to five weeks). As an ex vivo paradigm of inflammatory cellular activity in response to SP during chronic Hypo-Mg, we showed that neutrophils from the five-week Hypo-Mg rats displayed not only heightened basal activity, but also elevated PMA-stimulated superoxide anion-generating activity; this suggests that the neutrophil NADPH oxidase system was upregulated in addition to being endogenously activated. Because NEP is readily susceptible to inactivation by reactive oxygen species and peroxynitrite (21,22), we suggest that the neutrophil-derived reactive oxygen species, and perhaps reactive nitrogen species (23), may inactivate NEP, and that this process of autoinactivation is attenuated by the SP receptor blockade. When neutrophil NEP activity was at least 80% intact, as seen in neutrophils from one-week Hypo-Mg rats (Figure 1), no significant elevation in superoxide production was observed (Figure 2). However, as shown in our previous study (8), when rats were treated with the NEP inhibitor phosphoramidon (5 mg/kg/day for one week), which inhibited the neutrophil NEP activity by 50%, these one-week Hypo-Mg neutrophils displayed a fourfold higher basal superoxide-producing activity (8). The combined results support the notion that NEP on the neutrophil surface plays an important role in controlling the functional level of SP that is accessible to its NK-1 receptor and thereby modulates SP stimulatory activity.

We also found that five weeks of Hypo-Mg led to prominent WBC infiltration in the ventricles, which was associated with a 50% loss in cardiac NEP protein (24). This elevated infiltration may be a reflection of SP (chemoattractant)-mediated recruitment of WBCs to the tissue inflammatory/lesion sites during chronic Hypo-Mg. We propose that this enhanced WBC infiltration contributed, in part, to significant impairment of contractile function. A causal role for the SP/NK-1 receptor activation and the subsequent development of cardiomyopathy and ventricular dysfunction was confirmed by the protective effects provided by L-703,606. In summary, the following related scheme of events is depicted below (Appendix 1):

Appendix 1)

Sequences of events leading to substance P (SP)-mediated neutral endopeptidase (NEP) inactivation, neutrophil activation and eventual cardiac dysfunction during chronic hypomagnesemia. CGRP Calcitonin gene-related peptide; NK-1 Neurokinin 1; RNS Reactive nitrogen species; ROS Reactive oxygen species; WBC White blood cell