Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract.

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.

Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.

Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y(2) receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y(2) receptor gene disruption. Pharmacological data suggested the involvement of P2Y(4) and P2Y(6) receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y(4) and P2Y(6) receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca(2+) mobilization. The (m)P2Y(4) receptor was potently, but not selectively, activated by UTP (UTP > or = ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y(6) receptor was highly selective for UDP (UDP >> ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y(4) and (m)P2Y(6) receptors resemble those for nucleotide-promoted chloride transport in murine P2Y(2)(-/-) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (I(sc)) was impaired in the CF mouse, UDP-promoted I(sc) changes were not affected in CF gallbladder epithelium, suggesting that the P2Y(6) receptor is a target for treatment of CF gallbladder disease.

Distribution of ion transport mRNAs throughout murine nose and lung.

Evidence of absorptive or secretory ion transport in different respiratory regions of the mouse was sought by assessing the regional distribution of alpha-, beta-, and gamma-epithelial sodium channel (ENaC; Na(+) absorptive), cystic fibrosis transmembrane conductor regulator (CFTR), and Na(+)-K(+)-2Cl(-) cotransporter mRNAs. High levels of ENaC subunit expression were found in nasal surface epithelium and gland ducts. CFTR was expressed in both superficial nasal respiratory epithelium and glands. These results are consistent with basal amiloride-sensitive Na(+) absorption and cAMP-dependent Cl(-) secretion in murine nasal epithelia. Expression of all three ENaC subunits increased progressively from trachea to terminal bronchioles. Intermediate levels of CFTR and cotransporter expression in bronchial epithelium diminished in bronchioles. The low abundance of CFTR mRNA throughout murine pulmonary epithelium is consistent with functional data that attributes Cl(-) secretion predominantly to an alternative Cl(-) channel. alpha-ENaC as the only mRNA found in all regions of airway epithelia is consistent with the alpha-subunit as requisite for Na(+) absorption, and the increased expression of alpha-, beta-, and gamma-ENaC in distal airways suggests a greater absorptive capability in this region.

Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.

Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.

Spectroscopic studies of nitric oxide (NO) interactions with cobalamins: reaction of NO with superoxocobalamin(III) likely accounts for cobalamin reversal of the biological effects of NO.

Recent reports indicate that oxidized cobalamin, Cbl(III), can interfere with the biological effects of nitric oxide (NO) on vascular and visceral smooth muscle and in other systems. In attempting to elucidate the mechanism of these effects of Cbl(III), we reported that a Cbl(III)NO complex could be detected by electron paramagnetic resonance (EPR) spectroscopy, but not by ultraviolet/visible spectroscopy. Subsequently, others concluded that the alleged Cbl(III)NO complex is detectable by ultraviolet/visible, but not by EPR spectroscopy and provided ultraviolet/visible evidence for an alleged Cbl(III)NO complex. We report further investigation of the interaction of NO with Cbl, using both techniques, Fourier transform infrared (FTIR) spectroscopy and mass spectrometry. Our EPR results and the UV/VIS results of others appear to be experimental artifacts that can now, at least in part, be explained. Under conditions where FTIR measurements readily detect a N-O stretching frequency of NO bound to Fe(II), we do not detect a similar signal that can be ascribed to either Cbl(III)NO or Cbl(II)NO, indicating that neither Cbl(III) nor Cbl(II) form a stable complex with NO. Loss of the Cbl(II) EPR signal and mass spectral detection of N2O upon addition of NO to Cbl(II) solutions, demonstrates that Cbl(II), which is present in aerobic Cbl(III) solutions, reduces NO; however, this reaction does not appear to be fast enough to account for the observed biological effects in aerated media. Nitric oxide also reacts rapidly and irreversibly with the superoxo complex of Cbl(III), Cbl(III)O2-, which is always present in aerated solutions of Cbl(III). We believe that this latter reaction accounts for the observed inactivation of NO by Cbl(III) in biological systems. Because Cbl(III)O2- is spontaneously regenerated from Cbl(II) and O2 in aerated solutions, this may constitute a cyclic mechanism for the rapid elimination (oxidation) of NO. Thus, several physicochemical techniques fail to provide convincing evidence for the existence of stable Cbl(III)NO or Cbl(II)NO complexes but do provide evidence that Cbl species participate in redox reactions with NO under aerobic conditions, thereby inhibiting its physiological roles.

Concurrent production of reactive oxygen and nitrogen species by airway epithelial cells in vitro.

Intracellularly generated reactive species of both oxygen (ROS) and nitrogen (RNS) have been implicated in signaling responses in airway epithelial cells, but these radicals have not been measured directly in such cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithelial (GPTE) cells maintained in primary culture. ROS and RNS were quantified under basal (constitutive) conditions and in response to different stimuli: LPS and TNFalpha [activators of inducible nitric oxide synthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bradykinin, ionophore A23187, and thapsigargin); and exogenous oxidant stress generated by addition of xanthine oxidase to purine (p + XO). Studies with LPS and TNFalpha also were performed using the murine macrophage cell line, RAW 264.7, as a positive control. Intracellular oxidant production was detected from oxidation of dihydrorhodamine to rhodamine. NOx was quantified by either chemiluminescent or fluorescent detection. NOS activity was measured as citrulline production from arginine. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhodamine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodamine). TNFalpha and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activators affected production of oxidants in these cells. Concentrations of NO2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NOx in GPTE and 62% in RAW cells. TNFalpha and LPS both increased NO2 in GPTE cells, but none of the Ca++-mobilizing agents nor p + XO significantly affected intracellular RNS. The results suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in these cells in response to different stimuli.

The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates.

Epidemiologic and occupational studies indicate adverse health effects due to inhalation of particulate air pollutants, but precise biologic mechanisms responsible have yet to be fully established. The tracheobronchial epithelium forms the body's first physiologic barrier to such airborne pollutants, where ciliary movement functions to remove the offending substances caught in the overlying mucus layer. Resident and infiltrating phagocytic cells also function in this removal process. In this paper, we examine the role of reactive oxygen and nitrogen species (ROS/RNS) in the response of airway epithelium to particulates. Some particulates themselves can generate ROS, as can the epithelial cells, in response to appropriate stimulation. In addition, resident macrophages in the airways and the alveolar spaces can release ROS/RNS after phagocytosis of inhaled particles. These macrophages also release large amounts of tumor necrosis factor alpha (TNF-alpha), a cytokine that can generate responses within the airway epithelium dependent upon intracellular generation of ROS/RNS. As a result, signal transduction pathways are set in motion that may contribute to inflammation and other pathobiology in the airway. Such effects include increased expression of intercellular adhesion molecule 1, interleukin-6, cytosolic and inducible nitric oxide synthase, manganese superoxide dismutase, cytosolic phospholipase A2, and hypersecretion of mucus. Ultimately, ROS/RNS may play a role in the global response of the airway epithelium to particulate pollutants via activation of kinases and transcription factors common to many response genes. Thus, defense mechanisms involved in responding to offending particulates may result in a complex cascade of events that can contribute to airway pathology.

Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators.

Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).

Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide variety of respiratory diseases. We investigated mechanisms of ROS-induced mucin secretion by guinea pig tracheal epithelial (GPTE) cells in primary culture, and ROS-induced activation of the second messenger-producing enzyme phospholipase C (PLC), in GPTE cells and in a virally transformed cell line (BEAS-2B) derived from human bronchial epithelium. Mucin secretion was measured by a monoclonal antibody-based enzyme-linked immunosorbent assay, and PLC activation was assessed by anion exchange chromatography. ROS generated enzymatically by xanthine oxidase (XO, 500 microM) in the presence of purine (500 microM) enhanced release of mucin by GPTE cells and activated PLC in GPTE and BEAS cells. Hypersecretion of mucin and activation of PLC in response to purine + XO appeared to occur via an intracellular pathway(s) dependent on endogenously produced nitric oxide and possibly intracellularly generated oxidants. Both responses could be blocked or attenuated by preincubation of the cells with NG-monomethyl-L-arginine, an inhibitor of the enzyme nitric oxide synthase, or with dimethylthiourea, a compound that can react with a variety of intracellular oxidant species. Reactive nitrogen species generated chemically also stimulated secretion of mucin and activated PLC via a mechanism dependent (at least in part) on intracellular oxidant-mediated process(es). The results suggest that intracellularly generated radical species of nitrogen and oxygen may be important modulators of the response of airway epithelial cells to external oxidant stress.

Interactions between hydroxocobalamin and nitric oxide (NO): evidence for a redox reaction between NO and reduced cobalamin and reversible NO binding to oxidized cobalamin.

Interactions of nitric oxide (NO) with various cobalamin species have been examined, apparently for the first time, with both absorption and electron paramagnetic resonance spectroscopy. Only slight shifts in the absorption spectrum of hydroxocobalamin, B12a [Cb(III)], were produced by NO, but dramatic changes in the spectrum of B12r [Cb(III)] were found on addition of NO. The addition of NO shifted the spectrum of Cb(II) to one very similar to that of Cb(III), indicating the oxidation of Cb(II). The addition of NO to Cb(III) resulted in a novel, weak and previously undescribed electron paramagnetic resonance signal. Although it has not been fully characterized, this appears to represent a reversible complex in which NO is liganded to the Cb(III). When NO was added to Cb(II), its strong electron paramagnetic resonance spectrum was replaced by that of this novel species, consistent with oxidation of Cb(II) by NO and then binding of additional NO by the resulting Cb(III). Porcine, aortic endothelial cells were able to partially reduce Cb(III), and release to the supernatant a previously characterized superoxide cobalt(III) complex, but some Cb(II) remained with the cell fraction. These reactions of Cb species could play a role in altering intracellular and intratissue levels of NO.

Bioactivation of nitroprusside by porcine endothelial cells.

Sodium nitroprusside (Na2[(CN)5FeNO], SNP), which is stable, diamagnetic, and not detectable by electron paramagnetic resonance (EPR) spectroscopy, can be activated by one-electron reduction. The initial product, which retains the five cyanides and is here called penta, has a distinctive EPR signal. Penta spontaneously dissociates the trans-cyanide ligand resulting in a second paramagnetic species called tetra, which has a different and distinctive EPR signal. Tetra is able to transfer its NO ligand to a suitable acceptor, and all four equatorial cyanides subsequently dissociate. However, excess free cyanide shifts the tetra-penta equilibrium in the direction of penta and prevents NO release. This study was an attempt to extend the above results on SNP reduction, which were obtained in a model hemoglobin system, to intact porcine cells by characterizing all EPR-detectable intermediates. When porcine aortic endothelial or smooth muscle cells in culture were incubated under anaerobic conditions with SNP, an EPR spectrum was obtained, which could be resolved into the signal for penta and a signal previously described as a nonheme iron-nitrosyl-sulfur complex, Fe-NOSR. Tetra was not detected. This FeNOSR has some differences in its stability and location from that described by others in activated macrophages. When incubations were carried out under air, penta could not be detected, but a somewhat diminished signal for FeNOSR was still detectable. When incubations were carried out in the presence of excess free cyanide, conditions under which reduced SNP does not nitrosylate hemoglobin, the penta signal became stronger and the FeNOSR signal, though decreased, was still observed. Depletion (95%) of intracellular reduced glutathione in endothelial cells had no effect on the FeNOSR signal strength. We conclude that SNP is activated in porcine endothelial cells by a one-electron reduction to penta, which apparently dissociates its trans-cyanide to form tetra which then goes on to form FeNOSR upon reaction with a membrane-bound thiol. Glutathione is not involved in any of these reactions.

An impurity present in some samples of SIN-1 oxidizes it to nitric oxide in anaerobic solutions.

N-Morpholino-N-nitrosoaminoacetonitrile (SIN-1), a nitrovasodilator metabolite of the drug, molsidomine, is widely used in studies on the pharmacology and toxicology of nitric oxide (NO) because solutions of SIN-1 'spontaneously' release NO in a pathway involving molecular oxygen. Preliminary results, however, suggested that SIN-1 could react with hemoglobin in anaerobic solutions to release NO and form NO-hemoglobin. Electron paramagnetic resonance (EPR) studies showed that heme(III) of methemoglobin was not being reduced, thereby not serving as the oxidant in the reaction generating NO-hemoglobin. When anaerobic solutions of SIN-1 and hemoglobin kept in the light and in the dark were compared, substantially more NO-hemoglobin was eventually generated in the dark, indicating that SIN-1 did not undergo photochemical decomposition to NO under the conditions used. Solutions of NO-hemoglobin were equally stable under these same conditions of light and dark. The initial pH (7.0) of stirred, unbuffered solutions of SIN-1 decreased at nearly the same rate whether or not oxygen was present. Anaerobic and aerobic solutions plateaued at the same pH, namely 5.4. Anaerobic solutions of SIN-1 in phosphate buffer, pH 7.4, released NO to the gas phase, where it was identified by trapping it with hemoglobin on agarose beads and deriving the characteristic NO-hemoglobin EPR spectrum. High pressure liquid chromatography revealed the presence of an unknown species with a retention time between that of SIN-1 and molsidomine. Samples from two different lots of SIN-1 contained this impurity which appears to oxidize SIN-1 to products that release NO in the absence of oxygen. This unknown impurity may be unstable toward light.

Distribution of chlordecone to liver plasma membranes and recovery from hepatobiliary dysfunction in rats.

Chlordecone (CD) impairs biliary excretion of organic anions (including phenolphthalein glucuronide (PG), imipramine polar metabolites, and taurocholate) without evidence of hepatocellular necrosis in rats. In this study we investigated the hypothesis that CD-induced hepatobiliary dysfunction is dependent on CD concentration in liver plasma membranes where it inhibits active transport in vitro. Rats were treated by gavage (0 or 60 mg CD/kg in corn oil) 24 or 72 h prior to bile duct cannulation. Biliary excretion of PG, a marker of hepatic organic anion transport, and [14C]mannitol, a marker of passive transcellular permeability, was determined. Biliary excretion of PG decreased approximately 25% in rats 24 h after CD treatment, however rats recovered control PG excretion rates 72 h after CD treatment. Recovery of PG excretion occurred despite higher liver homogenate [14C]CD concentrations at 72 h than at 24 h after [14C]CD treatment. Biliary clearance of [14C]mannitol decreased both 24 h and 72 h after treatment. Even though the amount of [14C]CD retained in the liver was greater at 72 h than at 24 h after treatment, the concentration of [14C]CD in isolated liver plasma membranes (LPM) was the same (3.5-3.9 nmol/mg protein) at both times. There was a significant reduction in 5'-nucleotidase activity of LPM at 24 h but not at 72 h after CD. This study demonstrated no correlation between recovery from CD-induced hepatobiliary dysfunction and whole liver accumulation. Altered subcellular [14C]CD distribution (reduced LPM-to-homogenate concentration ratio was coincident with recovery.

Active calcium absorption in primary cultures of cortical collecting duct cells.

Primary cultures of rabbit cortical collecting duct (CCD) cells demonstrated accumulation of Ca at the basolateral (BL) side when cultured on either impermeable or permeable supports. Cell monolayers cultured on impermeable plastic surfaces absorbed Ca with such avidity that hydroxyapatite crystals formed. When cultured on a permeable difference. A steady-state BL/A [Ca] ratio of 120 developed across monolayers in 24 h on days 6 through 8 postseeding. Initial rates of unidirectional 45Ca fluxes on days 6 through 8 indicated a negligible BL to A flux (5.4 +/- 2.6 nmol.h-1 x cm-2) compared with A to BL 45Ca flux (99.4 +/- 19.4 nmol.h-1 x cm-2). Parathyroid hormone applied to the BL side had no significant effect on either unidirectional 45Ca flux, but the second messenger analog, 8-bromoadenosine cyclic monophosphate, increased the A to BL flux by 65%. Inhibiting the Na(+)-K+ ATPase with ouabain (10(-4) M) reduced the A to BL flux by 77%; however, a significant net A to BL flux still remained. Apical addition of amiloride (2 x 10(-5) M) did not affect either unidirectional 45Ca flux. In addition, the inorganic Ca channel blockers Ni2+ (100 microM and 1 mM), La3+ (100 microM and 1 mM), and Cd2+ (20 and 50 microM) did not significantly inhibit either unidirectional 45Ca flux. These results demonstrate that CCD monolayers actively absorb Ca and this can be stimulated by cyclic AMP, raising the possibility that apical Ca entry does not involve amiloride-sensitive channels, or typical Ca channels.

Effects of temperature, oxygen, heme ligands and sulfhydryl alkylation on the reactions of nitroprusside and nitroglycerin with hemoglobin.

Nitrovasodilators react with hemoglobin (Hb) to form heme(III) and nitric oxide (NO)Hb. These reactions can be exploited as models for events that take place at the cellular level leading to the biological effects of the prodrugs. Sodium nitroprusside (SNP) is known to undergo a one-electron reduction in its reaction with heme(II), resulting in the labilization of the cyanide ligand trans to the NO ligand. This reduced form is here called "penta." Upon dissociation of the trans-cyanide, the resulting species is here called "tetra." Dissociation of the trans-cyanide is obligatory for transfer of the NO to a heme(II) group. NO release from penta is blocked by excess free cyanide in solution, which prevents the formation of tetra. As reported here, both penta and tetra had unique EPR signals when frozen at -196 degrees, but only tetra gave an EPR signal at 22 degrees. NOHb also has a unique EPR signal, but it could not be detected when SNP was incubated with Hb in air or 10 or 5% oxygen. NOHb was detected in similar incubations under 1% oxygen, but the levels were 3- to 10-fold lower than those found under 100% nitrogen. The concentration of tetra was also much lower under 1% oxygen and penta was not detectable, suggesting that oxygen may either shift the penta-tetra equilibrium towards tetra or that penta may be susceptible to oxidation by molecular oxygen. Nitroglycerin (GTN) also generated much less NOHb but more heme(III) under 1% oxygen than under nitrogen. Carbon monoxide (CO), which binds to heme(II), completely blocked the reactions of SNP and GTN with Hb, whereas N-ethylmaleimide (NEM) alkylation of globin sulfhydryl groups increased both NOHb and heme(III) formation. 13C NMR studies on uniformly 13C-labeled SNP suggested that oxygen had little effect on the concentrations of the NMR-detectable species in the reaction. In summary, the most oxygen-sensitive step in the nitrosylation of Hb by SNP was probably the transfer of NO to heme(II). However, the penta-tetra equilibrium was affected by oxygen, temperature and cyanide. No evidence was found for the involvement of the globin sulfhydryl groups in either the GTN or the SNP reaction with Hb.

Identification of increased nitric oxide biosynthesis during pregnancy in rats.

We reported previously that plasma levels, urinary excretion, and metabolic production of cyclic guanosine 3',5'-monophosphate (cGMP) are increased in gravid rats, and postulated that endogenous nitric oxide (NO), a potent vasodilator and immune modulator, may mediate this change. Four lines of evidence are now presented demonstrating increased biosynthesis of NO during pregnancy in rats: 1) Urinary excretion and plasma levels of the stable NO metabolite, nitrate, are elevated in pregnant rats; urinary excretion of nitrate is increased in pseudopregnant rats. 2) The urinary excretion of cGMP also increases during pregnancy and pseudopregnancy, paralleling the rise in urinary nitrate excretion. 3) Chronic treatment with the NO synthase inhibitor, NG-nitroarginine methyl ester (NAME), inhibits the increase in urinary nitrate excretion. 4) Nitric oxide hemoglobin is detected by electron paramagnetic resonance spectroscopy in the blood of pregnant, but not in nonpregnant, rats. The results show endogenous NO production is increased in gravid rats. This finding raises the possibility that NO may contribute to maternal vasodilation and uterine immune suppression of normal pregnancy.

Chlordecone impairs Na(+)-stimulated L-[3H]glutamate transport and mobility of 16-doxyl stearate in rat liver plasma membrane vesicles.

Chlordecone (CD) treatment of rat liver plasma membranes (LPM) provided in vitro evidence for mechanisms of in vivo liver dysfunction caused by CD. LPM preparations enriched 14- to 19-fold in the bile canalicular markers gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were isolated from male Sprague-Dawley rats. CD inhibited the bile canalicular-specific active transport of Na(+)-stimulated L-[3H]glutamate in LPM vesicles. CD (0.08 and 0.5 mumol/mg protein) reduced both the initial velocity and the maximum level of Na(+)-stimulated L-[3H]glutamate uptake without significantly reducing Na(+)-independent uptake. In vitro treatment of LPM with CD (0.2-1.0 mumols/mg protein) also reduced the mobility of a 16-doxyl stearate spin label probe in a concentration-dependent manner. No change in mobility was apparent at CD concentrations below 0.2 mumol/mg protein. These results demonstrated that CD impaired a bile canalicular-specific transport system and induced liver plasma membrane perturbation. Na(+)-stimulated L-[3H]glutamate uptake was more sensitive to CD than was detectable immobilization of the spin label probe.