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PubMed1984

Photoaffinity labelling of dopamine receptors. Synthesis and binding characteristics of azapride.

A new compound, azapride, i.e. 4-azido-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benza mide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation-induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation-induced inactivation by azapride remained unchanged in the presence of the scavenger p-aminobenzoic acid. These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.

Affinity Labels↗

PubMed1971

Comparison of two plating media for the isolation of Erysipelothrix rhusiopathiae from enrichment broth culture.

Sodium azide-crystal violet-agar and a modified blood-azide (MBA)-agar were used to isolate Erysipelothrix rhusiopathiae from swine tissues. There was no significant difference in the number of isolations. However, 78% of the isolants from modified blood-azide medium required only 24 hr of incubation, whereas all of the isolants obtained on sodium azide-crystal violet medium required 48 hr.

Agar↗

PubMed2006

Critical role of 5'-AMP-activated protein kinase in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

5'-AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptation under conditions of ATP depletion, such as hypoxia and inhibition of oxidative phosphorylation. Whether activation of AMPK is critical for stimulation of glucose transport in response to inhibition of oxidative phosphorylation is unknown. Here we found that treatment of Glut1-expressing Clone 9 cells with sodium azide (5 mM for 2 h) or the AMPK activator 5'-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 2 mM for 2 h) stimulated the rate of glucose transport by two- to fourfold. Use of small interference RNA (siRNA) directed against AMPKalpha(1) or AMPKalpha(1) + AMPKalpha(2) (total AMPKalpha) resulted in a significant inhibition of the glucose transport response and the content of phosphorylated AMPKalpha(1) + phosphorylated AMPKalpha(2) (total p-AMPKalpha) and phosphorylated acetyl-CoA carboxylase (p-ACC) in response to azide. Transfection with siRNA directed against AMPKalpha(2) did not affect the glucose transport response. The efficacy of transfection with siRNAs in reducing AMPK content was confirmed by Western blotting. Incubation of cells with compound C, an inhibitor of AMPK, abrogated the glucose transport response and abolished the increase in total p-AMPK in azide-treated or hypoxia-exposed cells. Simultaneous exposure to azide and AICAR did not augment the rate of transport in response to AICAR alone. There was no evidence of coimmunoprecipitation of total p-AMPKalpha with Glut1. However, LKB1 was associated with total p-AMPKalpha. We conclude that activation of AMPK plays both a sufficient and a necessary role in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

AMP-Activated Protein Kinase Kinases↗

PubMed2006

Characterization of cell clones isolated from hypoxia-selected renal proximal tubular cells.

Under hypoxia, some cells survive and others are irreversibly injured and die. The factors that determine cell fate under stress remain largely unknown. We recently selected death-resistant cells via repeated episodes of hypoxia. In the present study, 80 clones were isolated from the selected cells and their response to apoptotic injury was characterized. Compared with the wild-type cells, the isolated clones showed a general resistance to apoptosis: 13 were extremely resistant to azide-induced apoptosis, 10 to staurosporine, and 9 to cisplatin. The cell clones that most consistently demonstrated resistance or sensitivity to injury were further studied for their response to azide treatment. Azide induced comparable ATP depletion in these clones and wild-type cells. Hypoxia inducible factor-1 (HIF-1) was upregulated in several clones, but the upregulation did not correlate with cell death resistance. The selected clones maintained an epithelial phenotype, showing typical epithelial morphology, forming "domes" at high density, and expressing E-cadherin. Azide-induced Bax translocation and cytochrome c release, two critical mitochondrial events of apoptosis, were abrogated in death-resistant clones. In addition, cell lysates isolated from these clones showed lower caspase activation on addition of exogenous cytochrome c. Bax, Bak, and Bid expression in these clones was similar to that in wild-type cells, whereas Bcl-2 expression was higher in all the selected clones and, interestingly, Bcl-xL was markedly upregulated in the most death-resistant clones. The results suggest that apoptotic resistance of the selected clones is not determined by a single factor or molecule but, rather, by various alterations at the core apoptotic pathway.

Adenosine Triphosphate↗

PubMed1973

Degradation of thyroid hormones by phagocytosing human leukocytes.

Thyroxine (T(4)) and triiodothyronine (T(9)) are rapidly degraded by a purified preparation of myeloperoxidase (MPO) and H(2)O(2) with the formation of iodide and material which remains at the origin on paper chromatography. Deiodination by MPO and H(2)O(2) occurs more readily at pH 7.0 than at pH 5.0 in contrast to iodination by this system which is known to occur more readily at pH 5.0 than at pH 7.0. Degradation is inhibited by azide, cyanide, ascorbic acid, and propylthiouracil. Methimazole stimulates deiodination by MPO and H(2)O(2) but inhibits this reaction when MPO is replaced by lactoperoxidase or horseradish peroxidase.Intact human leukocytes, in the resting state, degrade T(4) and T(3) slowly: degradation, however, is increased markedly during phagocytosis of preopsonized particles. Serum inhibits this reaction. T(3) can be detected as a minor product of T(4) degradation. Proteolytic digestion of the reaction products increases the recovery of monoiodotyrosine. The fixation of iodine in the cytoplasm of leukocytes which contain ingested bacteria was detected radioautographically. Chronic granulomatous disease leukocytes, which are deficient in H(2)O(2) formation, degrade T(4) and T(3) poorly during phagocytosis. MPO-deficient leukocytes degrade the thyroid hormones at a slower rate than do normal leukocytes although considerable degradation is still observed. Azide, cyanide, ascorbic acid, and propylthiouracil which inhibit certain peroxidasecatalyzed reactions inhibit degradation by normal leukocytes; however, inhibition is incomplete. Formation of iodinated origin material is inhibited to a greater degree by azide, cyanide, and propylthiouracil than is deiodination. Methimazole inhibits the formation of iodinated origin material by both normal and MPO-deficient leukocytes. However, deiodination by normal leukocytes is stimulated and that of MPO-deficient leukocytes is unaffected by methimazole. Hypoxia inhibits the degradation of T(4) and T(3) by untreated normal or MPO-deficient leukocytes and by normal leukocytes treated with azide or methimazole. These data suggest that both MPO-dependent and MPO-independent systems are involved in the degradation of T(4) and T(3) by phagocytosing leukocytes. The role of leukocytic degradation of T(4) and T(3) in thyroid hormone economy and in leukocytic microbicidal activity is considered.

Ascorbic Acid↗

PubMed2004

Involvement of sulfide:quinone oxidoreductase in sulfur oxidation of an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1.

The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined. The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO. Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO. Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells. Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol. The transcription of three genes, encoding an aa(3)-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction. The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells. A model is proposed for the oxidation of reduced inorganic sulfur compounds in A. ferrooxidans NASF-1 cells.

Acidithiobacillus↗

PubMed1999

Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II.

The Chinese hamster V79 lung cell in vitro micronucleus assay was adapted to detect and quantify phototoxicity and photogenotoxicity of fluoroquinolones. Using this assay, the quinolones were ranked in terms of decreasing phototoxicity: clinafloxacin >> lomefloxacin, sparfloxacin >> trovafloxacin, nalidixic acid, ofloxacin, ciprofloxacin > enoxacin, norfloxacin. This rank order agrees well with published studies utilizing various other phototoxicity models and establishes this approach as a fast and sensitive way to characterize the phototoxic potential of quinolones. Nearly complete inhibition of phototoxicity was observed if the cells were pretreated for as little as 1 min with 10-20 mM sodium azide prior to the addition of quinolone. An identical azide effect was seen in unirradiated quinolone- and etoposide-treated cells. These findings are consistent with a model in which sodium azide renders DNA topoisomerase II catalytically inactive. In this state, topoisomerase II cannot initiate DNA strand cleavage and the DNA/topoisomerase complex becomes insensitive to quinolones and other topoisomerase II inhibitors. The fact that azide reduces both UV-dependent and UV-independent toxicity and clastogenicity strongly suggests a common mechanism of toxicity dependent on the formation of topoisomerase-induced DNA double-strand breaks.

Animals↗

PubMed1999

New insights on flow-independent mechanisms of 99mTc-HMPAO retention in nervous tissue: in vitro study.

UNLABELLED: SPECT using 99mTc-hexamethyl propyleneamine oxime (HMPAO) mainly reflects regional cerebral blood flow, however metabolic abnormalities also affect the retention of 99mTc-HMPAO. METHODS: To rule out any flow factor, a test-tube model was used to evaluate the effects of metabolic alterations both on intracellular trapping of 99mTc-HMPAO and on extracellular glutamate and lactate dehydrogenase (LDH) outflow from rat brain slices. RESULTS: Under control conditions, slices took up 7.0%+/-1.4% of 99mTc-HMPAO contained in the medium, whereas prelabeled slices released 10.8%+/-2.6% of their radioactive content; glutamate and LDH outflow were 49.1+/-21.6 pmol/mg protein/ min and 4.8+/-0.9 U/L/mg protein/min, respectively. The control medium was altered by adding a metabolic poison (5 mmol/L azide), removing glucose and replacing O2 with N2 to mimic ischemia (in vitro ischemia) and replacing Krebs solution with hypotonic medium to evoke cell lysis. Both azide and in vitro ischemia induced a significant increase in 99mTc-HMPAO release (15.8%+/-3.3% and 18.3%+/-6.2%, respectively), without any modification in LDH efflux. However, only azide reduced the uptake of the tracer. Conversely, glutamate outflow was massive during in vitro ischemia and was far lower during azide treatment. Under hypotonic medium conditions, the release of 99mTc-HMPAO, glutamate and LDH were dramatically increased. Surprisingly, a two-fold increase of 99mTc-HMPAO uptake was also found. When 1 mmol/L glutathione was added to the medium, to convert native lipophilic 99mTc-HMPAO into hydrophilic derivatives, tracer uptake was inhibited both under control and hypotonic medium conditions. CONCLUSION: This study provides evidence that not only poisoning of the tissue but also in vitro ischemia induced a reduction of 99mTc-HMPAO retention. Moreover, we demonstrated that injuries causing cell membrane disruption led to hyperfixation of 99mTc-HMPAO.

Animals↗

PubMed2001

Protein kinase C agonists enhance phagocytosis of P. aeruginosa by murine alveolar macrophages.

Pulmonary alveolar macrophages (AMphis) are incompetent to phagocytose unopsonized Pseudomonas aeruginosa, but ingestion by other macrophage phenotypes (i.e., peritoneal macrophages) occurs efficiently. The purpose of this study was to explore factors that might control such phenotypic differences. Our laboratory has demonstrated that AMphis exposed to sodium azide display enhanced phagocytosis of P. aeruginosa. Here we report that the phagocytic-enhancing effect of sodium azide was abrogated by inhibitors of protein kinase C (PKC). Furthermore, the addition of PKC agonists, such as phorbol myristate acetate (PMA), and tumor necrosis factor alpha (TNF-alpha), mimicked the phagocytic enhancing effect of sodium azide. We conclude that AM4phis are normally incompetent to phagocytose P. aeruginosa. Factors that up-regulate AMphi function (azide, PMA, TNF-alpha) can reverse the phagocytic incompetence in vitro. Although these compounds are not appropriate candidate therapeutic agents, their effects provide insights for understanding of the pathways responsible for regulation of P. aeruginosa phagocytosis.

Animals↗

PubMedDate not supplied

[Modelling of Mg2+, ATP-dependent mitochondrial Ca ions transport in smooth muscle cells using protonophore CCCP-sensitive fluorescent tetracycline].

Mg2+, ATP-dependent Ca2+ accumulation in the rat myometrial mitochondria was investigated in complex experiment using Ca2+ isotope (45Ca2+) and Ca(2+)-sensitive label tetracycline. Monotonous increase of the fluorescence signal, insensitive to thapsigargin (100 nM) was observed with following establishing the stationary state of incubation at 2 min. which correlates with results obtained using isotope technique. Experiments with isotope label signify, that protonophore CCCP, ruthenium red and sodium azide, in concentration 1 microM, 10 microM and 10 mM respectively, totally inhibits the accumulation of the Ca ions in mitochondria. At the same time, in conditions of Mg2+, ATP-dependent Ca2+ accumulation modeling in these cellular structures, CCCP and sodium azide, used in the same concentration, diminished tetracycline fluorescence signal increase. In the same conditions, the introduction of the CCCP (1 mM) into the incubation medium at 75 sec. after initiation of the transport process induced reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP presence in the medium. According to data obtained in the experiment, using Ca2+ isotope, Ca(2+)-ionophore A-23187 induces both the reversible release of previously accumulated Ca ions, and cause reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP (1 mM) and sodium azide (10 mM) presence in the incubation medium. Conclusion was drawn that the thapsigargin-insensitive and CCCP, sodium azide and A-23187-sensitive tetracycline fluorescence increasing in case of modeling of Mg2+, ATP-dependent Ca2+ accumulation in myometrial mitochondria reflect the Ca2+ uniporter functioning in those subcellular structures.

Animals↗

PubMed1992

Accumulation of Golgi-processed secretory proteins in an organelle of high density upon reduction of ATP concentration in rat hepatocytes.

We have previously shown that when rat hepatocytes are incubated with 4 mM azide, which reduces the intracellular ATP concentration to about 30% of its normal level, secretory proteins are reversibly arrested within the cell. Analysis of haptoglobin after 150 min of azide incubation shows that its carbohydrates have been processed by Golgi enzymes (Persson, R., Ahlström, E., and Fries, E. (1988) J. Cell Biol. 107, 2503-2510). Here, we have further characterized the site of arrest. Subcellular fractionation by density gradient centrifugation showed that albumin and haptoglobin fractionated like a marker for the endoplasmic reticulum. Localization of albumin by immunoelectron microscopy showed, however, that it occurred in flattened cisternae and that the endoplasmic reticulum was devoid of the protein. A possible explanation for these results is that the azide treatment blocks transport through the Golgi complex, leading to an accumulation of secretory proteins in a pre- or early Golgi compartment of high density. This compartment could contain sufficient amounts of Golgi enzymes to carry out the observed carbohydrate processing upon prolonged incubation or possibly acquire them as an effect of the azide treatment.

Adenosine Triphosphate↗

PubMed1979

Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.

Affinity Labels↗

PubMed1986

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase.

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.

Animals↗

PubMed1982

[Faecal streptococci as indicator organisms of drinking water].

The rules of the European Economic Community to harmonize the hygienic quality of drinking water write out the faecal streptococci as indicator organisms. This operating definition of the faecal streptococci prohibits its taxonomic limitation to enterococci or group D streptococci. Therefore, we have undertaken studies concerning the methods for routine examinations. Comparative investigations of selective enrichment broths showed no significant differences of the end concentration of glucose in the range of 0.5-1.0% and of sodium azide in the range of 0.02-0.05%. 36 degrees C was a better incubation temperature than 44 degrees C and the results were better after 24 h than after 48 h. Comparative investigations of selective plating media showed less selectivity of the bile-aesculin agar and gentamicinthallous-carbonate agar than that on the one hand of citrate-azide-tween-carbonate agar, KF streptococcal agar or Slanetz-Bartley enterococcal agar, and on the other hand of kanamycin-aesculin-azide agar. All together the kanamycin-aesculin-azide agar seems to be a little better than the other media (Table 2) 356 strains of faecal streptococci were isolated from the studied media. More than 20 characteristics were investigated and, there upon, the strains were differentiated (Table 3). S. faecalis predominates in human materials, i.e. urine, S. faecium and S. durans were isolated specially from sewage and waste water (Table 4). The single species of faecal streptococci grow specificly on the different selective media, and therefore, each agar specimen prefers the one or other species more or less selectively (Table 5).

Azides↗

PubMed1994

Multiple mechanisms for the targeting of photosystem I subunits F, H, K, L, and N into and across the thylakoid membrane.

The photosystem I (PSI) complex in higher plants contains eight nuclear-encoded subunits, of which two (PSI-F and -N) are synthesized with bipartite presequences containing cleavable thylakoid transfer sequences. Previous studies on four other chloroplast proteins bearing bipartite presequences have shown that they are transported across the thylakoid membrane by two distinct mechanisms. One mechanism is delta pH-dependent and hence sensitive to uncouplers, whereas the other is inhibited by azide. We show that PSI-F is targeted by the latter pathway, since its translocation across the thylakoid membrane is inhibited by azide but not by nigericin. Translocation is furthermore unaffected by the presence of high concentrations of the lumenal 23-kDa photosystem II (PSII) protein, which is known to be transported by the delta pH-dependent pathway. In contrast, translocation of PSI-N across the thylakoid membrane is completely blocked by saturating concentrations of pre-23-kDa protein. Three proteins are now known to be synthesized with thylakoid transfer signals in both higher plants and cyanobacteria (PSI-F, plastocyanin, and the 33-kDa PSII protein), and all three are transported by the azide-sensitive, possibly sec-dependent pathway. In contrast, PSI-N and the 23-kDa and 16-kDa PSII proteins (transported by the delta pH-driven pathway in higher plants) are all absent in cyanobacteria. These data suggest that the delta pH-dependent translocation mechanism for these proteins may also have arisen relatively recently during the evolution of the chloroplast. Three additional PSI proteins (PSI-H, -K, and -L) are synthesized in the cytosol with stroma-targeting presequences and hence integrate into the thylakoid membrane by means of information in the mature proteins. We show that the integration mechanisms are insensitive to azide in each case, and nigericin causes only a slight inhibition of integration in each case. We therefore suggest that these proteins are targeted into the thylakoid membrane by a separate pathway(s).

Azides↗

PubMed1993

Chemiluminescence in activated human neutrophils: role of buffers and scavengers.

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the result of the interaction among H2O2, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H2O2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and Co2+ very markedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by superoxide dismutase (SOD) and by deferoxamine LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.

Acridines↗

PubMed1988

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium.

Proton secretion in the urinary bladder of the freshwater turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo- and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15 degrees C. In addition, serosal application of 0.5 mM acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15 degrees C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1 mM serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.

Acetazolamide↗

PubMed1976

Ligand-induced spectral changes in cytochrome c oxidase and their possible significance.

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.

Animals↗