Characterization of oxidation of glutathione by cytochrome c.

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis-elevated pH, Ca2+ level and Mg2+-increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

17-ß-estradiol Decreases Neutrophil Superoxide Production through Rac1.

Neutrophil granulocytes form the biggest free radical producing system of the human body. The importance of this system in atherosclerotic plaque formation and other free radical mediated disorders is confirmed by both in vivo and in vitro studies. Estrogen's effect on free radical production involves multiple estrogen receptors and occurs both on transcriptional and on protein phosphorylational level. Estrogen decreases the superoxide production of neutrophil granulocytes in such a short time frame it is unlikely to be mediated by transcription regulation. We investigated the underlying mechanism through which the mentioned estrogen effect takes place using an immunabsorption-based method. Phosphorylation data of 43 different messenger proteins were used for pathway analysis. The newly identified pathway involved largely second messengers from previously described non-genomic estrogen effects and affected superoxide production via Rac1 - an important regulator of free radical production and chemotaxis. Selective inhibition of the participating second messengers altered superoxide production in the predicted direction confirming that this pathway is at least partly responsible for the effect of 17-ß-estradiol on chemoattractant induced superoxide production.

Regulation of nitric oxide synthase activity by tetrahydrobiopterin in human placentae from normal and pre-eclamptic pregnancies.

The possible regulatory role of tetrahydrobiopterin (BH(4)) in Type III nitric oxide synthase (NOS III) activity of human placentae from first trimester, term and pre-eclamptic pregnancies was investigated. In homogenates of first-trimester or term placentae, BH(4)stimulated NOS III activity up to 2.5-fold in a concentration dependent manner from 20 n m to 1 microm BH(4), and half-maximal stimulation (EC(50)) was observed at 100-110 n m. No significant further stimulation was detectable over an extended concentration range from 1 microm to 50 microm BH(4). NOS III present in microsomal and gel-filtered cytosol fractions exhibited similar BH(4)-activation patterns, with an identical EC(50)value of 50 n m. Remarkably, tissue concentrations of BH(4)showed a marked decrease in term placentae (57+/-23 n m, mean+/-s.d., n=26) relative to first-trimester placentae (189+/-79 n m, mean+/-s.d., n=17), suggesting that alterations in cellular BH(4)concentrations may play a more significant role in the regulation of NOS III activity in late pregnancy. Placental homogenates from 10 pre-eclamptic pregnancies exhibited two distinct types of response to BH(4). In seven placental homogenates, addition of physiological concentrations of BH(4)(20 n m to 1 microm) elicited no increase whatsoever in basal NOS III activity, and only high BH(4)concentrations (50 microm) caused notable stimulation (BH(4)resistant group). In contrast, in three of 10 placental homogenates both physiological and 50 microm BH(4)concentrations stimulated NOS III to levels similar to that of normal placentae (BH(4)responsive group). There were no appreciable differences in the clinical presentation of pre-eclampsia between the two groups. Importantly, BH(4)concentrations in pre-eclamptic placentae were comparable with those of normal, control placentae. Taken together, the observations suggest that BH(4)controls NOS III activity in the human placenta, and a defect in BH(4)regulation of NOS III may contribute to the development of pre-eclampsia. A model implicating the malfunction of placental NOS III rather than its actual tissue level in the pathogenesis of pre-eclampsia is discussed.

Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta.

We have found in a previous study that 5-50 microM tetrahydrobiopterin (BH4) stimulated 1.2-2.5-fold Ca(2+)-dependent nitric-oxide synthase (NOS) activity in homogenates prepared from primordial human placentae. Now we report on the dramatic, about sixfold, activating effect of BH4 on this activity measured in microsomal preparations. Firstly, both in the absence and presence of BH4, arginine bound to kinetically homogeneous sites, with no significant change between the apparent KM values for arginine (3.12 +/- 1.99 microM and 2.06 +/- 1.13 microM in the absence and presence of 50 microM BH4 respectively, mean +/- s.d., n = 3). On the other hand, the Vmax values measured in different pools of placenta tissue varied between 2.5-7.55 (no BH4 added) and 13.3-58.5 (with BH4 added) pmol/min/mg protein. Secondly, the microsomal preparations responded sensitively to BH4 addition. A dose-response study indicated that as low as 79 nM final BH4 concentration stimulated NOS activity half-maximally, and 1 microM BH4 resulted in an almost maximal effect. Thirdly, immunoblot analysis combined with laser densitometric evaluation demonstrated that BH4 efficiently promoted the aggregation of microsomal NOS type III isozyme into a protein having the characteristics (electrophoretic mobility, resistance of SDS) of the dimeric form. Half-maximal dimerizing activity was reached at 148 +/- 33 nM BH4 (mean +/- s.d., n = 3), whereas 1 microM BH4 led to almost, maximal aggregation of monomers. This is the first time that BH4-induced dimerization of a NOS type III isoform has been demonstrated. Considering that human placenta predominantly expresses NOS type III isoform and BH4 concentration in this tissue is 207 +/- 87 nM, the present results strongly suggest that the dimerizing effect of BH4 is a crucial physiological mechanism for the assembly of active Ca(2+)-dependent NOS in the human primordial placenta.

Calcium-dependent nitric oxide synthesis is potently stimulated by tetrahydrobiopterin in human primordial placenta.

Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (> 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.

Ca(2+)-dependent and Ca(2+)-independent NO-synthesizing activities of human primordial placenta.

In order to localize the site of production of nitrogen monoxide (NO) in first trimester human pregnancy, the cytosol and microsome fractions prepared from homogenized primordial placentas were tested for NO-synthase (NOS) activities by measuring the NADPH-dependent conversion of [3H]arginine to [3H]citrulline. Our results demonstrate that Ca(2+)-dependent enzyme activities are present in both fractions, whereas microsomes exhibit significant Ca(2+)-independent enzyme activity too. The highest specific activity is measurable in the presence of Ca2+ with microsomes, this activity is about 2-fold higher than the Ca(2+)-dependent specific activity of the cytosol. The Ca(2+)-independent specific NOS activity is about 30% of the Ca(2+)-dependent microsomal activity. The microsomal Ca(2+)-dependent NOS activity is inhibited by 50% in the presence of 0.5 mM aminoguanidine (AG), whereas the Ca(2+)-independent activity does not respond to this concentration of AG, suggesting that it is not the inducible isoform of NOS. Our results indicate that (I) NOS activity is present from an early phase of placental development, (II) the NOS activity is of trophoblastic origin, since the primordial placenta is avascular and (III) NO-production by the primordial placenta can proceed in the absence of any Ca(2+)-mobilizing agonist.

Tetrahydrobiopterin preferentially stimulates activity and promotes subunit aggregation of membrane-bound calcium-dependent nitric oxide synthase in human placenta.

Type III nitric oxide synthase (NOS III) is responsible for > 90% of nitric oxide (NO) synthesizing activity in first trimester placentae. Enzyme activity is distributed between cytosolic (30%) and membrane-bound forms (70%), with highest specific activity observed in microsomal fractions. In the present study, the effect of tetrahydrobiopterin (BH4) on subunit structure and activity of microsomal and cytosolic NOS III was compared. As revealed by immunoblot analysis, incubation of microsomal membranes with 50 microM final concentration BH4 for 10 min at 37 degrees C resulted in a striking conversion of monomeric NOS III into a protein having the characteristics (electrophoretic mobility, resistance to sodium dodecyl sulphate) of the homodimeric form. In contrast, BH4 induced significantly less marked changes in the NOS III dimer content of cytosolic fractions. Enzyme activity in microsomes is stimulated approximately 6-fold upon addition of 50 microM BH4, while only a 2-fold activation is detectable in cytosolic fractions. Taken together, the observations suggest that BH4 activates NOS III in the primordial human placenta by promoting its subunit assembly in the membrane, while cytosolic NOS III is relatively insensitive to BH4. Compartment-specific action of BH4 represents a novel mechanism which is implicated in the regulation of placental NOS activity.

Differential response of basal and tetrahydrobiopterin-stimulated activities of placental type III nitric oxide synthase to sodium dodecyl sulphate: relation to dimeric structure.

The major enzyme isoform that synthesizes nitric oxide (NO) in first trimester human placentae is endothelial or type III NO-synthase (NOS III) which exhibits high specific activity in the microsomal fraction. In the present study, we investigated the possible protective and enzyme-stabilizing role of tetrahydropterin (BH4). The anionic detergent, sodium dodecyl sulphate (SDS) and thermal stress (freeze-thaw) were used as non-specific 'subunit-dissociating' agents, and alterations in enzyme activity and subunit structure were investigated. SDS (> or =0.05% w/v) resulted in significant inhibition both of basal and BH4-stimulated activities of NOS III, but the latter responded more sensitively. Preincubation of microsomes with SDS (> or =0.1%, w/v), followed by incubation in an SDS-depleted reaction mixture led to an inhibition of BH4-stimulated enzyme activity, while no change in the basal activity was noted. This indicated that the SDS effect is only fully reversible in the case of basal activity. Considering that basal activity is due to the presence of endogenous BH4 tightly bound to the enzyme, this differential sensitivity of basal and BH4-stimulated enzyme activities to SDS may be related to a putative differential protective effect of BH4 on the two subunits of the NOS III dimer. Western blot analysis revealed that the SDS-induced inhibition of enzyme activity could not be ascribed to disruption of the dimeric structure. This finding confirms the view that SDS may affect NOS III activity without necessarily deteriorating quaternary protein structure. Nevertheless, BH4 is essential in maintaining dimeric structure under denaturing conditions, e.g. SDS treatment and freezing/thawing; it is even able to reverse the dissociation caused by SDS. A model describing the interaction between BH4 and NOS III, and its implications on the physiology and pathology of the human placenta, is discussed.