Early life hypoxic or hypoxic/hypercapnic stress alters acute ventilatory sensitivity in adult mice.

In this study we investigated the effect of early life conditioning (hypoxia ± hypercapnia) on adult acute ventilatory sensitivity to hypoxia and hypercapnia. Mice were exposed to either hypoxia (5% O(2)) or hypoxia/hypercapnia (5% O(2)/8% CO(2)) in a normobaric chamber for 2 h at postnatal day 2 (P2), and then returned to normoxia. At 3 months of age, hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR) were measured using a plethysmograph system. Results showed that HVR was significantly decreased in the P2-hypoxia mice but not in the P2 hypoxia/hypercapnia mice as compared to the P2-normoxic mice, respectively. However, HCVR was significantly decreased in the P2 hypoxia-hypercapnia group but not in the P2-hypoxia group. These data suggest early postnatal hypoxic stress vs. hypoxic/hypercapnic stress plays different roles in fetal programming of the respiratory control system as shown by altered adult acute ventilatory sensitivity.

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Role of the conserved arginine pair in proton and electron transfer in cytochrome C oxidase.

A hydrogen-bonded network is observed above the hemes in all of the high-resolution crystal structures of cytochrome oxidases. It includes water and a pair of arginines, R481 and R482 (Rhodobacter sphaeroides numbering), that interact directly with heme a and the heme a(3) propionates. The hydrogen-bonded network provides potential pathways for proton release. The arginines, and the backbone peptide bond between them, have also been proposed to form part of a facilitated electron transfer route between Cu(A) and heme a. Our studies show that mutations of R482 (K, Q, and A) and R481 (K) retain substantial activity and are able to pump protons, but at somewhat reduced rates and stoichiometries. A slowed rate of electron transfer from cytochrome c to Cu(A) suggests a change in the orientation of cytochrome c binding in all but the R to K mutants. The mutant R482P is more perturbed in its structure and is altered in the redox potential difference between heme a and Cu(A): +18 mV for R482P and +46 mV for the wild type (heme a - Cu(A)). The electron transfer rate between Cu(A) and heme a is also altered from 93000 s(-1) in the wild type to 50 s(-1) in the oxidized R482P mutant, reminiscent of changes observed in a Cu(A)-ligand mutant, H260N. In neither case is the approximately 2000-fold change in the rate accounted for by the altered redox potentials, suggesting that both cause a major modification in the path or reorganization energy of electron transfer.

pH dependence of the donor side reactions in Ca2+-depleted photosystem II.

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.

Direct electrochemistry of cytochrome C at nanocrystalline boron-doped diamond.

Direct electron transfer of horse heart cytochrome c is measured at a nanocrystalline boron-doped diamond thin-film electrode. A quasi-reversible, diffusion-controlled cyclic voltammetric response is observed for untreated diamond. The peak currents change linearly with the concentration, and importantly, there is no electrode fouling. The results, observed for a hydrogen-terminated and uncharged surface, (i.e., no ionizable carbon-oxygen functional groups), raise interesting questions about the necessary surface interactions of cytochrome c for relatively rapid electrode kinetics.

Resonance Raman detection of the Fe-S bond in endothelial nitric oxide synthase.

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.

High-field EPR study of tyrosyl radicals in prostaglandin H(2) synthase-1.

Various tyrosyl radicals generated by reaction of both native and indomethacin-inhibited ovine prostaglandin H synthase-1 with ethyl hydrogen peroxide were examined by using high-field/high-frequency EPR spectroscopy. The spectra for the initially formed tyrosyl radical commonly referred to as the "wide-doublet" species and the subsequent "wide-singlet" species as well as the indomethacin-inhibited "narrow-singlet" species were recorded at several frequencies and analyzed. For all three species, the g-values were distributed. In the case of the wide doublet, the high-field EPR spectra indicated that dominant hyperfine coupling was likely to be also distributed. The g(x)-values for all three radicals were found to be consistent with a hydrogen-bonded tyrosyl radical. In the case of the wide-doublet species, this finding is consistent with the known position of the radical and the crystallographic structure and is in contradiction with recent ENDOR measurements. The high-field EPR observations are consistent with the model in which the tyrosyl phenyl ring rotates with respect to both the protein backbone and the putative hydrogen bond donor during evolution from the wide-doublet to the wide-singlet species. The high-field spectra also indicated that the g-values of two types of narrow-singlet species, self-inactivated and indomethacin-inhibited, were likely to be different, raising the possibility that the site of the radical is different or that the binding of the inhibitor perturbs the electrostatic environment of the radical. The 130 GHz pulsed EPR experiments performed on the wide-doublet species indicated that the possible interaction between the radical and the oxoferryl heme species was very weak.