Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sodium cations localized at crystallographically distinct cation sites in dehydrated zeolites were characterized using 23Na double rotation, two-dimensional nutation, and magic-angle-spinning nuclear magnetic resonance spectroscopy. The new DOR NMR technique has been applied at different magnetic field strengths to determine the quadrupole parameters of the overlapping quadrupole patterns. In the NMR spectra of dehydrated NaY and NaEMT two signals of sodium cations were identified, a low-field gaussian line at -12 +/- 1 ppm and a high-field quadrupole pattern, with an isotropic chemical shift of -8 +/- 1 ppm and a quadrupole coupling constant of about 4 MHz. By comparison of the 23Na MAS NMR intensities of these signals with the population of the cation sites determined by XRD and by calculation of the electric field gradients, the former signal was attributed to sodium cations at the sites SI and the latter one to sodium cations at the sites SI' as well as SII in faujasite and zeolite EMT. This assignment has further been confirmed by 23Na MAS NMR studies of dehydrated HNaY and BaNaY zeolites.
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PMID:Characterization of sodium cations in dehydrated faujasites and zeolite EMT by 23Na DOR, 2D nutation, and MAS NMR. 781 49

The extracellular pH (pHe) in solid tumors is frequently lower than the pHe in normal tissues, but the intracellular pH (pHi) is regulated to physiological levels. Cell killing can be achieved in an acidic environment in tissue culture by nigericin, which acidifies cells by transporting H+ from the extracellular space into the cytoplasm; this cell killing can be enhanced when used with 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of membrane-based Na+/H+ exchange, which plays a major role in the regulation of pHi (R. P. Maidorn; E. J. Cragoe; I. F. Tannock, Br. J. Cancer 67:297-303; 1993). We have therefore assessed the ability of nigericin and EIPA to kill cells in two murine solid tumors (the KHT fibrosarcoma and the EMT-6 sarcoma). Hydralazine, which reduces tumor blood flow, or glucose, which stimulates glycolysis leading to accumulation of lactate, were also administered to mice to lower pHe in the tumors. We observed only a small decrease in the surviving fractions of cells in the tumors when tolerated doses of nigericin and EIPA were given IP to tumor-bearing mice. When nigericin and EIPA were combined with administration of hydralazine, the surviving fraction of cells in both tumors was reduced by a factor of 0.01, but there were minimal effects on growth delay. Administration of glucose with nigericin and EIPA led to a smaller reduction in surviving fraction of the KHT tumor (by approximately 0.1), although glucose was more effective than hydralazine in lowering the mean tumor pHe. When KHT tumors were treated with 15 Gy X-rays followed immediately by nigericin, EIPA, and hydralazine, a reduced surviving fraction as well as an increase in tumor growth delay was observed compared to radiation alone; however, there was little evidence to suggest that these agents were selectively toxic to the cells that survived radiation. Nigericin and EIPA, with or without hydralazine, had minimal effects on normal tissues, as assessed by changes in body weight, number of leukocytes, and serum creatinine levels. We conclude that pharmacological effects to acidify cells and to prevent regulation of pHi under the acidic conditions that exist in solid tumors can lead to moderate levels of cell killing, if additional strategies are used to lower tumor pHe.
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PMID:Antitumor activity of nigericin and 5-(N-ethyl-N-isopropyl)amiloride: an approach to therapy based on cellular acidification and the inhibition of regulation of intracellular pH. 786 1

The viability of cells within the acidic microenvironment found in solid tumours is expected to depend on the regulation of intracellular pH (pHi). 5-(N,N-hexamethylene) amiloride (HMA) is a potent inhibitor of the Na+/H+ antiport, a major mechanism for the regulation of pHi. We have therefore studied the cytotoxicity of HMA in combination with nigericin, a cell-acidifying agent, for EMT-6 cells in monolayer cell culture, in spheroids and in a murine tumour model. The combination of nigericin and HMA was toxic to cells in tissue culture at extracellular pH (pHe) < or = 6.8 (as may be found in tumours) but not at pH 7.0 or above (as in most normal tissues). Compared with amiloride, the relative potency of HMA in causing in vitro cytotoxicity (approximately 100-fold) was similar to that for inhibition of the Na+/H+ antiport. The fluorescent probe Hoechst 33342 was used with flow cytometry to study the cytotoxicity of HMA and nigericin at different depths in multicellular tumour spheroids. Only small differences in the level of cell survival were observed, but higher concentrations of HMA were required as compared with those giving equal levels of survival in monolayer culture. The pharmacokinetics of HMA in mice was studied by using high-performance liquid chromatography: after intraperitoneal injection of 20 micrograms g-1, the plasma level of HMA peaked at 8 microM after about 15 min and decreased to 1 microM at 120 min; the half-life was 35 min. Nigericin and HMA, at doses of 1.25 micrograms g-1 and 10 micrograms g-1 respectively, failed to cause significant cell killing in the EMT-6 murine tumour, but the surviving fraction was reduced to approximately 0.004 when hydralazine was administered with nigericin and HMA. Local tumour irradiation (15 Gy), followed by treatment with these drugs, led to cell killing that was additive to the effects of drugs and radiation alone, so that hypoxic cells which survived radiation did not appear more sensitive to pH-dependent drug treatment. Acid-mediated therapy can lead to cell death in murine solid tumours, but further measures will be required before the strategy can be exploited clinically.
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PMID:Inhibition of the regulation of intracellular pH: potential of 5-(N,N-hexamethylene) amiloride in tumour-selective therapy. 791 6

Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.
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PMID:Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. 792 40

Solid tumours are known to develop regions of extracellular acidity and survival of tumour cells in such regions depends on membrane-based mechanisms which regulate intracellular pH (pHi). We have therefore developed a method, based on dual staining of cells and flow cytometry, to study the regulation of pHi in subpopulations of tumours and spheroids. The activity of membrane-based pHi regulating transporters was studied in EMT-6 and MGH U1 cells grown in monolayer culture, spheroids, and tumours. pHi was measured with the fluorescent pH probe 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, and Hoechst 33342 was used to identify cells from different regions of tumours and spheroids. In monolayer culture, incubation of cells for 18 h at pHe 6.6 led to a 1.3-1.5-fold enhancement in the activity of both the Na+/H+ exchanger and the Na(+)-dependent Cl-@HCO3- exchanger. This effect was prevented by the protein synthesis inhibitor cycloheximide. Cells from the centre of EMT-6 spheroids had increased activity of the Na+/H+ exchanger compared to cells from the periphery, when spheroids were grown in medium at pH 6.6, but not at 7.4. By contrast, in MGH U1 spheroids, cells from the centre had increased activity of the Na+/H+ antiport under both sets of conditions. There was no significant difference in the activity of the Na+/H+ exchanger in cells derived from different subpopulations of EMT-6 tumours or MGH U1 xenografts in nude mice. Although upregulation of Na+/H+ exchange occurs after exposure to acidic conditions in vitro, the microenvironmental conditions found within solid tumours do not appear to cause this effect. Our results suggest the feasibility of pharmacological inhibition of Na+/H+ exchange activity as an approach to therapy directed against nutrient-deprived tumour cells.
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PMID:Regulation of intracellular pH in subpopulations of cells derived from spheroids and solid tumours. 821 5

The mean extracellular pH (pHe) within solid tumours has been found to be lower than in normal tissues. Agents which cause intracellular acidification at low pHe might have selective toxicity towards cells in tumours. Weak acids (or their anions) with pKa values in the range of 4-6 have a higher proportion of molecules in the uncharged form at low pHe and can diffuse more rapidly into cells. The effects of organic acids including succinate, monomethyl succinate and malonate to acidify cells have been evaluated under conditions of different pHe in the acidic range. These weak acids caused intracellular acidification of murine EMT-6 and human MGH-U1 cells in a concentration and pHe dependent fashion. At concentrations of 10 mM and above, these acids also caused in vitro cytotoxicity to these cells at low pHe (< 6.5). The rate and extent of cellular acidification caused by these weak acids, and their cytotoxicity at low pHe, were enhanced by exposure to amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA), agents which inhibit Na+/H+ exchange, and hence the regulation of intracellular pH. Acid dependent cytotoxicity was also investigated in a murine solid tumour using the endpoints of growth delay and colony formation in vitro following treatment in vivo. Agents were tested alone or with 15 Gy X-rays to select a population of hypoxic (and presumably acidic) cells. Achievable serum concentrations of succinate were about 1 mM and no antitumour activity of succinate was detected when used in this way. It is concluded that weak acids are selectively taken up into cells, and can cause selective cellular acidification and toxicity, at low pHe in culture. Weak acids that are normal cellular metabolites are not toxic in vivo, but weak acids carrying cytotoxic groups offer the potential for selective uptake and toxicity under the conditions of low pHe that exist in many solid tumours.
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PMID:Selective cellular acidification and toxicity of weak organic acids in an acidic microenvironment. 826 Mar 58

We evaluated a new analyzer designed for point-of-care testing of blood gases, sodium, potassium, ionized calcium, and hematocrit. The Gem Premier (Mallinckrodt) system has two components: the analyzer and a disposable cartridge. Analysis takes place in the cartridge, which contains the electrochemical sensors, the calibrants, the reagents, the sampling stylus, and the waste container. The system was evaluated for imprecision and accuracy. With aqueous control materials, total imprecision (CV) was: pH, 0.10-0.18%; PCO2, 3.16-5.78%; PO2, 2.92-4.85%; sodium, 0.82-1.44%; potassium, 1.35-1.48%; ionized calcium, 0.75-1.45%; and hematocrit, 1.13-1.83%. Accuracy of the system was assessed by split-sample comparison with the Radiometer ABL 330 blood gas analyzer for pH and blood gases, the Nova Stat Profile 5 for whole-blood electrolyte and hematocrit analysis, and the IL Phoenix for plasma electrolyte analysis. After outlier correction, regression statistics were excellent for all analytes except sodium, which demonstrated Sy[x values between 1.80 and 2.30 mmol/L and 0.85 < or = r < 0.90.
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PMID:Performance of Gem Premier blood gas/electrolyte analyzer evaluated. 837 66

The extracellular pH (pHe) in solid tumours is frequently lower than the pHe in normal tissues. Cells within an acidic environment depend on mechanisms which regulate intracellular pH (pHi) for their survival, including the Na+/H+ antiport which exports protons in exchange for Na+ ions. Amiloride and its analogues DMA (5-(N,N-dimethyl)amiloride), MIBA (5-(N-methyl-N-isobutyl)amiloride) and EIPA (5-(N-ethyl-N-isopropyl)amiloride) are known to inhibit the Na+/H+ antiport and therefore decrease the cells ability to regulate pHi. All three analogues were found to be potent inhibitors of the antiport in human MGH-U1 and murine EMT-6 cells, with DMA being approximately 20, MIBA 100 and EIPA 200-fold as potent as amiloride; EIPA also gave more complete suppression of the Na+/H+ antiport. These agents were not toxic to cells when used alone; however, in combination with nigericin, an agent which acidifies cells, all three analogues were toxic to cells at pHe < 7.0, and markedly enhanced the toxicity of nigericin alone. Cell killing was greatest for nigericin used with EIPA or MIBA. None of the agents were toxic to cells at pHe 7.0 or above. When used against variant cells lacking the Na+/H+ antiport (PS-120 cells) EIPA did not enhance the cytotoxicity of nigericin alone, suggesting that the observed effect was due to inhibition of Na+/H+ exchange, rather than due to non-specific effects. The combination of EIPA and nigericin gave similar cell killing in previously dissociated and intact MGH-U1 spheroids, suggesting that the agents have good penetration of solid tissue. Preliminary experiments using EMT-6 tumours in mice suggested that EIPA and nigericin were able to enhance the toxicity of radiation in vivo, presumably through selective effects against the hypoxic (and probably acidic) subpopulation of cells that is resistant to radiation.
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PMID:Therapeutic potential of analogues of amiloride: inhibition of the regulation of intracellular pH as a possible mechanism of tumour selective therapy. 838 57

A temperature-sensitive mutant of the v-abl oncoprotein has previously been shown to have markedly reduced tyrosine protein kinase activity in interleukin 3 (IL-3)-dependent cells grown at restrictive (39 degrees C), compared to permissive (32 degrees C) temperatures. Transfection of this mutant v-abl into the IC2.9 cell line, generated the IC.DP subclone which was dependent on IL-3 for survival at 39 degrees C, but not at 32 degrees C. Furthermore, IC.DP cells cultured at 32 degrees C exhibited IL-3-independent thymidine incorporation, which was not apparent at 39 degrees C. Switching cells from the restrictive to the permissive temperature resulted in an increase in cellular inositol-1,4,5-trisphosphate, choline phosphate and diacylglycerol levels in the IC.DP cell line. These increases were only observed after a lag period of 4 h. Within 2 h of switching IC.DP cells previously maintained at 32 to 39 degrees C, there was a significant decrease in all three metabolites. Temperature switches had no effect upon these metabolites in the parent IC2.9 cell line. Down-regulation of protein kinase C inhibited v-abl-stimulated DNA synthesis in IC.DP cells cultured at 32 degrees C. IC.DP cells cultured at 32 degrees C were found to have a constitutively activated Na+/H+ antiport, although this activation was inhibited by the down-modulation of protein kinase C. These data indicate a role for phospholipid hydrolysis and protein kinase C activation in V-ABL-mediated abrogation of IL-3 dependence.
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PMID:Cellular signaling events elicited by v-abl associated with growth factor independence in an interleukin-3-dependent cell line. 839 52

N-dodecylimidazole is a compound which acquires detergent properties under acidic conditions and might be useful in killing selectively cells in those regions of solid tumours which have a reduced extracellular pH (pHe). We have therefore studied the effects of N-dodecylimidazole against malignant cells in tissue culture. N-dodecylimidazole displayed pHe-dependent cytotoxicity against EMT-6 and MGH U1 cells; cell killing was dose dependent and was 100-fold greater at pHe 6.0 than pHe 7.0. Reduced toxicity of N-dodecylimidazole was observed at higher cell concentrations (> 10(6) cells ml-1), and only minor effects were observed against multicellular tumour spheroids. Potential mechanisms of action of N-dodecylimidazole include detergent-mediated lysis of the cell membrane at low pHe, and selective uptake into lysosomes where detergent activity leads to rupture of the lysosomal membrane and release of cytolytic enzymes. Inhibition of activity of cysteine proteases by the inhibitor E-64 did not protect cells against the toxicity of N-dodecylimidazole, suggesting that these lysosomal enzymes do not play a major role in the mechanism of action of this compound. Lysis of erythrocytes (which contain no lysosomes) was observed with low concentrations of N-dodecylimidazole. Dependence of cell lysis on cell concentration was similar to that observed for two other detergents that act on the plasma membrane, Triton X-100 and sodium dodecyl sulfate. We conclude that N-dodecylimidazole causes pHe dependent cell killing in two cultured tumour cell lines, and that its mechanism of action is probably due to acid mediated production of detergent activity which acts primarily on the cell plasma membrane.
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PMID:pH dependent cytotoxicity of N-dodecylimidazole: a compound that acquires detergent properties under acidic conditions. 842 83


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