Suppression of neutrophil and endothelial activation by substance P receptor blockade in the Mg‐deficient rat

ARTICLE

Auteur(s) : I. Tong Mak, Jay H. Kramer, William B. Weglicki.

The George Washington University Medical Center, Department of Physiology and Experimental Medicine, Washington, DC 20037, USA

Introduction

Increased oxidative stress is involved in cardiovascular injury during Mg-deficiency in rodent models [1, 2]. Recent studies from our laboratory [3], as well as from others [4], suggest that neutrophils are activated to produce reactive oxygen species which may play a key role in contributing to the oxidative stress during Mg-deficiency. In addition, increased nitric oxide (NO) production suggested endothelial activation in response to Mg-deficiency [5, 6]. However, the signal pathway mediating the white cell and endothelial responses to Mg-deficiency remains to be clarified. We previously reported that dietary Mg-deficiency resulted in early elevation of the neuropeptides, substance-P (SP) and calcitonin gene-related peptide (CGRP), in the circulation followed by increased tissue accumulation within the myocardium. [7] We also presented evidence that SP may play a key role in modulating the inflammatory events leading to cardiac lesion development during the progression of Mg-deficiency. [7, 8] In the present study, the direct contribution of SP towards neutrophil and endothelial activation was examined in Mg-deficient (MgD) rats. The subsequent effects on systemic indices of oxidative stress and post-ischemic heart NO production were also studied.

Materials and methods

Animal model and Neutrophil isolation: All animal experiments were guided by the principles for the care and use of laboratory animals as recommended by the US Department of Health and Human Services and approved by the The George Washington University Animal Care and Use Committee. Male Sprague-Dawley rats (180 gm) were fed either a modified diet (Teklad Laboratory, Madison, Wisconsin) containing 2 mmole Mg²⁺/kg diet (Mg-deficient group, 9% Recommended daily allowance[RDA]); [1-3, 6] or the same diet supplemented with 25 mmoles magnesium oxide/kg diet (Mg-sufficient group, 100% RDA). Animals were placed on the diets for up to three weeks. The SP-receptor blocker (SPB) specific for the neurokinin-1 (NK-1) receptor, L-703,606 (RBI, Natick, MA) was administered s.c. as implanted sustained-release pellets (formulated by Innovative Research, Sarasota, Fl) at a dose of 1 mg/kg/day for 21 days. Neutrophils were isolated from the whole blood using a modified ficoll-hypaque reagent (NIM.2 neutrophil isolation media, Cardinal Associates Inc. Santa Fe, NM) which is essentially a step-gradient centrifugation procedure. [9] The neutrophil band (the lower band) was retrieved, washed and recentrifuged in PBS. Based on morphological assessment, the purity of the isolated neutrophils were found to be > 85%. Superoxide anion production by neutrophils, with or without challenge by 0.25 µg/ml phorbol myristate acetate (PMA), was assayed in the neutrophil suspensions (0.5-0.75 × 10⁶ cells/ml) in Krebs-Ringer phosphate buffer (pH 7.8) containing 5 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, 75 uM cytochrome c ± 50 ug superoxide dismutase. The release of superoxide anion was estimated as superoxide dismutase (SOD)-inhibitable reduction of cytochrome c using the extinction coefficient:E₅₅₀ = 2.1 × 10⁴M ^–1cm^–1.

Measurements of Plasma NO and PGI₂ products: Total plasma NO products (nitrate + nitrite) were first converted to nitrite by E. coli nitrate reductase as described, [6] and nitrite was determined by the Griess reagent method (0.1% naphthylethylenediamine 2HCl and 1% sulfanilamide in 5% phosphoric acid). Plasma PGI₂ was measured by its stable breakdown product, 6-keto-PG-F_1α, which was determined by competitive inhibition of ³H-6-keto-PG-F_1α binding to anti-6-keto-PG-F_1α using labeled tracers, standards and antiserum from Amersham [10]. Total RBC glutathione was determined enzymatically by the DTNB-GSSG reductase method as described. [6, 11]

Isolated Post-ischemic Rat Heart Model: The perfused working heart and global ischemia/reperfusion (I/R) models have been described. [12] Hearts (non-paced) from L-703,606 -treated and -untreated animals were exposed to 30 min of non-recirculating stabilization perfusion with physiologic Krebs-Henseleit buffer containing 1.2 mM MgSO₄ and 5.0 mM glucose (gassed with 95% O₂: 5% CO₂, pH 7.4, 37°C). Hearts were then subjected to global low-flow ischemia (0.2 ml/min coronary flow rate) for 30 min, and then 15 min of reperfusion. During reperfusion, 0.5 ml from sequentially-collected effluent samples were pooled (5.5 ml total), the volume was reduced to 1.0 ml (HetoVAC VR-1, High Technology of Scandinavia, Denmark), and then analyzed for NO formation (as oxidation product nitrite) using the Griess reagent method. After normalizing to preischemic levels, values were reported as net nitrite content due to 15 min of reperfusion after adjustment for total postischemic effluent volume.

Results were expressed as means with standard deviations unless otherwise stated. The statistical significance (p < 0.05) of differences between means was determined by Student's t test.

Results

Neutrophil activation and effect of the SP-receptor blocker

Previously, we observed that neutrophils isolated from the MgD rats were endogenously activated and that maximum activation occurred during the 3^rd week of the MgD diet. [3] The results in Figure 1A confirm that neutrophils isolated from MgD rats (3 weeks) displayed a 10-fold higher (p < 0.001) basal superoxide generating activity compared with the MgS controls. More importantly, in vivo treatment with L-703,606 effectively attenuated this elevated basal activity of neutrophils by 75%. However, the L-703,606 had no effect on the activity of the MgS samples.

As expected, neutrophils from MgS rats were quite responsive to the addition of PMA (0.25 µg/ml) and generated an 11-fold higher superoxide producing activity (15 ± 3 nmol/10 min/10⁶ cells, Figure 1B); however, under our assay conditions, the neutrophil samples from MgD rats did not respond further to stimulation by the PMA challenge (17 ± 4 nmol). Nevertheless, the response of the MgD neutrophils to PMA challenge, after the SP-receptor blockade was comparable to that of the MgS cells (Figure 1B).

L-703,606 effects on nitric oxide formation in plasma and in perfused hearts

Our previous study indicated that Mg-deficiency resulted in increased systemic nitric oxide production, as suggested by increased plasma nitrate + nitrite levels. [3, 6] In the present study, 3 week MgD plasma samples displayed a nitric oxide product level of 33.6 ± 4 µM, which was 2.2-fold higher than the level of MgS animals (15.7 ± 3 µM) (Figure 2A). Treatment with the SPB reduced the elevated nitrate + nitrite level to 22 ± 3.3 µM in the MgD animals; the SPB had minimal effects on the NO product level of the MgS group.

L-703,606 effects on plasma 6-keto-PGF_1α and red cell glutathione

Increased NO synthesis is suggestive of endothelium activation during MgD. When synthesis of NO and PGI₂ by the endothelium occur together, optimal vasodilation results. In this study, we found that PGI₂ production, as assayed by the stable breakdown product 6-keto-PGF_1α, was elevated > 10-fold during the 3^rd week of the MgD diet (Figure 3); more strikingly, we found that treatment with the SPR blocker attenuated this prostanoid product to the same degree observed for the NO products in the plasma. (Figure 2A).

Dietary Mg-deficiency results in oxidative depletion of RBC glutathione in rodent and swine models. [2, 7, 8, 14, 15] In the present study, RBCs obtained from MgD rats exhibited a 55% decrease in total glutathione (MgS: 1.85 ± 0.32 vs MgD: 0.83 ± 0.25 umol/ml packed RBCs, p < 0.01); SP-receptor blockade substantially limited the glutathione loss to about 16% (1.55 ± 0.23 umol/ml RBCs, p < 0.025 vs MgD untreated). Again, treatment with the SPB had no effect on the glutathione level of the MgS controls (1.83 ± 0.35 umol/ml RBCs).

Discussion

During the progression of Mg-deficiency, dramatic increases in circulating levels of inflammatory cytokines (IL-1, IL-6, TNF-α) begin at day 12 and reach their highest levels during the 3^rd week of deficiency; however, time course studies demonstrated significant elevations of circulating levels of SP and CGRP after only 3 days on the MgD diet. [7, 8, 16, 17] A second and more sustained SP peak also appeared during the second and third weeks on the MgD diet [7]. SP is known to have multiple pro-inflammatory properties including stimulation of NO production from the endothelium and promotion of oxy-radical production from a number of cells including leukocytes, macrophages and endothelial cells. [18] In an earlier study, [19] it was described that SP also promoted chemotactic activity in neutrophils isolated from rats. We have previously reported that neutrophils are activated during the 2^nd and 3^rd weeks of MgD and that NO products were elevated 2-3-fold during the same time frame [3]. We postulated that both cellular free radical generation and nitric oxide production occurred subsequent to SP stimulation. [20] SP may directly invoke its effects on the neutrophil by binding to the NK1 receptor which is linked to the G-protein/phospholipase C- mediated IP3 and diacyglycerol generation (from PIP2) resulting in biphasic increases in [Ca]i. [21] Superoxide production by the NADPH oxidase is then stimulated in a Ca-dependent manner. [22] A previous study using human neutrophils indicated that the SP- stimulated.O₂^– production was blocked by either a NK1 receptor blocker or by an intracellular Ca²⁺ chelator, suggesting that an increase in [Ca]i is a prerequisite. [21] In the present study, we report that treatment of the MgD animals with the SP-receptor blocker, L-703,606, effectively prevented the activation of the neutrophils supporting the notion that the neuropeptide SP may directly activate the neutrophil through the NK1 receptor/IP3/Ca signaling pathway. [21, 22] In data not shown, we observed that incubation of MgS neutrophils with 25 µM of SP for 30 min resulted in a modest stimulation (2.3-fold increase) in superoxide anion production. However, SP at concentrations up to 50 µM did not further stimulate the MgD neutrophils. Presumably, the MgD cells, which were already activated, could not respond further to any applied SP stimulation.

Mg-deficiency also promotes increased NO production which could be completely blocked by L-NAME, strongly suggesting that the increased levels of nitrite + nitrate were due to new NO synthesis from NOS. [6] SP may directly stimulate endothelium-dependent vasodilation through NO production involving endothelial cell surface receptors and/or membrane bound G protein signaling pathways. [23] In the present study, the inhibition of the NO product formation by the SPB suggests that increased NO synthesis during Mg-deficiency results from SP/SP-receptor signal transduction similar to that of the neutrophil. The isolated heart perfusion studies showed that MgD hearts had heightened nitric oxide production during imposed I/R stress compared to MgS (Figure 2B), and that nearly all of this enhanced NO formation was linked (directly or indirectly) to a SP receptor mechanism. Previously, we also demonstrated that SP-receptor blockade in vivo substantially decreased the severity of mechanical dysfunction and oxidative tissue injury in I/R-stressed MgD rat hearts; [12] but not in equally-stressed MgS hearts. This is reminiscent of the ineffectiveness of SP-receptor blockade on NO production from I/R-stressed MgS hearts in the present study. The ability of SP-receptor blockade in vivo to substantially reduce cardiovascular inflammatory lesion development in MgD rats, implicates an involvement of SP in the subsequent tissue accumulation of inflammatory cells during dietary Mg-restriction.[7, 8] Under these conditions, subsequent exposure to I/R stress may promote both endothelial-derived NO production, [24] as well as NO formation from tissue resident inflammatory cells (macrophages, neutrophils) [25].

Magnesium may affect vascular tone through its effects on calcium handling; MgD can produce vasoconstriction by allowing excess entry and intracellular release of calcium [26]. We demonstrated that the plasma PGI₂ product was elevated > 10-fold during MgD (Figure 3) which, together with the elevated NO production, may represent compensatory responses to counter the vasoconstrictive effect of MgD. Both PGI2 and PGE2, which was previously shown to be elevated in MgD rats, [7] may be similarly induced in the endothelium, and are subjected to the same cascade of events governed by SP. Since PGE2 and PGI2 are predominately COX-2 (cyclooxygenase-2) products, [27] the data suggest that SP up-regulates COX-2 during MgD and that this event might be prevented by SP-receptor blockade. Further study is required to confirm these specific events.

RBC glutathione, a key index of systemic oxidative stress, was depleted significantly in the MgD rats [6, 15]. Since excessive oxy-radical production (superoxide) alone or in combination with NO (leading to the generation of peroxynitrite) can oxidatively deplete blood glutathione, [6, 15] it is not surprising to observe that SP-receptor blockade attenuated the loss of the RBC glutathione. Conceivably, oxidative stress generated by neutrophil activation and elevated NO production may also contribute to cardiomyopathic lesion formation during Mg-deficiency. [1, 2] Previously, we reported the treatment of the MgD rats with CP-96345, another SPB analog of L703-606, effectively retarded cardiac lesion formation associated with MgD. The present findings reveal the direct role of SP in mediating neutrophil and endothelial activation and further emphasize its overall pivotal role in contributing to the cardiomyopathic process during the course of Mg-deficiency.

Acknowledgement

The authors wish to thank Mr. Wallid Al-Sharif and Ms. Lucie F. Nedelec for their excellent technical assistance and to Dr. Andrei Komarov, Department of Physiology and Experimental Medicine and Mr. Darren Sidney, for their assistance with the effluent NO product assessment.

This study was supported by NIH RO1 grants: HL-65178 and HL-62282.

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