Endogenous reactive oxygen species content and modulation of tyrosine phosphorylation during sperm capacitation.

Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine (Tyr) residues are two main cellular changes involved in sperm capacitation. This study examined the relationship between tyrosine-phosphorylation (Tyr-P) and endogenous ROS production during sperm capacitation, and correlated them with both sperm motility and functionality expressed as acrosome-reacted cells. Immediate ROS generation was observed to peak after a 45-min incubation, followed by a rapid decrease in ROS content and successive regeneration of the ROS peak in 3 h and later. These two peaks were directly correlated with both the Tyr-P process involving sperm heads and tails, and the acrosome reaction (69 ± 8% and 65 ± 4%, respectively). The period of low-ROS content resulted in low Tyr-P patterns, located exclusively in the cell midpiece, and drastic reduction in acrosome-reacted cells. Ascorbic acid addition inhibited both Tyr-P patterns and acrosome reactions, whereas NADPH induced high ROS generation, with Tyr-P patterns located only on sperm tails, and prevented the acrosome reaction. Sperm hyperactivation was insensitive to ROS content. This is an important parameter for evaluation of sperm capacitation, which is achieved only when both ROS generation reaches a peak and Tyr-P involves the sperm head.

Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function.

Cannabinoids and endocannabinoids negatively influence sperm functions. These substances have been demonstrated in many mammalian tissues, including male and female reproductive tracts, and previous studies have shown the presence of functional receptors for cannabinoids in human sperm. The present study, by means of RT-PCR and Western blot techniques, demonstrates that human sperm express the CB(1), but not CB(2), cannabinoid receptor (CB-R) subtype located in the head and middle piece of the sperm. The activation of this receptor by anandamide reduces sperm motility and inhibits capacitation-induced acrosome reaction. Activation of the CB(1)-R did not induce any variation in sperm intracellular calcium concentrations, but produced a rapid plasma membrane hyperpolarization that was reduced by the K(+) channel blocker tetraethylammonium. The effects of anandamide on human sperm motility were dependent on the reduction of sperm mitochondrial activity as determined by rhodamine 123 fluorescence. The specificity of anandamide effects in human sperm were confirmed by the effects of the CB(1)-R antagonist SR141716. These findings provide additional evidence that human sperm express functional CB(1)-R, the activation of which negatively influences important sperm functions, and suggest a possible role for the cannabinoid system in the pathogenesis of some forms of male infertility.

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria.

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, (32)P-poly(Glu-Tyr)(4:1) and (32)P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.

Fatty acids and cytokine mRNA expression in human osteoblastic cells: a specific effect of arachidonic acid.

Epidemiological, clinical and experimental evidence suggests that fatty acids have a modulatory effect on bone metabolism in animals and humans. To investigate this hypothesis, we evaluated the effects of three different fatty acids, arachidonic acid (AA), eicosapentaenoic acid (EPA) and oleic acid (OA), on the expression of cytokines involved in bone remodelling. Cytokine mRNAs in the human osteoblast-like cell line MG-63 were quantified by reverse transcription-PCR. AA induced increased expression of interleukin-1alpha, interleukin-1beta, tumour necrosis factor-alpha and macrophage colony-stimulating factor mRNAs in a time- and dose-dependent manner. EPA and OA had no stimulatory effects, but instead caused a significant inhibition of AA-induced cytokine mRNA expression. Cell treatment with calphostin C, an inhibitor of protein kinase C (PKC), and cellular PKC down-regulation experiments independently resulted in significant inhibition of AA-induced cytokine expression, suggesting that a PKC-dependent mechanism accounts for the effects of AA on cytokine production. In conclusion, our study demonstrates specific effects of fatty acids on cytokine gene expression in human osteoblast-like cells. The clinical relevance of our findings requires further investigation.

Aluminum as an inducer of the mitochondrial permeability transition.

Treatment of rat liver mitochondria with aluminum in the presence of Ca2+ results in large amplitude swelling accompanied by loss of endogenous Mg2+ and K+ and oxidation of endogenous pyridine nucleotides. The presence of cyclosporin A, ADP, bongkrekic acid, N-ethylmaleimide and dithioerythritol prevent these effects, indicating that binding of aluminum to the inner mitochondrial membrane, most likely at the level of adenine nucleotide translocase, correlates with the induction of the membrane permeability transition (MPT). Indeed, aluminum binding promotes such a perturbation at the level of ubiquinol-cytochrome c reductase, which favors the production of reactive oxygen species. These metabolites generate an oxidative stress involving two previously defined sites in equilibrium with the glutathione and pyridine nucleotides pools, the levels of which correlate with the increase in MPT induction. Although the above-described phenomena are typical of MPT, they are not paralleled by other events normally observed in response to treatment with inducers of MPT (e.g., phosphate), such as the collapse of the electrochemical gradient and the release of accumulated Ca2+ and oxidized pyridine nucleotides. Biochemical and ultrastructural observations demonstrate that aluminum induces a pore opening having a conformation intermediate between fully open and closed in a subpopulation of mitochondria. While inorganic phosphate enhances the MPT induced by ruthenium red plus a deenergizing agent, aluminum instead inhibits this phenomenon. This finding suggests the presence of a distinct binding site for aluminum differing from that involved in MPT induction.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites.

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Dietary manipulation of delta-6-desaturase modifies phospholipid arachidonic acid levels and the urinary excretion of calcium and oxalate in the rat: insight in calcium lithogenesis.

An anomalous n-6 polyunsaturated fatty acid composition in plasma and erythrocyte membrane phospholipids, namely increased levels of arachidonic acid (AA), has been reported in calcium nephrolithiasis and has been proposed to play an important role in its pathogenesis. To confirm this, in rats we modified phospholipid AA levels by dietary manipulation of the delta-6-desaturase, the rate-limiting enzyme of the fatty acid biosynthetic pathway, and evaluated the effect on cellular and renal functions predisposing to lithogenesis. Increased AA levels led to conditions at risk for nephrolithiasis: higher oxalate flux and lower sodium cotransport in erythrocytes and a rise in urinary prostaglandin E2, calcium, sodium, and oxalate levels; reduced AA levels reversed these changes. In vitro, in human erythrocytes the incorporation of exogenous AA into membranes increased band 3 protein phosphorylation directly activating the Ser/Thr protein kinase CK1 and induced a parallel raise in band 3-mediated oxalate transport. These findings demonstrate the pivotal role of phospholipid AA in modulating erythrocyte and renal transport of calcium and oxalate.

Specific modulatory effect of arachidonic acid on human red blood cell oxalate transport: clinical implications in calcium oxalate nephrolithiasis.

Greater arachidonic acid (AA) contents, which were correlated with erythrocyte transmembrane oxalate (Ox) transport, were observed in plasma and erythrocyte membrane phospholipids of patients with idiopathic calcium renal stones, suggesting a link between membrane phospholipid fatty acid composition and cellular Ox transport. To confirm this hypothesis, the effects of exogenous red blood cell incorporation of three different fatty acids (i.e., oleic acid, AA, and eicosapentaenoic acid) on Ox transport and the phosphorylation status of band 3 protein, which has been shown to mediate red blood cell Ox flux, were investigated. Preincubation of erythrocytes with AA induced a dose-dependent increase in the phosphorylation level of band 3 protein and an increase in transmembrane Ox self-exchange. In contrast, inhibitory effects on both parameters were observed after the incorporation of oleic and eicosapentaenoic acids. These data, together with previous observations of dietary effects on erythrocyte Ox transport and urinary Ox excretion, indicate that genetic and/or nutritional changes in membrane phospholipid fatty acid composition play a crucial role in modulating cellular Ox transport in idiopathic calcium Ox nephrolithiasis.

Effect of protein kinase C and insulin on Na+/H+ exchange in red blood cells of essential hypertensives.

The kinetic properties of sodium-proton exchange are abnormal in human red blood cells of hypertensive patients and it has been demonstrated that the transport protein undergoes post-translational modifications able to affect its kinetic properties. Protein kinase C (PKC) activation decreases the affinity constant for intracellular protons while insulin increases the maximal rate of proton translocation. The present study therefore aimed to examine the relationships among PKC activity, fasting insulin levels and the kinetic behaviour of sodium-proton exchange in red blood cells from 20 normotensives and 36 hypertensives. In comparison with normotensive subjects, hypertensive patients had higher body mass index (26.2 +/- 0.7 vs 23.6 +/- 0.6 kg/m2, P < 0.05), higher fasting insulin levels (93.2 +/- 10.8 vs 38.6 +/- 2.9 pmol/L), increased maximal velocity of proton translocation (37.9 +/- 2.7 vs 27.6 +/- 1.9 mmol/L per cell x h, P < 0.05), and reduced Hill's coefficient (1.6 +/- 0.1 vs 2.0 +/- 0.1, P < 0.01) of sodium-proton exchange. Basal PKC activity of the cytosol and membrane was similar in the study groups. However, after treatment with 1 micromol/L phorbol 12-myristate 13-acetate (PMA) for 10 min, membrane PKC activity was stimulated to a larger extent in hypertensives (to 181 +/- 8 pmol/min/mg protein) than in normotensives (to 136 +/- 6 pmol/min/mg protein, P < 0.01). The PMA stimulated PKC activity was positively correlated to fasting insulin levels (r = 0.59, P < 0.01). Stimulation of membrane PKC by PMA corrected the low Hill's coefficient for H(i)+ activation of sodium-proton exchange in the hypertensives, while the constant for half maximal activation for intracellular protons (ie, the affinity for intracellular protons) decreased to a similar extent in both groups. The maximal transport rate was unaffected by PMA. These results indicate that the abnormal proton activation of red blood cell sodium-proton exchange in hypertensives reflects an abnormal regulation of PKC translocation to the cell membrane, associated to hyperinsulinaemia and probably insulin resistance. Therefore, post-translational modifications of the transport protein(s) account for the altered kinetic behaviour of sodium-proton exchange in hypertensives.

Protein kinase C and insulin regulation of red blood cell Na+/H+ exchange.

Insulin activation of red blood cell (RBC) Na+/H+ (NHE) and Na+/Li+ (NLiE) exchanges is mimicked by okadaic acid, thus suggesting that it may change the state of phosphorylation of serine/threonine NHE residues. To investigate the role of the serine/threonine protein kinase C (PKC) in insulin regulation, we evaluated the effect of phorbol 12-myristate 13-acetate (PMA; 1 microM) and insulin on PKC activity, membrane protein phosphorylation, and the activation kinetics of both exchangers. Our studies revealed that PMA decreased cytosolic PKC activity (4.1 +/- 0.6 to 2.3 +/- 0.5 pmol x mg protein(-1) x min(-1), n = 9, P < 0.001), increased membrane PKC activity (42.3 +/- 5 to 132 +/- 12 pmol x mg protein(-1) x min(-1), n = 11, P < 0.001), and enhanced serine phosphorylation of bands 3, 4.1, and 4.9 membrane proteins. PMA markedly reduced the Michaelis constant (Km) for intracellular H+ (415 +/- 48 to 227 +/- 38 nM, n = 11, P < 0.01) but had no effect on the maximal transport rate (Vmax) of NHE and the Km for Na+ of NLiE. NHE activation and PKC activity were affected differently by insulin (100 microU/ml) and PMA. Insulin increased the Vmax of NHE and the Km for Na+ of NLiE but had no effect on the Km for intracellular H+ and membrane PKC activity. These findings lead us to conclude that in the human RBC, NHE is modulated by PKC and insulin through different biochemical mechanisms.

Casein kinase 2 phosphorylates recombinant human spermidine/spermine N1-acetyltransferase on both serine and threonine residues.

Casein kinase 2 purified from human erythrocyte cytosol has been found to phosphorylate human spermidine/spermine N1-acetyltransferase (SSAT) expressed as a fusion protein in E. coli and purified to homogeneity with a specific activity similar to that reported for pure human SSAT. The amino acid sequence of the protein revealed not less than four phosphorylable residues, optimal target for protein kinase 2 phosphorylation being flanked by acid residues in position +1 and +3. Our results indicate that most 32P-phosphate is taken up by Ser residues, as evidenced by HCl hydrolysis and electrophoresis and that the phosphorylation extent is modulated by the physiological polyamine concentration. Partial digestion with trypsin at a low concentration for less than one hour preferentially hydrolyzes Lys-Arg-Arg in position 141-143 of the SSAT suggesting that the Ser-phosphorylated residues are located in the C-terminus of the protein, probably Ser 146 and 149.

The Lyn-catalyzed Tyr phosphorylation of the transmembrane band-3 protein of human erythrocytes.

Band-3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr-protein kinases on different Tyr residues at its N and C cytoplasmic domains. Both the added p36syk (catalytic domain of p72syk) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts). However, the two Tyr-protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36syk markedly Tyr phosphorylates the band-3 protein, whereas the added Lyn phosphorylates it very poorly. It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p36syk, i.e. the p36(syk)-catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn-catalyzed phosphorylation of different band-3 Tyr sites.

Anomalous phospholipid n-6 polyunsaturated fatty acid composition in idiopathic calcium nephrolithiasis.

Anomalies in the erythrocyte transport of anions and cations have been described in idiopathic calcium oxalate nephrolithiasis and seem to play a pathogenetic role in this disease. In consideration of the hypothesis that the complex array of ion flux cell abnormalities is an epiphenomenon of an anomaly in the composition of cell membranes, this study investigated cell-membrane lipid composition. In idiopathic calcium oxalate renal stone formers, in which ion transport abnormalities were present, and in healthy control subjects, plasma and erythrocyte membrane lipid composition, the erythrocyte oxalate exchange, and Na/K/2Cl cotransport activity were evaluated. Furthermore, in stone formers, the effect of a 30-day fish-oil diet supplementation on plasma lipids, erythrocyte oxalate exchange, oxaluria, and calciuria was investigated. The effect of archidonic acid released by phospholipase A2 on anion-carrier phosphorylation and activity in erythrocytes was evaluated as well. Patients had a lower content of linoleic and higher concentration of archidonic acids in both plasma and erythrocyte membrane phospholipids, and an increased archidonic/linoleic acid ratio. The archidonic acid level correlated with the erythrocyte oxalate exchange and sodium cotransport activity. Fish-oil supplementation lowered calcium and oxalate urine excretion, and normalized the erythrocyte oxalate exchange. Phospholipase A2 increased the erythrocyte anion-carrier protein phosphorylation and the oxalate exchange. This study shows that idiopathic calcium nephrolithiasis in the patient group reported here is characterized by a systemic defect in phospholipid archidonic acid levels that might provide an answer to the link between genetic background, dietary habits, and renal lithiasis.

Posttranslational effects of protein kinase C and insulin on red cell membrane phosphorylation and cation heteroexchange in hypertension.

In the red blood cell membrane, sodium-proton exchange (NHE-1) exchanges intracellular H(+), Li(+), and Na(+) with extracellular Na(+). In hypertensives (HT), the maximal velocity of translocation (V max)of Na(+)/H(+) and of Na(+)/Li(+) exchange modes are higher, while apparent affinity for external Na(+) of Na(+)/Li(+) exchange and Hill's coefficient for H(+) activation of Na(+)/H(+) exchange are lower than in normotensive subjects (NT). We have therefore examined the effects of protein kinase C (PKC) and insulin on red blood cell membrane phosphorylation and on the kinetic properties of cation heteroexchange. In red cell from NT, PMA-induced activation of PKC reduced K(m) for H(+) of NHE but it did not affect V(max) and K(m) for Na(+). In red cell from HT, PMA-induced a greater PKC stimulation and membrane phosphorylation of band 3,4.1,4.9 than in NT and it did not significantly reduced K(m) for H(i). On the contrary, in HT PKC activation significantly increased Hill's coefficient of NHE. The larger activation of PKC in HT could be due to downregulation secondary to higher membrane calpain activity. Incubation of red cells with insulin decreases K(m) for external Na(+) and increases V(max) of Na(+)/Li(+) exchange. Therefore, we have examined the relationships between Na(+)-activation kinetics of Na(+)/Li(+) exchange and fasting insulin levels. Na(+)-stimulated Li(+) efflux was studied by raising Na(+)up to 300 mM isoosmotically to measure K(m) for Na(+) and V (max). Li(+) efflux saturated at 150 mM external Na(+)in NT but not in HT because in HT it exhibited a two fold higher Na(+) Km. V(max) was higher in HT than in NT. In hyperinsulinemic (fasting insulin > 10 mu U/ml) HT, V(max) and Na(+) Km were higher than in normoinsulinemic HT. In NT, hyperinsulinemia was not associated to abnormal kinetic properties of Na(+)/Li(+)exchange. Stepwise multiple regression analysis confirmed that the main determinants of a high Km were blood pressure and insulin. Our results show that posttranslational effects of PKC and insulin affect the kinetic properties of NHE-1 in red blood cells and suggest that the differences observed between hypertensives and normotensive subjects can be accounted for by PKC activation and insulin exposure.

Functional link between phosphorylation state of membrane proteins and morphological changes of human erythrocytes.

The Tyr-phosphorylation of the cytoplasmic domain of the major membrane-spanning band 3, rather than the Ser/Thr-phosphorylation of the membrane proteins (spectrin and band 3 itself), might be functionally related to certain morphological changes of human erythrocytes. This view is supported by the following lines of evidence: a) vanadate or its derivative pervanadate (vanadyl hydroperoxide), which markedly increase the Tyr-phosphorylation of band 3 (without practically affecting the Ser/Thr-phosphorylation of spectrin) promotes a crenation of human erythrocytes; b) okadaic acid, which selectively increases the Ser/Thr-phosphorylation of spectrin and other membrane proteins, does not promote any shape change, at least at a level detectable with scanning electron microscopy.

Spermine effect on the binding of casein kinase I to the rat liver mitochondrial structures.

The results indicated here, together with those previously reported, show that spermine, ubiquitous polyamine, while promoting the transmembrane translocation of casein kinase II (CKII) across the outer membrane to more internal compartments of rat liver mitochondria, promotes the binding of casein kinase I (CKI) to the external surface of outer mitochondrial membrane but inhibits its spontaneously occurring binding to more internal structures.

Relationship between membrane protein phosphorylation and intracellular translocation of casein kinase in human erythrocytes.

The present paper shows that an increased phosphorylation of the membrane proteins, promoted by the okadaic acid (strong inhibitor of P-Ser/Thr-protein phosphatase(s)), is accompanied by a release of casein kinase from the membrane into cytosol. Such an intracellular translocation might provide a feedback mechanism for the regulation of the casein kinase catalyzed phosphorylation of membrane proteins in the human erythrocytes.

Spermine-mediated casein kinase II-uptake by rat liver mitochondria.

Spermine, ubiquitous intracellular polyamine, is able to promote the transmembrane translocation of casein kinase CKII through the outer membrane of rat liver mitochondria and its binding to more internal mitochondrial structures. These findings suggest that spermine may play a critical role in regulating the subcellular distribution of casein kinase CKII.

Effect of okadaic acid on membrane protein phosphorylation in human erythrocytes.

Okadaic acid, penetrating the human erythrocytes, almost completely inhibits P-Ser-protein phosphatase activity, whereas it unaffects Ser/Thr-protein kinase activity (casein kinases CKI and CKII), thus promoting a marked increase of the endogenous Ser-phosphorylation level of membrane proteins, such as cytoskeletal spectrin beta-subunit (band 2) and transmembrane band 3 protein. By contrast, the Tyr-phosphorylation state of band 3 protein is practically unaffected by okadaic acid, being unaffected both Tyr-protein kinase and P-Tyr-protein phosphatase activities.

Functional correlation between the Ser/Thr-phosphorylation of band-3 and band-3-mediated transmembrane anion transport in human erythrocytes.

In human erythrocytes, okadaic acid, a potent inhibitor of certain protein phosphatases, promotes a marked increase of Ser/Thr-phosphorylation of membrane proteins, including band-3 protein. Moreover, okadaic acid also increases the band-3-mediated oxalate transport across the membranes, thus suggesting that this process is regulated by Ser/Thr-phosphorylation of transporter band-3 protein.