Production of nitric oxide by human salivary peroxidase and by bovine lactoperoxidase.

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO(2) and the other implying the generation of peroxynitrite. The products of nitrite (NO(2) (-) ) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO(2) and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H(2) O(2) , convert nitrite into nitrate and form some NO, with a molar ratio of 10(3) . In our experimental conditions, no NO(2) was detectable among the products of nitrite oxidation. SCN(-) inhibits NO formation and so does I(-) , although at higher concentrations. No effects are observed with Cl(-) or Br(-) . We conclude that SPO and LPO transform NO(2) (-) into nitrate-forming small amounts of NO in the presence of H(2) O(2) as an intermediate or a by-product, synthesized through the peroxynitrite pathway.

Effect of FSH and progesterone on human spermatozoa cytosolic calcium.

Ejaculated spermatozoa must undergo a number of modifications before fertilizing the oocyte: among these the capacitation and the acrosome reaction. Calcium signals play an essential role in these functional and structural modifications. Mature spermatozoa have few organelles and a very small cytoplasmic volume but maintain the homeostasis of [Ca(2+)](c) with great accuracy. We study Ca(2+) mobilization in human spermatozoa exposed to FSH and progesterone by measuring the [Ca(2+)](c) with the FURA-2AM method and report for the first time that the exposure to FSH (up to 98ng/ml) produced an increase of [Ca(2+)](c) to an extent comparable to that observed with 1muM progesterone. FSH and progesterone increase the spermatozoa [Ca(2+)](c) by acting primarily on calcium entry from the external medium. The effects of the two hormones on [Ca(2+)](c) were similar but not identical; the pre-treatment of progesterone blocks the effects of FSH, but not vice-versa. The increase of [Ca(2+)](c) due to FSH was more sensitive to nifedipine (VOCCs inhibitor) than that of progesterone. The effects of these hormones on calcium homeostasis may be relevant for sperm activation.

Blood lipids in Antarctic and in temperate-water fish species.

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 omega-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, omega-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes.

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.

Role of cholesterol, DOTAP, and DPPC in prostasome/spermatozoa interaction and fusion.

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5-8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R(18)) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.

Cytosolic calcium levels in spermatozoa are modulated differently in healthy subjects and patients with varicocele.

OBJECTIVE: To study parameters connected to fertility in the semen of patients with varicocele. DESIGN: We examine the ability of spermatozoa obtained from patients with varicocele to respond with an increase of cytosolic Ca2+ ([Ca2+]i) to some stimuli that are connected with spermatozoa activation. SETTING: An academic research environment. PATIENT(S): Ten healthy volunteer donors and 10 patients affected by II or III grade left varicocele. INTERVENTION(S): Spermatozoa and prostasomes (vesicles of prostatic origin obtained from semen) were prepared according to standard procedures. Spermatozoa were stimulated with 1 microM P. The [Ca2+]i was evaluated with the FURA II method. MAIN OUTCOME MEASURE(S): The level of [Ca2+]i. RESULT(S): In resting cells, the level of [Ca2+]i was 120 +/-15 nmol/L (10 determinations). This value increases by > or =100 nmol/L upon stimulation with P. No difference was observed between spermatozoa obtained from healthy donors or from patients with varicocele. S-nitrosocysteine, a nitric oxide donor, and the fusion between spermatozoa and prostasomes increased the effect of P on [Ca2+]i in control spermatozoa but not in spermatozoa obtained from patients with varicocele. CONCLUSION(S): Different responsiveness of varicocele patients' spermatozoa to S-nitrosocysteine and/or to fusion with prostasomes may be among the possible causes of reduced fertility.

Antagonism between olive oil phenolics and nitric oxide on lymphomonocyte cytosolic calcium.

Some biological actions of olive oil phenolics (inhibition of platelet aggregation, decrease of LDL-oxidation, inhibition of bacterial growth and hypertensive action) have been attributed to NOS stimulation in endothelial cells through an increase of cytosolic calcium, notwithstanding the scavenging activity of phenolics on NO and superoxide. In this paper, we determine the concentration of cytosolic calcium in human lymphomonocytes incubated with high concentrations of NO-donors (CysNO) and we evaluate the effects of olive oil phenolics on this parameter. CysNO induces a marked decrease of cytosolic calcium; both olive oil phenolics oppose this action of CysNO. The effects of phenolics and CysNO are independent and additive.

Activity of olive oil phenols on lymphomonocyte cytosolic calcium.

Phenols, present in the Mediterranean diet, have antioxidant properties and are free radical scavengers; however, the molecular mechanisms of their beneficial effects are not yet fully understood. The level of cytosolic calcium ([Ca2+]i) is an important signal also in nonexcitable cells, including immune cells, and regulates fundamental processes. In this paper, we determine [Ca2+]i in human lymphomonocytes incubated with two olive oil phenols: 3,4-(dihydroxyphenyl)ethanol and p-(hydroxyphenyl)ethanol. Both tested phenols increase [Ca2+]i in a dose-dependent way. This effect is antagonized by nifedipine and is noticeable both in the presence and in the absence of calcium in the extracellular medium.

Selective liberation of NO from S-nitrosocysteine with potassium thiocyanate, as monitored by an amperometric sensor.

S-Nitrosocysteine (CysNO) releases either NO (in the presence of divalent cations) or NO+ (in the presence of chelating agents). NO+ is then transferred to peptides or protein SH groups to form high-mass nitrosothiols. The aim of this work was the development of a specific reaction between thiocyanate (SCN-) and CysNO. This reaction selectively liberates NO from CysNO in the presence of high-mass nitrosothiols. Free NO is measured with an amperometric sensor. We examine with this system the transnitrosylation reaction between CysNO and BSA at low molecular ratios and could assay nitrites, SNO-BSA, and CysNO in the incubation mixture without any preliminary purification steps.

Electrochemical assay for determining nitrosyl derivatives of human hemoglobin: nitrosylhemoglobin and S-nitrosylhemoglobin.

Nitric oxide (NO) is an important biological regulator. It can bind to heme iron and form NO+, involved in the synthesis of S-nitrosothiols (-SNOs). NO reacts with human hemoglobin (Hb) to produce the derivatives: S-nitrosylhemoglobin (-SNOHb) and nitrosylhemoglobin (HbNO). At neutral pH values, free NO does not react directly with the -SH groups of Hb. The reductive nitrosylation of Fe(III) heme upon reaction with NO has long been studied, but it is not yet completely known. To quantify the reaction of NO with Hb, we developed a new, sensitive (nanomolar concentration range) electrochemical assay to selectively measure HbNO and -SNOHb. The assay also allows the monitoring of free NO during the reaction with human Fe(III)Hb and Fe(II)HbO(2).

Fusion of prostasomes to human spermatozoa stimulates the acrosome reaction.

OBJECTIVE: To determine the effect of the fusion of prostasomes to spermatozoa on the acrosome reaction. DESIGN: In vitro study of human spermatozoa. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Healthy volunteer men, 25 to 35 years old. INTERVENTION(S): Human semen was fractionated into spermatozoa and prostasomes. Fusion of prostasome to spermatozoa was performed at pH 5.5. Progesterone (1 microM) was added when required. MAIN OUTCOME MEASURE(S): Evaluation of the acrosome reaction by fluorescence microscopy. RESULTS(S): The percentage of spontaneously acrosome-reacted cells was very low unless the Ca(2+)-ionophore A 23187 was added. The treatment of spermatozoa with 1 microM of progesterone scarcely affected the acrosome reaction; a pretreatment in conditions permitting fusion increased it. The addition of progesterone to prostasome-fused spermatozoa further increased the extent of the acrosome reaction. CONCLUSION(S): The H(+)-dependent fusion with prostasomes makes spermatozoa more sensitive to the effect of progesterone on acrosome-reaction induction.

Electrochemical determination of nitric oxide and of its derivatives.

Nitric oxide (NO) is one of the simplest odd electron species. Furthermore, it is relatively hydrophobic, which is consistent with its role as a diffusible intracellular messenger or as an immune effector. NO is generated in biological systems and plays important roles as a regulatory molecule. The main problem in NO analysis is its extreme reactivity; in aerated water solution it is transformed into nitrite and nitrate, inactive biological forms. Moreover, it may lose an electron forming the NO(+) ion, involved in the synthesis of nitrosothiols (RSNOs). The main problems encountered in the analytical determination of free NO and of RSNOs in biological systems are the low stability and the very low concentration of these compounds. The determination of nitrates and nitrites may also be difficult when their concentration is in the nmolar range. We describe an electrochemical assay for the determination in the same sample of free NO and of its derivatives in nmolar range. Owing to its high sensitivity, the procedure could also be applied to environmental analyses.

Formation of nitrosothiols from gaseous nitric oxide at pH 7.4.

Nitric oxide (NO) is generated in biological systems and plays important roles as a regulatory molecule. Its ability to bind to haem iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of S-nitrosothiols (SNOs). It has been suggested that S-nitrosohaemoglobin (-SNO Hb) and low molecular weight SNOs may act as reservoirs of NO. SNOs are formed in vitro, at strongly acidic pH values; however, the mechanism of their formation at neutral pH values is still debated. In this paper we report the anaerobic formation of SNOs (both high- and low-molecular weight) from low concentrations of NO at pH 7.4, provided Hb is also present. We propose a reaction mechanism entailing the participation of Fehaem in the formation of NO(+) and the transfer of NO(+) either to Cysbeta(93) of Hb or to glutathione; we show that this reaction also occurs in human RBCs.

Nitric oxide and fusion with prostasomes increase cytosolic calcium in progesterone-stimulated sperm.

Spermatozoa must undergo a number of reactions before they are able to fertilize the oocyte. Among these is the acrosome reaction, which is related to an increase in cytosolic Ca2+ concentration ([Ca2+]i). It has been reported in the literature that progesterone may achieve this effect through the intervention of extragenomic receptors. Nitric oxide (NO) has been reported to affect spermatozoa; the nature of the effect depends on the concentration of the radical. In a previous paper, we reported that the fusion of spermatozoa with prostasomes may also produce a transient increase in spermatozoa [Ca2+]i; in addition, this phenomenon causes a long-lasting effect that influences the action of progesterone. In this paper, we test the effects of a NO donor (CysNO) and of fusion of the prostasome to spermatozoa on progesterone-induced [Ca2+]i increase. No effect at all was noticed in the absence of progesterone stimulation. In the presence of the hormone, both CysNO and fusion increased the progesterone effect. This phenomenon was much more evident if the two treatments were used together. We conclude that both NO and fusion with prostasomes act on the progesterone-dependent pathway additively. Probably the effects are independent.

Nitric oxide in ischemic and reperfused human muscle.

BACKGROUND: Biochemical events explaining the pathology of ischemia-reperfusion in the muscle are still debated. Nitric oxide (NO) has been postulated to be implicated in these phenomena, but the short half-life of this compound makes it difficult to measure. METHODS: In this paper, we used an amperometric solid-sate sensor to measure NO concentrations in frozen human muscles before, during and after a period of ischemia. We also measured cytochrome oxidase activity and malondialdehyde (MDA). RESULTS: NO increased during ischemia but it soon returned to normal values upon reperfusion. On the other hand, cytochrome oxidase that also decreased in ischemic muscle did not increase during the reperfusion and malondialdehyde only increased during reperfusion, indicating the occurrence of peroxidative reactions in this situation. CONCLUSIONS: NO is implicated in the ischemia/reperfusion pathology, but it is difficult to relate whether this is connected to cytochrome oxidase activity and malondialdehyde formation, also modified in this ischemia-reperfusion model.