Nitric oxide is involved in heat-induced HSP70 accumulation

Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock-induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO-synthase inhibitor (L-NNA). L-NNA also markedly attenuated the heat shock-induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock-induced activation of HSP70 synthesis.     

Cross-talk between nitric oxide and HSP70 in the antihypotensive effect of adaptation to heat

In this work, we evaluated the effect of adaptation to heat on the fall of blood pressure (BP) induced by heat shock (HS) and the interrelation between nitric oxide (NO) and heat shock protein, HSP70. Experiments were carried out on Wistar rats. It was shown that HS resulted in a generalized and transient increase in NO production (the electron paramagnetic resonance method) and a fall of BP from 113+/-3 to 88+/-1 mm Hg (p<0.05). Adaptation to heat itself did not affect BP, but completely prevented the NO overproduction and hypotension induced by HS. The adaptation simultaneously increased the brain NO-synthase content and induced HSP70 synthesis (the Western blot analysis) in various organs. Both the antihypotensive effects of adaptation and HSP70 accumulation were completely prevented by L-NNA, an inhibitor of NO synthesis, or quercetin, an inhibitor of HSP70 synthesis. The data suggest that adaptation to heat stimulates NO synthesis and NO activates synthesis of HSP70. HSP70, which hampers NO overproduction, thus restricts the BP fall induced by heat shock.     

Selective inhibition of inducible NO-synthase by nonselective inhibitor

The study has shown that Nw-nitro-L-arginine, a nonselective nitric oxide (NO) inhibitor, in low non-vasoactive doses (10 mg/kg) exerted a protective effect in heat shock as demonstrated by a decrease in the mortality rate and prevention of acute hypotension in rats. The L-NNA in the same dose inhibited the basal NO production but left unaffected a carbachol-activated NO production. The findings suggest a possibility in principle of preferential inhibition of inducible NO-synthase in pathological conditions related to the NO overproduction using non-vasoactive doses of L-NNA the nonselective NO-synthase inhibitor.     

Antitumor activity of dinitrosyl iron complexes with glutathione

The antitumor dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on a solid tumor in the mouse, Lewis lung carcinoma, was detected. The complexes being injected at doses of 21, 42, 105 mg/kg daily for 10 days blocked completely the development of the tumor for the first week after tumor cell implantation into animals. After that, the part of tumor cells which remained in intact alive state began to grow at a rate equal to that for control animals. The effect was proposed to be caused via formation of an antinitrosative defense system in the cells as a response to NO attack on cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. Data were obtained which were in line with the hypothesis.     

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

Protonation of nitrite is an obligatory stage in the generation of nitric oxide from nitrite in biological systems

The yield of nitric oxide from 1 mM sodium nitrite differs 200 times when the process was initiated by 10 mM sodium dithionite in the solution of 5 or 150 mM HEPES-buffer (pH 7.4). Dithionite acted both as a strong reductant and an agent that induced a local acidification of solutions without notable change in pH value. The amount of nitric oxide was estimated by the EPR method by measuring the incorporation of nitric oxide to water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which led to the formation of EPR-detectable mononitrosyl iron complexes with MGD (MNIC-MGD). Ten seconds after dithionite addition, the concentration of MNIC - MGD complexes reached 2 microM in 5 mM HEPES-buffer in contrast to 0.01 microM in 150 mM HEPES-buffer. The difference was suggested to be due to a higher life-time of zones with decreased pH values in a weaker weak buffer solution. The life-time was high enough to ensure the protonation of a part of nitrite. The resulting nitrous acid was decomposed to form nitric oxide. The difference in the formation of nitric oxide from nitrite was also observed in weak and strong buffer solutions in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, the ratios of nitric oxide yields in weak and strong buffer did not exceed 3-4 times. The increase in the formation of nitric oxide from nitrite was characteristic for the solutions containing both proteins. Large amounts of nitric oxide formed from nitrite was observed in mouse liver preparation subjected to freezing-thawing procedure followed by incubation in 150 mM HEPES-buffer (pH 7.4) and addition of dithionite. The proposition was made that the presence of zones with low pH value in cells and tissues can ensure the predominant operation of the acid mechanism formation of nitric oxide from nitrite. The contribution of the formation of nitric oxide from nitrite catalyzing with heme-containing proteins nitrite reductases can be minor one under these conditions.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

L-arginine--an endogenous source of nitric oxide in animal tissues in vivo]

According to EPR data, NG-mononitro-L-arginine (MNA) being intraperitoneally injected to inbred albino mice in the dose of 70-700 mg/kg strongly decreases the formation of mononitrosyl iron complexes (MNIC) with the exogenous ligand, diethyldithiocarbamate (DETC) in liver cells. Simultaneous injections of experimental mice with MNA (70 mg/kg) and L-arginine (700 mg/kg) are unaccompanied by the formation of MNIC-DETC complexes. It is concluded that nitric oxide (NO) which is produced in mouse liver in vivo and which provides for the formation of MNIC complexes with DETC is generated by L-arginine via an enzymatic reaction which is competitively inhibited by MNA. Besides, MNA causes reversible inhibition and augmented synthesis of NO formed in mouse liver after the injection of the exogenous lipopolysaccharide of E. coli.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.