Investigations of methoxyflurane-induced nephrotoxicity in man have been extensively aided by the use of an animal model. To be of value the animal model must share similar metabolic pathways with man and have the same clinical manifestations of the diseases process. The Fischer 344 rat appears to meet these criteria. The predominant factors in the production of methoxyflurane nephrotoxicity appear to be high methoxyflurane dosage and serum inorganic fluoride concentration. It is likely that secondary factors include: (1) a high rate of methoxyflurane metabolism and sepsitivity of the kidney to inorganic fluoride toxicity: (2) concurrent treatment with other nephrotoxic drugs; (3) preexisting renal disease; (4) surgery of the urogenital tract, aorta, or renal vasculative; (5) repeat administration of methoxyflurane due to accumulation of inorganic fluoride and, perhaps, methoxyflurane induction of its own metabolism: and (6) concurrent treatment with enzyme-inducing drugs such as phenobarbital. (+info)
Inhibition of myosin ATPase by metal fluoride complexes.
(2/1729)
Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics. (+info)
The thrombospondin receptor integrin-associated protein (CD47) functionally couples to heterotrimeric Gi.
(3/1729)
Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with beta3 integrins resulting in enhanced alphavbeta3-dependent cell spreading and chemotaxis and, in platelets, alphaIIbbeta3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its beta3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Gialpha is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPgamma35S to membranes from cells expressing IAP and alphavbeta3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems. (+info)
Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens.
(4/1729)
1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays. (+info)
The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.
(5/1729)
Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes. (+info)
Agonist-induced translocation of Gq/11alpha immunoreactivity directly from plasma membrane in MDCK cells.
(6/1729)
Both Gsalpha and Gqalpha are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) alpha-subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11alpha immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 microM) caused Gq/11alpha, but not Gialpha, to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11alpha immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11alpha immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11alpha is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity. (+info)
A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity.
(7/1729)
It has been reported that substitution of Arg258, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) alpha-subunit (alphas), to alanine (alphas-R258A) results in decreased activation by receptor or aluminum fluoride (AlF4-) and increased basal GDP release. Arg258 interacts with Gln170 in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF4- and increased thermolability. In this study, we mutate Gln170 to alanine (alphas-Q170A) and demonstrate that this mutant, like alphas-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike alphas-R258A, alphas-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of alphas-R258A was 14-fold higher than normal whereas that of alphas-Q170A was unaffected. Examination of the alphas crystal structure suggests that Arg258, through interactions with Glu50, might constrain the position of Arg201, a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction. (+info)
De novo expression of the class-A macrophage scavenger receptor conferring resistance to apoptosis in differentiated human THP-1 monocytic cells.
(8/1729)
The class-A macrophage scavenger receptor (MSR) is a trimeric multifunctional protein expressed selectively in differentiated monomyeloid phagocytes which mediates uptake of chemically modified lipoproteins and bacterial products. This study investigated whether MSR plays a role in the regulation of apoptosis, a model of genetically programmed cell death. De novo expression of MSR occurred in human THP-1 monocytic cells differentiated with phorbol esters, which activated a nuclear transcription factor binding to the Ap1/ets-like domain of the MSR promoter. The phorbol ester-stimulated THP-1 cells also expressed increased levels of the pro-apoptotic gene products, caspase-3 and Fas ligand, but the cells exhibited no change in apoptosis. Global activation of GTP-binding proteins with fluoride anions triggered apoptosis of THP-1 cells in a time- and concentration-dependent manner, demonstrated by nuclear shrinkage and fragmentation and internucleosomal DNA fragmentation. However, the MSR-expressing THP-1 macrophage-like cells showed a significant reduction in apoptosis compared to undifferentiated control THP-1 cells, which produce MSR at undetectable levels. Fluoride stimulation also triggered apoptosis of human Jurkat T cells. Stimulation with phorbol ester made no difference in apoptosis between treated and untreated Jurkat cells. Finally, Chinese hamster ovary (CHO) cells overexpressing the class-A MSR type I by cDNA transfection showed markedly increased resistance to G-protein-coupled apoptosis. Thus, de novo expression of MSR associated with monocyte maturation into macrophages appears to confer the resistance of macrophages to apoptotic stimulation by G-protein activation. (+info)