ABSTRACT
Recent studies suggest that hydrogen sulfide (H2S) exhibits potent antioxidant capacity and improves vascular and tissue functions. Thus we aimed to compare the antioxidant efficacy of H2S to that of superoxide dismutase (SOD).Isometric force of isolated rat carotid arteries and gracilis veins was measured with a myograph. The vasomotor effect of the superoxide-generator pyrogallol (10-5M) was obtained in control conditions, and then in the presence of SOD (120 U/ml) or H2S (10-5M or 10-4M), respectively. Spectrophotometric measurements were performed to detect the effect of SOD and H2S on the auto-oxidation of pyrogallol.Pyrogallol increased the isometric force of carotid arteries (9.7 ± 0.8 mN), which was abolished by SOD (5.3 ± 0.8 mN), was not affected by 10-5M H2S (9.1 ± 0.5 mN), whereas 10-4M H2S slightly, but significantly reduced it (8.1 ± 0.7 mN). Pyrogallol significantly increased the isometric force of gracilis veins (1.3 ± 0.2 mN), which was abolished by SOD (0.9 ± 0.2 mN), whereas 10-5M (1.3 ± 0.2 mN), or 10-4M H2S (1.2 ± 0.2 mN) did not affect it. Pyrogallol-induced superoxide production was measured by a spectrophotometer (A420 = 0.19 ± 0.0). SOD reduced absorbance (A420 = 0.02 ± 0.0), whereas 10-5M H2S did not (A420 = 0.18 ± 0.0) and 10-4M H2S slightly reduced it (A420 = 0.15 ± 0.0).These data suggest that H2S is a less effective vascular antioxidant than SOD. We propose that the previously described beneficial effects of H2S are unlikely to be related to its direct effect on superoxide.
Subject(s)
Antioxidants/pharmacology , Biological Assay/methods , Hydrogen Sulfide/pharmacology , Pyrogallol/pharmacology , Superoxide Dismutase/metabolism , Animals , Antioxidants/chemistry , Carotid Arteries/drug effects , Carotid Arteries/enzymology , Free Radicals/chemistry , Free Radicals/metabolism , Hydrogen Sulfide/chemistry , Male , Oxidative Stress/drug effects , Oxidative Stress/physiology , Pyrogallol/chemistry , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Veins/drug effects , Veins/enzymologyABSTRACT
The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.
Subject(s)
Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , 3T3 Cells , Actins/metabolism , Animals , Blotting, Western , COS Cells , Catalytic Domain , Cell Line , Cell Nucleus/metabolism , Cytoskeleton/metabolism , DNA, Complementary/metabolism , Enzyme Activation , Escherichia coli/metabolism , Gene Deletion , Glutathione Transferase/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Insecta , Mice , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Precipitin Tests , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Stress Fibers/metabolism , Time Factors , Transfection , cdc42 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
The dbl oncogene product (onco-Dbl) is the prototype member of a family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. The Dbl homology (DH) domain of onco-Dbl is responsible for the GEF catalytic activity, and the DH domain, together with the immediately adjacent pleckstrin homology (PH) domain, constitutes the minimum module bearing transforming function. In the present study, we demonstrate that the onco-Dbl protein exists in oligomeric form in vitro and in cells. The oligomerization is mostly homophilic in nature and is mediated by the DH domain. Mutagenesis studies mapped the region involved in oligomerization to the conserved region 2 of the DH domain, which is located at the opposite side of the Rho GTPase interacting surface. Residue His556 of this region, in particular, is important for this activity, since the H556A mutant retained the GEF catalytic capability and the binding activity toward Cdc42 and RhoA in vitro but was deficient in oligomer formation. Consequently, the Rho GTPase activating potential of the H556A mutant was significantly reduced in cells. The focus-forming and anchorage-independent growth activities of onco-Dbl were completely abolished by the His556-to-Ala mutation, whereas the abilities to stimulate cell growth, activate Jun N-terminal kinase, and cause actin cytoskeletal changes were retained by the mutant. The ability of onco-Dbl to oligomerize allowed multiple Rho GTPases to be recruited to the same signaling complex, and such an ability is defective in the H556A mutant. Taken together, these results suggest that oligomerization of onco-Dbl through the DH domain is essential for cellular transformation by providing the means to generate a signaling complex that further augments and/or coordinates its Rho GTPase activating potential.
Subject(s)
Cell Transformation, Neoplastic , Retroviridae Proteins, Oncogenic/chemistry , Retroviridae Proteins, Oncogenic/metabolism , 3T3 Cells , Actins/metabolism , Animals , Cell Division , Cytoskeleton/metabolism , Enzyme Activation , Guanine Nucleotide Exchange Factors/metabolism , JNK Mitogen-Activated Protein Kinases , Kinetics , Mice , Microscopy, Fluorescence , Mitogen-Activated Protein Kinases/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutation/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Fusion Proteins , Retroviridae Proteins, Oncogenic/genetics , Signal Transduction , Transfection , cdc42 GTP-Binding Protein/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Retroviridae Proteins, Oncogenic/metabolism , rho GTP-Binding Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Binding Sites , Blood Proteins/metabolism , Catalysis , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoproteins/metabolism , Protein Conformation , Retroviridae Proteins, Oncogenic/genetics , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The effect of metabolites accumulating in phenylketonuria (PKU) was investigated on carnitine metabolism in rats and in patients with PKU. Of phenylacetic acid (PEAA), phenylpyruvic acid and homogentisic acid the PEAA was found to be the most effective in inhibiting carnitine biosynthesis in rats. Following 60 min, a single intraperitoneal dose of PEAA the relative conversion rate, i. e. the hydroxylation, of tracer [Me-(3)H]butyrobetaine to [Me-(3)H]carnitine decreased from 62.2+/-6.00% to 39.4+/-5.11% (means+/-S.E.M., P<0.01) in the liver, in the only organ doing this conversion in rats. The conversion of loading amount of unlabeled butyrobetaine to carnitine was also markedly reduced. The impaired hydroxylation of butyrobetaine was reflected by a reduced free and total carnitine levels in the liver and a reduced total carnitine concentration in the plasma. PEAA decreased the hepatic level of glutamic acid and alpha-ketoglutaric acid (alpha-KG), suggesting a mechanism for the reduced flux through the butyrobetaine hydroxylase enzyme, because alpha-KG is an obligatory co-enzyme. In the plasma and urine of PKU patients on unrestricted diet, markedly decreased total carnitine levels were detected. In the liver of PEAA-treated rats and urine of PKU patients, a novel carnitine derivative, phenacetyl-carnitine was verified by HPLC and gas chromatography-mass spectrometry.
Subject(s)
Carnitine/metabolism , Phenylacetates/pharmacology , Phenylketonurias/metabolism , Adult , Animals , Betaine/analogs & derivatives , Betaine/metabolism , Carnitine/analogs & derivatives , Carnitine/analysis , Carnitine/blood , Carnitine/urine , Female , Glutamic Acid/metabolism , Homogentisic Acid/pharmacology , Humans , Ketoglutaric Acids/metabolism , Liver/drug effects , Liver/metabolism , Male , Mass Spectrometry , Phenylketonurias/urine , Phenylpyruvic Acids/pharmacology , Rats , Rats, WistarABSTRACT
The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.
Subject(s)
Carrier Proteins/metabolism , GTP Phosphohydrolases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , cdc42 GTP-Binding Protein/metabolism , ras GTPase-Activating Proteins , 3T3 Cells , Animals , COS Cells , GTP Phosphohydrolases/chemistry , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Wiskott-Aldrich Syndrome Protein , cdc42 GTP-Binding Protein/chemistry , p21-Activated KinasesABSTRACT
The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.
Subject(s)
GTP-Binding Proteins/physiology , GTPase-Activating Proteins , Mitogen-Activated Protein Kinases , Nerve Regeneration/physiology , Neurites/physiology , Animals , Blotting, Northern , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Differentiation/drug effects , Cell Division/physiology , Cell Membrane/enzymology , Cells, Cultured , Clone Cells , Enzyme Activation/physiology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Mutation , Nerve Growth Factors/pharmacology , Nerve Regeneration/genetics , Neurites/ultrastructure , PC12 Cells , Plasmids , RatsABSTRACT
There exists a considerable controversy in the literature with regard to the effect of either opiate receptor blockade or that of morphine in different gastric and intestinal ulcer models in the rat. We performed experiments to evaluate the effects of naloxone and morphine on gastric acid secretion and gastric mucosal damage in different experimental models of gastric mucosal injury, namely in indomethacin-, HCl (0.6N)- and ethanol (96%)-models. We found that: 1) 10 mg/kg naloxone i.p. given twice, effectively protected gastric mucosa against indomethacin (30 mg/kg i.p.) and against the acid-dependent injury caused by 0.6 N HCl (1 mL i.g.), but not against the non acid-dependent injury caused by 96% ethanol (1 mL i.g.); 2) morphine (10 + 10 mg/kg i.p.) increased ulcers in the HCl-model, but had no effect in the two other models; 3) this ulcer-aggravating effect of morphine in the HCl-model was blocked by pretreatment of 2 mg/kg i.p. naloxone; and 4) both naloxone (5 + 5 and 10 + 10 mg/kg i.p.) and morphine (10 + 10 mg/kg i.p.) significantly decreased gastric acid secretion in 1-h pylorus ligated rats. We conclude that: 1) naloxone dose-dependently protects against the indomethacin- and HCl-, but not against the ethanol-induced gastric mucosal damage; 2) morphine aggravates the HCl-induced ulcerogenesis; and 3) both opioid receptor agonist and antagonist decrease gastric acid secretion.
Subject(s)
Gastric Acid/metabolism , Morphine/pharmacology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Narcotics/pharmacology , Stomach Ulcer/prevention & control , Animals , Anti-Inflammatory Agents, Non-Steroidal , Central Nervous System Depressants , Ethanol , Female , Hydrochloric Acid , Indomethacin , Male , Rats , Rats, Sprague-Dawley , Stomach Ulcer/chemically inducedABSTRACT
The relationship between the hypothalamic catecholamine and serotonin level as well as the activation of the pituitary-adrenal axis was investigated after administration or morphine (MO) in the rat. Five mg/kg b. wt. of MO induced a significant increase in norepinephrine and a 78%, but insignificant, increase in dopamine level of the hypothalamus within 60 min without changing corticosterone secretion. Electric footshock, in addition to elevating hypothalamic norepinephrine and dopamine levels, significantly increased the pituitary-adrenocortical response in the MO pretreated rats. Five mg/kg b. wt. of MO, or electric footshock alone did not influence the hypothalamic serotonin level within 60 min, but the hypothalamic serotonin level decreased significantly in the MO pretreated, electrically shocked animals. We conclude, that 1) low dose of MO may induce changes of the hypothalamic catecholamine levels without influencing pituitary-adrenocortical activation. 2) enhanced hypothalamic catecholamines by MO did not prevent increasing pituitary-adrenocortical response elicited by stress. It appears, that the hypothalamic catecholaminergic mechanism which may inhibit ACTH release during stimulation does not function in the MO treated rats.