Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.

Fluorescence photobleaching recovery methods reveal marked changes in lateral mobilities of rabbit lymphocyte membrane components during the course of stimulation with succinyl concanavalin A (S Con A). The diffusion constant of S Con A receptors on T lymphocytes falls from 1.6×10^?10 cm²/sec to 6.5×10^?11 cm²/sec within 4 hr after stimulation, remains constant for 14 hr, and returns to its former value. The mobility of B cell receptors similarly falls from 1.4×10^?10 cm²/sec to 5.5×10^?11 cm²/sec but regains its unstimulated value much more slowly. In contrast, a fluorescent phospholipid analog shows constant mobilities of 1.9×10^?8 cm²/sec and 1.5×10^?8 cm²/sec in T and B cells, respectively, throughout the experiment.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O₂ concentrations and mitochondrial O₂ consumption by high-resolution respirometry over a range of O₂ concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC₅₀ on [O₂] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O₂ and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O₂-dependent oxidation. It also explains the non-linear dependence of IC₅₀ on O₂ concentration, and the hyperbolic increase of c₅₀ as a function of NO concentration.     

Critical role of mac-1 sialyl lewis x moieties in regulating neutrophil degranulation and transmigration

Leukocyte cell surface sialyl Lewis x (sLe^x) and related epitopes play an important role in cell rolling and adhesion during diapedesis via interaction with E-selectin. Here, we present evidence that Mac-1 (CD11b/CD18, CR-3) is a major neutrophil glycoprotein decorated with sLe^x and ligation of these carbohydrate moieties by anti-sLe^x antibody significantly impairs neutrophil functions. First, Western blot analysis shows that both CD11b and CD18 subunit of purified Mac-1 are decorated with sLe^x moieties. A significant co-localization of CD11b and sLe^x moieties is observed at neutrophil secondary granules. With stimulation of formyl-Met-Leu-Phe (fMLP), neutrophil surface labeling with anti-sLe^x antibody follows an identical up-regulation pattern of Mac-1. Second, protein-binding assays indicate that sLe^x moieties on Mac-1 are critical for binding interaction of Mac-1 to E-selectin. Removal of sLe^x moieties completely abolishes Mac-1-E-selectin binding. Finally, ligation of Mac-1 sLe^x by anti-sLe^x antibody induces a significant degranulation of neutrophil secondary granules at the absence of chemoattractant stimulation. This “dysregulated” degranulation induced by anti-sLe^x antibody strongly inhibits neutrophil transmigration in response to fMLP. In summary, Mac-1 sLe^x moieties play a critical role in regulating β₂ integrin functions during neutrophil transmigration and degranulation.     

Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily

Human Hint3 (hHint3) has been classified as a member of the histidine triad nucleotide (Hint) binding protein subfamily. While Hint1 is ubiquitously expressed by both eukaryotes and prokaryotes, Hint3 is found only in eukaryotes. Previously, our laboratory has characterized and compared the aminoacyl-adenylate and nucleoside phosphoramidate hydrolase activity of hHint1 and Escherichia coli hinT. In this study, hHint3-1(Ala36) and its single nucleotide polymorphism, hHint3-2 (A36G variant), were cloned, overexpressed, and purified. Steady-state kinetic studies with a synthetic fluorogenic indolepropinoic acyl-adenylate (AIPA) and with a series of fluorogenic tryptamine nucleoside phosphoramidates revealed that hHint3-1 and hHint3-2 are adenylate and phosphoramidate hydrolases with apparent second-order rate constants (kcat/Km) ranging from 10(2) to 10(6) s(-1) M(-1). Unlike hHint1, hHint3-1 and hHint3-2 prefer AIPA over tryptamine adenosine phosphoramidate by factors of 33- and 16-fold, respectively. In general, hHint3s hydrolyze phosphoramidate 370- to 2000-fold less efficiently than hHint1. Substitution of the potential active-site nucleophile, His145, by Ala was shown to abolish the adenylate and phosphoramidate hydrolase activity for hHint3-1. However, 0.2-0.4% residual activity was observed for the H145A mutant of hHint3-2. Both hHint3-1 and hHint3-2 were found to hydrolyze lysyl-adenylate generated by human lysyl-tRNA synthetase (hLysRS) by proceeding through an adenylated protein intermediate. hLysRS-dependent labeling of hHint3-1 and hHint3-2 was found to depend on His145, which aligns with the His112 of the Hint1 active site. The extent of active-site His145-AMP labeling was shown to be similar to His112-AMP labeling of hHint1. In contrast to all previously characterized members of the histidine triad superfamily, which have been shown to exist exclusively as homodimers, wild type and the H145A of hHint3-1 were found to exist across a range of multimeric states, from dimers to octamers and even larger oligomers, while wild type and the H145A of hHint3-2 exist predominantly in a monomeric state. The differences in oligomeric state may be important in vivo, because unlike tetracysteine-tagged Hint1, which was found along linear arrays exclusively in the cytoplasm in transfected HeLa cells, tagged Hint3-1 and Hint3-2 were found as aggregates both in the cytosol and in the nucleus. Taken together, these results imply that while Hint3 and Hint1 prefer aminoacyl-adenylates as substrates and catalytically interact with aminoacyl-tRNA synthetases, the significant differences in phosphoramidase activity, oligomeric state, and cellular localization suggest that Hint3s should be placed in a distinct branch of the histidine triad superfamily.     

Setaria cervi: kinetic studies of filarial glutathione synthetase by high performance liquid chromatography

The bovine filarial worm Setaria cervi was found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzyme being involved in catalysing the final step of glutathione (GSH) biosynthesis. A RP-HPLC method involving precolumn derivatization with o-phthalaldehyde has been followed for the estimation of GS activity in crude filarial preparations. Subcellular fractionation of the enzyme was undertaken and it was confirmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determine the Km value for L-gamma-glutamyl-L-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively high dipeptide concentrations (>1 mM) extrapolate to a Km value of about 400 microM whereas data obtained at lower concentrations (<0.1 mM) extrapolate to a value of about 33 microM. Km was determined to be around 950 and 410 microM for ATP and glycine, respectively. The effect of various amino acids was studied on enzyme activity at 1mM concentration. L-cystine caused a significant enzyme inhibition of 11%. Preincubation with N-ethylmaleimide also resulted in significant inhibition of GS activity.