Andrei M. Komarov

Neurogenic Inflammation and Cardiac Dysfunction Due to Hypomagnesemia

Hypomagnesemia continues to be a significant clinical disorder that is present in patients with diabetes mellitus, alcoholism, and treatment with magnesuric drugs (diuretics, cancer chemotherapy agents, etc.). To determine the role of magnesium in cardiovascular pathophysiology, we have used dietary restriction of this cation in animal models. This review highlights some key observations that helped formulate the hypothesis that release of substance P (SP) during experimental dietary Mg deficiency (MgD) may initiate a cascade of deleterious inflammatory, oxidative, and nitrosative events, which ultimately promote cardiomyopathy, in situ cardiac dysfunction, and myocardial intolerance to secondary stresses. SP acts primarily through neurokinin-1 receptors of inflammatory and endothelial cells, and may induce production of reactive oxygen and nitrogen species (superoxide anion, NO•, peroxynitrite, hydroxyl radical), leading to enhanced consumption of tissue antioxidants; stimulate release of inflammatory mediators; promote tissue adhesion molecule expression; and enhance inflammatory cell tissue infiltration and cardiovascular lesion formation. These SP-mediated events may predispose the heart to injury if faced with subsequent oxidative stressors (ischemia/reperfusion, certain drugs) or facilitate development of in situ cardiac dysfunction, especially with prolonged dietary Mg restriction. Significant protection against most of these MgD-mediated events has been observed with interventions that modulate neuronal SP release or its bioactivity, and with several antioxidants (vitamin E, probucol, epicaptopril, d-propranolol). In view of the clinical prevalence of hypomagnesemia, new treatments, beyond magnesium repletion, may be needed to diminish deleterious neurogenic and prooxidative components described in this article.

Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase.

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angelis salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.

Epr detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes

Spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (·NO) production were compared in the ischemic-reperfused myocardium for the first time, using both aqueous-soluble and lipophilic complexes of reduced iron (Fe) with dithiocarbamate derivatives. The aqueous-soluble complex of Fe and N-methyl-D-glucamine dithiocarbamate (MGD) formed MGD2-Fe-NO complex with a characteristic triplet EPR signal (aN12.5 G and giso = 2.04) at room temperature, in native isolated rat hearts following 40 min global ischemia and 15 min reperfusion. Diethyldithiocarbamate (DETC) and Fe formed in ischemic-reperfused myocardium the lipophilic DETC2-Fe-NO complex exhibiting an EPR signal (g⊥ = 2.04 and g∥ = 2.02 at 77K) with a triplet hyperfine structure at g⊥. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the ·NO synthase inhibitor, NG-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provi ded similar values for tissue ·NO production, which were observed primarily during ischemia. Postischemic hemodynamic recovery of the heart was not affected by the trapping procedure. (Mol Cell Biochem 175: 91–97, 1997)

Iron potentiates nitric oxide scavenging by dithiocarbamates in tissue of septic shock mice

Vanin and co-workers (Kubrina et al., Biochim. Biophys. Acta 1176 (1993) 240-244; Mikoyan et al., Biochim. Biophys. Acta 1269 (1995) 19-24) reported that short term (30 min) iron (Fe) exposure potentiates nitric oxide (NO) production in tissues of septic shock mice, based on increased formation of NO complex by diethyldithiocarbamate (DETC). We have reexamined the effect of Fe administration in mice treated with Escherichia coli lipopolysaccharide (LPS) and have not found any changes in nitrosylhemoglobin (HbNO) or (NOs- + NO3-) levels in blood 30 min after Fe-citrate complex injection. However, Fe-citrate promotes NO complex formation by iron-dependent NO traps: DETC, pyrrolidinedithiocarbamate (PDTC) and N-methyl-D-glucamine dithiocarbamate (MGD), when given simultaneously at 6 h after LPS. Rather than potentiation of NO production, our data support that short-term iron treatment (30 min) enhances in vivo spin trapping ability of dithiocarbamate.

The nerve-heart connection in the pro-oxidant response to Mg-deficiency.

Magnesium is a micronutrient essential for the normal functioning of the cardiovascular system, and Mg deficiency (MgD) is frequently associated in the clinical setting with chronic pathologies such as CHF, diabetes, hypertension, and other pathologies. Animal models of MgD have demonstrated a systemic pro-inflammatory/pro-oxidant state, involving multiple tissues/organs including neuronal, hematopoietic, cardiovascular, and gastrointestinal systems; during later stages of MgD, a cardiomyopathy develops which may result from a cascade of inflammatory events. In rodent models of dietary MgD, a significant rise in circulating levels of proinflammatory neuropeptides such as substance P (SP) and calcitonin gene-related peptide among others, was observed within days (1–7) of initiating the Mg-restricted diet, and implicated a neurogenic trigger for the subsequent inflammatory events; this early “neurogenic inflammation” phase may be mediated in part, by the Mg-gated N-methyl-D-aspartate (NMDA) receptor/channel complex. Deregulation of the NMDA receptor may trigger the abrupt release of neuronal SP from the sensory-motor C-fibers to promote the subsequent pro-inflammatory changes: elevations in circulating inflammatory cells, inflammatory cytokines, histamine, and PGE2 levels, as well as formation of nitric oxide, reactive oxygen species, lipid peroxidation products, and depletion of key endogenous antioxidants. Concurrent elevations of tissue CD14, a high affinity receptor for lipopolyssacharide, suggest that intestinal permeability may be compromised leading to endotoxemia. If exposure to these early (1–3 weeks MgD) inflammatory/pro-oxidant events becomes prolonged, this might lead to impaired cardiac function, and when co-existing with other pathologies, may enhance the risk of developing chronic heart failure.

Mg-Gluconate provides superior protection against postischemic dysfunction and oxidative injury compared to Mg-sulfate

Cardioprotection by Mg Sulfate (MgSO4) during ischemia/reperfusion (I/R) is attributed largely to the Mg2+ cation. However, Mg-gluconate (MgGl2) may provide added benefit, possibly through its anions antioxidant properties. Protective effects of both Mg-salts and their anions during 30 min global I and 50 min R were assessed in Langendorff-perfused (Krebs-Henseleit buffer) rat hearts. Recovery of function was compared between untreated hearts and those receiving supplement (2.4 mM MgGl2, MgSO4, or Na2SO4, or 4.8 mM NaGl) for 5 min prior to I and during the initial 30 min R. The final 20 min R was conducted without supplement. End diastolic pressure (EDP, mmHg) of the 50 min reperfused MgGl2 group (2.6) was lower than MgSO4 (16.2) and untreated (35.6) groups, and the NaGl group (25.2) was considerably lower than Na2SO4 (38.8). Recovery of developed pressure (% preischemic DP) at the onset of R for MgGl2 (74.9) was greater than MgSO4 (37.9) and untreated (33.2). After 50 min, MgGl2 (77.9) and MgSO4 (66.9) provided protection compared to untreated (51.8). In separate studies, ESR spin trapping with α-phenyl-N-tert-butylnitrone (3 mM PBN) showed that I/R alkoxyl radical production was reduced with MgGl2 (0.0 vs. 2.4 vs. 3.6 mM: 184 vs. 97 vs. 54.8 nM/g tissue × min) to a greater extent than seen with MgSO4 (3.6 mM: 108). Additional studies suggest that Gl1−, unlike SO42−, may scavenge hydroxyl radicals, accounting for the added protection. MgGl2 treated hearts exhibited less postischemic dysfunction and oxidative injury compared to MgSO4, suggesting the contribution of Gl1− to cardioprotection.

Activation of the neutrophil and loss of plasma glutathione during Mg-deficiency — modulation by nitric oxide synthase inhibition

Sprague-Dawley rats (200 g) were fed either a Mg-deficient or Mg-sufficient diet for 3 weeks. An enriched neutrophil fraction (>85%) was isolated from the blood by sodium metrizoate/dextran gradient centrifugation. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, the basal activity of neutrophils isolated from the Mg-deficient rats were found elevated 5 fold after two weeks, and up to ∼7 fold after three weeks on the diet. Upon challenge by phorbol myristate acetate (PMA), unlike the Mg-sufficient cells, the Mg-deficient cells exhibited no significant activation. Treatment of the Mg-deficient rats with the nitric oxide (NO)-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water, significantly attenuated the basal superoxide producing activity of the neutrophils and partially restored its response to PMA challenge. In association with the neutrophil activation. Mg-deficiency resulted in 70% decrease in plasma glutathione and 220% increase in Fe-promoted, thiobarbituric acid reactive substance (TBARS) levels; both changes were significantly attenuated by L-NAME treatment. The results suggest that neutrophils from Mg-deficient rats are activated endogenously to generate oxy-radicals which might directly mediate the in vivo peroxidative indices during Mg-deficiency. Furthermore, the neutrophil activity was lowered by NO-synthase inhibition suggesting that NO overproduction during Mg-deficiency participates in the neutrophil activation process.

Effect of septic shock on nitrate, free amino acids, and urea in murine plasma and urine.

OBJECTIVES Concentration changes of free amino acids, urea and nitrate in plasma and urine were studied for the murine model of septic shock. METHODS After administration of a bolus dose of bacterial lipopolysaccharide (LPS), concentrations of amino acids and urea in plasma, and urea and nitrate in urine were determined. RESULTS For individual amino acids four different trends were observed: (1) no change ( e.g., taurine, histidine, phenylalanine, hydroxproline); (2) continuous increase (e.g., aspartate and glutamate); (3) continuous decrease (e.g., threonine, serine, asparagine, proline, methionine, tyrosine); and (4) decrease during the first 4 hours, but return to normal at 8 hours after the LPS treatment (e.g., all the other amino acids). The ratio of phenylalanine to tyrosine was increased to about 2x. In plasma, urea concentration was increased about 3x, but in urine it decreased about 4x. Nitrate levels were increased 3x in urine. CONCLUSION These early changes in the concentrations of amino acids as well as in the urea and nitrate may be useful as sensitive markers for the early and rapid diagnosis of septic shock.

Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice

The effect of exogenous Fe‐citrate complex (Fe doses of 120 and 240 μmol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin‐treated mice. Fe‐citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS). Iron‐dependent decrease in NO− 2/NO− 3 and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation. NO production in the liver tissue of LPS‐treated mice was decreased after Fe administration judging from the amount of mononitrosyl‐iron complexes formed in the tissue by diethyldithiocarbamate. The iNOS protein determination in the liver tissue of LPS‐treated mice demonstrated iron‐dependent inhibition of iNOS expression. We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e. after iNOS expression. This is a first report demonstrating iron‐dependent iNOS down‐regulation in endotoxin‐treated mice.

The role of magnesium deficiency in cardiovascular and intestinal inflammation.

Hypomagnesemia continues to cause difficult clinical problems, such as significant cardiac arrhythmias where intravenous magnesium therapy can be lifesaving. Nutritional deficiency of magnesium may present with some subtle symptoms such as leg cramps and occasional palpitation. We have investigated dietary-induced magnesium deficiency in rodent models to assess the pathobiology associated with prolonged hypomagnesemia. We found that neuronal sources of the neuropeptide, substance P (SP), contributed to very early prooxidant/proinflammatory changes during Mg deficiency. This neurogenic inflammation is systemic in nature, affecting blood cells, cardiovascular, intestinal, and other tissues, leading to impaired cardiac contractility similar to that seen in patients with heart failure. We have used drugs that block the release of SP from neurons and SP-receptor blockers to prevent some of these pathobiological changes; whereas, blocking SP catabolism enhances inflammation. Our findings emphasize the essential role of this cation in preventing cardiomyopathic changes and intestinal inflammation in a well-studied animal model, and also implicate the need for more appreciation of the potential clinical relevance of optimal magnesium nutrition and therapy.