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S M Thoroed

Publications and source records attributed to S M Thoroed.

7 recordsLinked to original sources
PubMed2000

Dual role of ATP in supporting volume-regulated chloride channels in mouse fibroblasts.

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.

Adenosine Triphosphate↗
PubMed2000

Hypotonic cell swelling induces translocation of the alpha isoform of cytosolic phospholipase A2 but not the gamma isoform in Ehrlich ascites tumor cells.

We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor cells. Both enzymes are almost uniformly distributed throughout the cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic cell swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich cells, was immediately and transiently increased on hypotonic cell swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic cell swelling is not accompanied by any detectable increase in intracellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.

Animals↗
PubMed1999

Protein phosphotyrosine phosphatase inhibitors suppress regulatory volume decrease and the volume-sensitive Cl- conductance in mouse fibroblasts.

The effects of the protein tyrosine phosphatase (PTP) inhibitors, pervanadate, monoperoxo(picolinato)- oxo-vanadate(V) [mpV(pic)] and dephostatin, on regulatory volume decrease (RVD) and the volume-sensitive Cl- current in mouse L-fibroblasts were studied with the aid of video microscopy and the whole-cell patch-clamp technique. The RVD induced by the hyposmotic shift from 300 to 150 mosmol/l, was strongly suppressed in cells that had been pre-incubated in pervanadate (25 microM) or in mpV(pic) (10 microM), or subjected to extracellular application of dephostatin (20 microM). The acceleration in RVD caused by gramicidin (0.5 microM) was also slowed down by pervanadate pre-treatment, suggesting that the PTP inhibitors affected the volume-sensitive Cl- conductance. Inhibition of the volume-sensitive Cl- current by pervanadate (25 microM) pre-treatment and by acutely applied dephostatin (20 microM) was confirmed in the whole-cell experiments (by @70% and by @50%, respectively). Both pervanadate and dephostatin inhibited the outward and inward Cl- currents equally, which suggests that only the number of open channels was affected. The amplitude of the Cl- current decreased slowly during application of dephostatin and did not recover after its termination. We conclude that in mouse L-fibroblasts, similar to bovine chromaffin cells, inhibition of PTPs results in the suppression of both RVD and the volume-sensitive Cl- current.

Animals↗
PubMed1997

Cell swelling activates phospholipase A2 in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells, loaded with 3H-labeled arachidonic acid and 14C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of 3H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP beta S or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that cell swelling activates a phospholipase A2--perhaps the cytosolic 85 kDa type--by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.

Arachidonic Acid↗
PubMed1994

The Na(+)-independent taurine influx in flounder erythrocytes and its association with the volume regulatory taurine efflux.

95% of the Na(+)-independent influx of taurine in flounder erythrocytes at normal osmolality (330 mosmol kg-1) and 0.30 mmol l-1 taurine was mediated by a saturable system (Vmax = 0.689 nmol g-1 dry mass min-1; Km = 0.47 mmol l-1). The influx was inhibited by taurine analogues, but was not significantly affected by reduced osmolality. This saturable influx of taurine was probably mediated by the so-called Na(+)-dependent influx system for taurine operating in the 0 Na+: 1 taurine mode. The remaining 5% of the Na(+)-independent influx was mediated by a diffusional pathway (Kd = 0.050 microliter g-1 dry mass min-1), since it did not show saturation kinetics, was not inhibited by taurine analogues and did not mediate counter-exchange. This non-saturable influx system for taurine was strongly, but transiently, stimulated by reduction of osmolality. The time course for this stimulatory effect was the same as that for the system that mediates the volume regulatory efflux of taurine. The relative inhibitory effect of bumetanide, furosemide, DIDS and quinine on the fluxes mediated by these two transport systems were also the same. We suggest that these unidirectional fluxes of taurine were mediated by only one transport system: a taurine channel. The effect of reduction of osmolality on the rate coefficient for efflux of beta-alanine was equal to the effect on the efflux of taurine, but greater than the effect on the efflux of choline. This difference probably reflects structural and/or electrical restrictions on the substrates to be transported by the taurine channel. The volume regulatory efflux of taurine was inhibited in the presence of the anti-calmodulin drug trifluoperazine and, in a Ca(2+)-free medium, added EGTA. The 5-lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked the volume regulatory efflux of taurine. We suggest that both Ca2+/calmodulin and leukotrienes contribute to the control of the transport mediated by the taurine channel.

Animals↗
PubMed1986

Taurine transport associated with cell volume regulation in flounder erythrocytes under anisosmotic conditions.

The taurine transport of flounder erythrocytes is associated with a cell volume regulation in anisosmotic media. An osmolality reduction leads to a cell volume increase, which is followed by a volume readajustment towards the original level. A 75 mosM reduction is accompanied by a 33 mumol g dry wt.-1 reduction in the cellular taurine content. The reduction in osmolality activates the taurine release mechanism by transiently increasing the rate coefficient for taurine efflux. The rate coefficient for taurine influx is similarly stimulated. This influx is mediated by a Na+-independent transport system. The concomitant activation of influx and efflux suggests a coupling between these two systems. Higher taurine efflux and influx rate coefficients which decayed more slowly with time were measured in cells suspended in Na+-free (choline replacement) media than in the presence of Na+. This suggests that Na+ may play a role in the taurine release mechanism. Noradrenaline induced a cellular swelling at normal osmolality (330 mosM), but had only a minor effect on the taurine efflux and influx and the cellular taurine content. Urea-induced cellular swelling at normal osmolality initiated a volume regulatory process and activated the taurine release mechanism, similarly to an osmolality reduction. These results show that osmolality reduction and cellular swelling are no prerequisites for the activation of the taurine release mechanism and the cell volume readajustment. It is suggested that the dimension of an intracellular solute compartment determines the activation level of this mechanism.

Animals↗