Possible roles of the AP-1 site in the cytosolic T3 binding protein promoter and insights into its physiological significance.

Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to µ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on µ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in µ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of µ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased µ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of µ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on µ-crystallin mRNA expression.

Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization.

Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera: The molluscan counterpart of the annelidan GGNG peptides.

The GGNG peptides are excitatory neuropeptides identified from earthworms, leeches and polychaeta. Two structurally related peptides were purified and characterized from a mollusk, Thais clavigera (prosobranch gastropod). The peptides designated as Thais excitatory peptide-1 (TEP-1) (KCSGKWAIHACWGGN-NH2) and TEP-2 (KCYGKWAMHACWGGN-NH2) are pentadecapeptides having one disulfide bond and C-terminal GGN-NH2 structures, which are shared by most GGNG peptides. TEP augmented the motilities of Thais esophagus and penial complex. TEP-like immunoreactivity is distributed in both the neurons of the central nervous system and nerve endings in the penial complex. Thus, the involvement of TEP in the contraction of the digestive and reproductive systems is suggested. Substitution of amino acids in TEP revealed that two tryptophan residues in TEP are important for maintaining bioactivity.

Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils.

We studied the action of iodinated thyronines on the superoxide (O2-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced O2- production with IC50 values of about 10(-6) M and 7.10(-6) M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the O2- production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O2- production stimulated with either the fifth component of the complement (C5a) or phorbol 12-myristate 13-acetate. L-Thyroxine and L-triiodothyronine were found to block [3H]FMLP binding to its own receptor with IC50 values similar to those for the inhibition of the O2- production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O2- production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.

Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C.

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.

Decrease in the phosphotyrosine phosphatase activity in the plasma membrane of human neutrophils on stimulation by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neurophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The PTPase activity was localized in the plasma membrane. The activity in the membrane with the optimum pH at 5.5 had a Km value for phosphotyrosine of 2.2 mM and Vmax of 2.0 mumol/min per mg of protein. No activity was observed against phosphoserine and phosphothreonine. Vanadate, molybdate, zinc and a sulfhydryl reagent, p-chloromercuribenzenesulphonic acid, inhibited the PTPase. The PMA-induced decrease in activity was almost completely recovered by treatment of the plasma membrane with Triton X-100 at low concentrations which did not solubilize the activity from the membrane. When the plasma membrane was treated with trypsin, the PTPase of the membrane from PMA-treated cells was mostly protected from the proteinase attack while that from the resting cells was not protected. Pretreatment of the plasma membrane with Triton X-100 enabled trypsin to gain access to all the PTPase in the membrane from both PMA-treated and resting cells. The PMA treatment affected neither subcellular localization of the PTPase nor the orientation of the plasma membrane vesicles. These findings suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.

Characterization of the NADPH-dependent superoxide production activated by sodium dodecyl sulfate in a cell-free system of pig neutrophils.

The NADPH-dependent superoxide production induced by sodium dodecyl sulfate (SDS) in the sonicates of unstimulated pig neutrophils required both membrane fraction and two components of cytosol fraction. The potency of the cytosol fraction in the activation of the superoxide production could be reconstituted dose dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa. Another low-molecular-mass component (1.3 kDa) could substitute the 50-kDa component. In the cell-free system consisting of the 300- and 50-kDa components and the membrane fraction, the superoxide production was markedly enhanced by FAD with a required concentration for half-maximal effect of 0.16 microM and inhibited by divalent cations such as Ca2+, Ba2+, Co2+, Zn2+ and Mn2+ and not Mg2+. ATP was not necessary for the activation, indicating that protein kinases such as protein kinase C are not involved in the SDS-dependent activation of NADPH oxidase. The NADPH oxidase activated by SDS in the cell-free system was recovered in the membrane fraction, and the superoxide formation by the SDS-activated membrane exhibited a Km value for NADPH of 46 microM and optimum pH at 7.0. The formation did not require the addition of SDS and FAD to the reaction mixture and was scarcely inhibited by the divalent cations.

ATP-induced calcium mobilization in human neutrophils.

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.

Phosphorylation of myosin light chain in intact pig leukocytes stimulated by phorbol 12-myristate 13-acetate.

Phosphorylation of myosin light chain was investigated in intact pig polymorphonuclear leukocytes. The labeling of the myosin light chain (Mr = 21000) was increased by exposure of the cells to phorbol 12-myristate 13-acetate. Two-dimensional gel electrophoresis revealed three forms of Mr = 21000 light chain, of which two were phosphorylated. The phosphorylation of the myosin light chain was inhibited by neither N-(6-aminohexyl)-5-chloro-1-naphthalene sufonamide nor trifluoperazine, suggesting that calmodulin is not involved in the phosphorylation.

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4.

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.

NADPH-dependent reduction of ubiquinone-1 associated with the superoxide-forming oxidase of pig polymorphonuclear leucocytes.

NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent Km-value of the reductase for NADPH was 29 microM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.

Protein phosphorylation in intact pig leukocytes.

The phosphorylation of proteins in intact pig polymorphonuclear leukocytes loaded with H3(32)PO4 was investigated by two-dimensional gel electrophoresis and subsequent autoradiography. The incorporation of 32P into at least 17 proteins began to increase and into one to decrease, relative to resting cells, upon exposure of the cells to phorbol 12-myristate 13-acetate. These changes in the autoradiographic patterns were accompanied by changes in the protein patterns obtained by staining with Coomassie brilliant blue, including the appearance, the acidic shift and the increase or decrease of the intensity of the spots. Among these proteins, Mr = 64 000, 31 000, 22 000, 21 000, 18 000 and 13 000 proteins were correlated well with the superoxide anion production of the cells in respect to the time-courses and the dose-responses. By taking the effects of EGTA into consideration, the phosphorylation of Mr 64 000 and 21 000 proteins, of which the latter was identified as the light chain of myosin, seemed to be involved in the signal-transmission mechanism of the induction of the NADPH oxidase responsible for the 'respiratory burst'. These two proteins were also phosphorylated in the cells stimulated by NaF or oil droplets opsonized with IgG.

NAD+-dependent oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by rat liver cytosol.

20-Hydroxyleukotriene B4 was converted by rat liver homogenates in the presence of NAD+ to a more polar product on reverse-phase high-performance liquid chromatography. The product was identified as 20-carboxyleukotriene B4 by straight-phase high performance liquid chromatography, ultraviolet spectrophotometry and gas chromatography-mass spectrometry. The oxidative activity of the homogenates was located in the cytosol with an optimal pH of 8.0. The activity was dependent on NAD+, and NADP+ could not substitute for NAD+. 1 mol of 20-carboxyleukotriene B4 was formed with the reduction of 2 mol of NAD+. The reaction was inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by various alcohols, such as ethanol, 12-hydroxylaurate, and 20-hydroxyprostaglandin E1. Disulfiram, an inhibitor of aldehyde dehydrogenase, also inhibited the activity. These results suggest that two discrete steps catalyzed by different enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, are involved in the oxidation of 20-hydroxyleukotriene B4 in rat liver cytosol. The enzyme system seems to be different from that of human neutrophils.

Characterization of the GTP-dependent activation of the superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We characterized the cell-free activating system of the superoxide (O2-)-producing NADPH oxidase of pig neutrophils. Activation of the oxidase required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resulted in separation of the cytosolic fraction into two fractions, the flow-through and bound fractions, which synergistically supported the O2- production with the membrane fraction in the absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas only the bound fraction besides the membrane fraction was required for the activation in the presence of GTP gamma S. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchange chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding native factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein responsible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-ADP-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of the ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding protein for the activation and the lost ability was recovered by the addition of GTP gamma S. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.

Characterization of NAD(P)H-dependent ubiquinone reductase activities in rat liver microsomes.

Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.

Preferential heme transport through endoplasmic reticulum associated with mitochondria in rat liver.

The transport of de novo synthesized protoheme into the conventional microsomal fraction and endoplasmic reticulum associated with mitochondria (MAER) was studied by injecting amino[14C]levulinic acid (ALA) into phenobarbital-treated rats to evaluate the role of MAER in the trafficking of heme between mitochondria and endoplasmic reticulum. In mitochondria, the specific radioactivity of the radiolabeled heme reached a maximum level at 4 min after the injection of 14C-ALA. The specific radioactivity in cytosol was about 2-fold lower than that in microsomes, suggesting that the cytosolic pathway of the heme transport from mitochondria to endoplasmic reticulum is not predominant, because the specific radioactivity of heme in cytosol should be higher than that in microsomes if heme is transported mainly through cytosol. MAER showed higher specific radioactivity than the conventional microsomal fraction up to 4 min and thereafter the specific radioactivities in MAER and the conventional microsomal fraction became nearly the same. The extents of decrease in cytochrome P-450 and the radioactivity in microsomes by the treatment with allylisopropylacetamide which destroyed cytochrome P-450 but not cytochrome b5, were essentially the same, suggesting that most of the radiolabeled heme in microsomes was incorporated into cytochrome P-450. These results suggest that MAER is a preferential site for the protoheme transport from mitochondria to endoplasmic reticulum.

Cytoplasmic pH change induced by leukotriene B4 in human neutrophils.

Leukotriene B4 induced a biphasic change in the cytoplasmic pH of human neutrophils: an initial rapid acidification followed by an alkalinization. The acidification was slightly reduced by the removal of extracellular Ca2+, but the subsequent alkalinization was not. The leukotriene B4-induced alkalinization was dependent on extracellular Na+ and pH, and was inhibited by amiloride and its more potent analogue, 5-(N,N-hexamethylene)amiloride. These characteristics indicate that the cytoplasmic alkalinization is mediated by the Na+-H+ exchange. Oxidation products of leukotriene B4, 20-hydroxyleukotriene B4, 20-carboxyleukotriene B4, and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) also stimulated the Na+-H+ exchange, but higher concentrations were required. Treatment of the cells with pertussis toxin inhibited both phases of the leukotriene B4-induced pHi change, while cholera toxin did not affect the pHi change. The alkalinization induced by leukotriene B4 was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, but was not inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide which has a less inhibitory effect on protein kinase C. Acidification was not affected by the drugs. These findings suggest that a GTP-binding protein sensitive to pertussis toxin and protein kinase C are involved in the activation of the Na+-H+ exchange stimulated by leukotriene B4.

Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP.

We have studied the activation of the Na+/H+ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca2+ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca2+. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca2+ concentration ([Ca2+]i) in the Ca2+ buffer, while only the rapid and transient increase of [Ca2+]i was observed in the absence of extracellular Ca2+. The Ca2+-depleted cells prepared by incubation in Ca2+-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca2+]i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA 1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na+/H+ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca2+]i is essential for the ATP-induced activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system.

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.