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Biomedical subjects

J A Maloney

Publications and source records attributed to J A Maloney.

11 recordsLinked to original sources
PubMed1999

Epidermal growth factor and angiotensin II regulation of extracellular signal-regulated protein kinase in rat liver epithelial WB cells.

Activation of extracellular signal-regulated protein kinase (ERK) is considered essential for mitogenesis. In the present study, rat liver epithelial WB cells were used to investigate the relative roles of Ca2+, protein kinase C (PKC), and protein tyrosine phosphorylation in mitogenesis and activation of the ERK pathway stimulated by epidermal growth factor (EGF) and angiotensin II (Ang II). The sensitivity of the ERK pathway to Ca2+ was studied by using 1,2-bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to chelate intracellular Ca2+ and a low extracellular Ca2+ concentration to prevent Ca2+ influx. Agonist-induced PKC activation was diminished by inhibition of PKC by GF-109203X (bisindolylmaleimide) or by down-regulation of PKC by long-term treatment of the cells with phorbol myristate acetate (PMA). Our results show that although activation of PKC was critical for mitogenesis induced by Ang II or EGF, the initial activation of ERK by both agonists in these cells was essentially independent of PKC activation and was insensitive to Ca2+ mobilization. This is in contrast to the findings in some cell types that exhibit a marked dependency on mobilization of Ca2+ and/or PKC activation. On the other hand, an obligatory tyrosine phosphorylation step for activation of ERK was indicated by the use of protein tyrosine kinase inhibitors, which profoundly inhibited the activation of ERK by EGF, Ang II, and PMA. Additional experiments indicated that tyrosine phosphorylation by a cytosolic tyrosine kinase may represent a general mechanism for G-protein coupled receptor mediated ERK activation.

Angiotensin II↗
PubMed1999

Activation of ERK by Ca2+ store depletion in rat liver epithelial cells.

In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+ pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+ stored in intracellular pools plays an important role in this regulation.

Angiotensin II↗
PubMed1998

Angiotensin II induces diverse signal transduction pathways via both Gq and Gi proteins in liver epithelial cells.

Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the Gi family as well as the Gq family are involved in angiotensin II-mediated mitogenic pathways in WB cells.

Angiotensin II↗
PubMed1998

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells.

The protein kinase C (PKC) family represents an important group of enzymes whose activation is associated with their translocation from the cytosol to different cellular membranes. In this study, the spatial distribution of PKC-alpha, -delta and -epsilon in rat liver epithelial (WB) cells has been examined by Western blot analysis after subcellular fractionation. Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtained from cells stimulated with phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II), or epidermal growth factor (EGF). PMA caused most of the PKC-alpha, -delta and -epsilon initially present in the cytosol to be transported to the membrane and nuclear fractions. In contrast, both ANG II and EGF induced only a minor translocation of PKC-alpha to the membrane fraction but caused a statistically significant membrane-directed movement of PKC-delta and -epsilon. Translocation of PKC-delta and -epsilon to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC-delta and -epsilon were present in the cytoskeleton of resting cells, but although PMA, ANG II, and EGF caused some changes in their content, these were variable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK). However, although PKC depletion inhibited EGF-induced mitogenesis, the maximum EGF-induced activation of the ERK pathway was only slightly retarded. We hypothesize that PKC-delta and -epsilon are involved in mitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Angiotensin II↗
PubMed1997

A new function for phospholipase C-gamma1: coupling to the adaptor protein GRB2.

Epidermal growth factor (EGF)-induced autophosphorylation of the EGF receptor results in high-affinity binding of the adaptor protein GRB2, which serves as a convergence point for multiple signaling pathways. Present studies demonstrate that EGF induces the co-immunoprecipitation of phospholipase C (PLC)-gamma1 with the adaptor protein GRB2 and the guanine nucleotide exchange factor Sos, but not with the adaptor protein SHC, in WB cells. Inhibition of PLC-gamma1 tyrosine phosphorylation by phenylarsine oxide reduces the co-immunoprecipitation of PLC-gamma1 with GRB2. Furthermore, angiotensin II, a G protein-coupled receptor agonist, also induces the tyrosine phosphorylation of PLC-gamma1 and its co-immunoprecipitation with GRB2 in WB cells. Interestingly, angiotensin II stimulation also causes tyrosine phosphorylation of the EGF receptor, suggesting that angiotensin II-induced PLC-gamma1 tyrosine phosphorylation in WB cells may be via EGF receptor tyrosine kinase activation. In addition, there is some level of association between PLC-gamma1 and GRB2 that is independent of the tyrosine phosphorylation of PLC-gamma1 in both in vivo and in vitro studies. In vitro studies further demonstrate that the Tyr771 and Tyr783 region of PLC-gamma1 and the SH2 domain of GRB2 are potentially involved in the tyrosine phosphorylation-dependent association between PLC-gamma1 and GRB2. The association of PLC-gamma1 with GRB2 and Sos suggests that PLC-gamma1 may be directly involved in the Ras signaling pathway and that GRB2 may be involved in the translocation of PLC-gamma1 from cytosol to the plasma membrane as a necessary step for its effect on inositol lipid hydrolysis.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Reduction in sarcoplasmic reticulum Ca2+-ATPase activity contributes to age-related changes in the calcium content and relaxation rate of rabbit aortic smooth muscle.

OBJECTIVE: Elevated blood pressure is a common effect of aging that results from alterations in the calcium (Ca2+) homeostatic mechanisms in vascular smooth muscle cells. The sarcoplasmic reticulum is a primary subcellular organelle involved in Ca2+ homeostasis in vascular smooth muscle. This study was therefore undertaken to delineate possible age-associated changes that occur in the sarcoplasmic reticulum Ca2+ homeostatic mechanisms. METHODS: Relaxation rates after phenylephrine-induced contractions in aortic smooth muscle from rabbits of increasing age were evaluated in the presence of thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase inhibitor. In addition, electron probe X-ray microanalysis (EPMA) was used to analyze the total calcium content of the sarcoplasmic reticulum and cytosol in aortic smooth muscle from rabbits of various ages. RESULTS: The relaxation rate of rabbit aorta contracted with phenylephrine declined with age, the decline being progressively reduced when Ca2+ uptake by the sarcoplasmic reticulum was abolished by thapsigargin. EPMA measurements demonstrated an increased cytosolic calcium content and possibly reduced sarcoplasmic reticulum calcium content in arteries from older animals compared with arteries from juvenile animals. CONCLUSIONS: Reuptake of Ca2+ by the sarcoplasmic reticulum is necessary for optimal relaxation of rabbit aorta after a maximal, agonist-induced contraction. The present data suggest that impaired activity of the sarcoplasmic reticulum Ca2+ pump associated with aging may contribute to the increased cytosolic calcium content and elevated resting tone of aortic smooth muscle obtained from older rabbits.

Aging↗
PubMed1995

Analysis of diclofenac and four of its metabolites in human urine by HPLC.

An HPLC method for the determination of diclofenac (DCF) and four of its metabolites (3'-hydroxydiclofenac, 4'-hydroxydiclofenac, 5-hydroxydiclofenac, and 3'-hydroxy-4'-methoxydiclofenac) in human urine is described. Following base hydrolysis, the samples were neutralized and extracted. Evaporated extracts were reconstituted in mobile phase containing ascorbic acid, and chromatographed, using flow-rate programming, on a reversed-phase column. Absolute recovery (average), was at least 78% for diclofenac and ranged from 75 to 85% for the four metabolites. Standard curves showed linearity over the range of concentrations of 0.2 to 40 ug/mL, using 0.25 mL of urine. Specificity was demonstrated by examining chromatograms of extracts of blank urine from 8 volunteers and 24 study subjects. Good accuracy was observed for all compounds over the concentration range of 0.2 to 40 ug/mL using 0.25 mL of urine. Based on accuracy and precision criteria, the limit of quantitation for all 5 analytes was 0.4 ug/mL, using 0.25 mL of urine. Analysis of urine from subjects with normal and reduced renal function who received diclofenac orally demonstrated that total diclofenac and metabolites excreted in the urine represented approximately 31% and 4% of an oral dose of diclofenac, respectively.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1990

High-performance liquid chromatographic determination of isepamicin in plasma, urine and dialysate.

A high-performance liquid chromatographic method for the measurement of isepamicin, a new aminoglycoside, in plasma, urine and dialysate is reported. The assay utilizes a simple extraction of isepamicin in plasma using commercially available Cyano solid-phase cartridges and dilution of urine and dialysate samples. The separation is performed on a Hypersil C18 column (15 cm X 4.6 mm I.D., 5 microns particle size) and utilizes a mobile phase consisting of 10% methanol and 90% buffer solution containing 0.01 M sodium hexanesulfonate, 0.1 M sodium sulfate and 17 mM acetic acid. The flow-rate is 1.1 ml/min. Dibekacin is used as the internal standard. Isepamicin is derivatized post-column with o-phthalaldehyde for spectrofluorometric detection. The method can also be used for the measurement of other aminoglycosides, i.e. tobramycin, kanamycin, netilmicin and gentamicin. The assay is fast, accurate and has a quantitation limit of 100 ng/ml isepamicin in plasma and 50 ng/ml in urine and dialysate.

Aminoglycosides↗
PubMed1988

Liquid-chromatographic determination of fleroxacin in serum and urine.

This highly sensitive, accurate, and reproducible HPLC method for determining fleroxacin in human serum and urine makes use of a common C18 column, a fluorescence detector, and an internal standard. Serum samples require a simple extraction procedure; urine must be diluted. The method, which we have used extensively for pharmacokinetic assessment of fleroxacin in patients, measures concentrations as low as 5 micrograms/L.

Chromatography, High Pressure Liquid↗