Biomedical subjects

P Doroshenko

Publications and source records attributed to P Doroshenko.

9 recordsLinked to original sources

PubMed2001

Cell cycle-related changes in regulatory volume decrease and volume-sensitive chloride conductance in mouse fibroblasts.

Cell cycle-related changes in the ability to regulate cell volume following hyposmotic swelling were studied in mouse fibroblasts using videomicroscopy and the whole-cell patch clamp technique. Regulatory volume decrease (RVD) and volume-sensitive Cl- conductance (G(Cl,vol)) were measured: (1) in proliferating cells of different sizes; (2) in cells arrested in defined phases of the cell cycle (G1, G1/S, S, and M phases) using mevastatin, mimosine, hydroxyurea, aphidicolin, cytosine beta-D-arabinofuranoside, and taxol; and (3) in serum-starved cells (G(0) state). Cells in all groups were able to undergo RVD, although the cells approaching mitosis (i.e., the largest cells in proliferating cultures and the taxol-treated cells) had the lowest rates of shrinkage during RVD. In agreement with this finding, the density of G(Cl,vol) was stable in proliferating and cell cycle-arrested cells for most of the cell cycle, with the exception of the cells approaching mitosis and the new daughter cells where the density was decreased to half. The impairment of RVD was greatest in serum-starved cells which also had the lowest density of G(Cl,vol). We conclude that proliferating cells maintain an ability to recover from osmotic swelling as they progress through the cell cycle, although this ability may be compromised during mitosis.

Animals↗

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↗

PubMed1999

High intracellular chloride delays the activation of the volume-sensitive chloride conductance in mouse L-fibroblasts.

1. The relationship between cell volume and volume-sensitive Cl- conductance during hyposmotic cell swelling of patched cells and the effects of intracellular chloride on the conductance have been studied in mouse L-fibroblasts. To this end, swelling-activated current and cell volume were measured simultaneously in cells dialysed with low-Cl- (16 mM) or high-Cl- (130 mM) solutions using the whole-cell patch-clamp technique and videomicroscopy. 2. The increase in cell volume of patched cells and the volume-sensitive conductance saturated during a 4-5 min exposure to mildly hyposmotic solutions (15-20 % less than isosmotic). The swelling of patched cells varied considerably and was greater than the swelling of intact cells. No correlation between the maximal values of the volume-sensitive conductance and the maximal volumes of swollen cells was evident for cells dialysed with the low-Cl- solutions. 3. The amplitude of the volume-sensitive conductance decreased with a reduction in either extracellular or intracellular Cl- concentration; the size of the maximal conductance was not modulated by intracellular Cl- ions. 4. The activation of the volume-sensitive conductance was slower in high-Cl- cells than in low-Cl- cells whether it was induced by hypotonic cell swelling or by cell inflation; in low-Cl- cells the conductance saturated before the cell volume had reached its maximal value. 5. It is concluded that in patched cells an increase in cell volume triggers activation of the volume-sensitive Cl- conductance but does not determine its amplitude and that the rate of activation of the conductance is affected by the intracellular Cl- concentration.

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↗

PubMed1998

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells.

The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl- current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5'-0-(3-thiotriphosphate) (GTP-[gamma-S], 100-250 microM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5-50 microM) and genistein (200 microM) did not inhibit the volume-sensitive Cl- current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 microM) applied intracellularly prevented activation of the current. Voltage-activated Na+ and Ca2+ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10-100 microM), phenylarsine oxide (10 microM), and ZnCl2 (500 microM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl- current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl- channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed.

Animals↗

PubMed1992

Volume-sensitive chloride conductance in bovine chromaffin cell membrane.

1. Bovine chromaffin cells were inflated by pressure applied through a pipette or swollen during intracellular perfusion with hypertonic solutions. Effects of such procedures on electrical properties of the membrane were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence measurements of free intracellular calcium concentration ([Ca2+]i). 2. Application of air pressure (about +5 cmH2O or 490 Pa) through the patch pipette caused an increase in the cell volume and concomitant development of an inwardly directed transient current at the holding potential of -60 mV. The current gradually increased to a peak value and subsequently decayed almost to its initial level within 5-10 min. A short pulse of pressure (5-10 s) was sufficient to elicit the whole sequence of events. 3. Intracellular free Ca2+ ion concentration, [Ca2+]i, steeply increased at the beginning of the pressure pulse to about 0.2 microM and either stayed at this level or decayed back to the more usual value of approximately 0.1 microM. 4. Similar changes in the transmembrane current and [Ca2+]i were observed during intracellular perfusion of cells with hypertonic solutions (30-50 mosM difference relative to the bath solution) or during extracellular application of hypotonic solution. 5. Swelling of non-perfused cells by extracellular application of hyposmotic solution caused the appearance of inward currents in cell-attached membrane patches held at a fixed potential -30 mV relative to the cell's resting potential. The kinetics of the current resembled those of the whole-cell current. 6. Intracellular introduction of guanosine triphosphate (GTP, 300 microM) significantly prolonged the duration (from 62 +/- 10 s, n = 5, to 98 +/- 8 s, n = 4, when measured at the level of half-amplitude), while introduction of the non-hydrolysable analogue of guanosine diphosphate (GDP), guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 300 microM), decreased the maximal rate of increase (from 11.4 +/- 2.6 pA/s, n = 6, to 3.2 +/- 2.1 pA/s, n = 10) of the current activated by pressure. 7. Lowering of the intracellular free Ca2+ ion concentration by introduction of 10 mM-EGTA did not significantly affect the current amplitude or time course. However, a rapid increase in the [Ca2+]i to micromolar levels (by activation of the voltage-operated calcium channels during membrane depolarization) could terminate development of the current activated by pressure and cause its fast decay to zero-current level.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells.

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABAb) receptor activation on K(+)-depolarization-induced signals and on voltage-activated Ca2+ currents were investigated. Both GABA (20 microM) plus bicuculline (20 microM) and (-)baclofen (20-100 microM), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10-20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1 micrograms/ml, for 4-6 h at 37 degrees C). Baclofen (50 microM) inhibited Ca2+ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5'-triphosphate GTP gamma S (100 microM), led to complete inhibition of the Ca2+ conductance and of voltage-evoked intracellular Ca ([CA]i) transients within 2 min. In paired cells intracellularly perfused with GTP gamma S-free solution, the Ca2+ current amplitude decreased by only about 30% for 5-6 min. It is concluded that bovine chromaffin cells have functional GABAb receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Novel chloride conductance in the membrane of bovine chromaffin cells activated by intracellular GTP gamma S.

1. The effects of introducing the non-hydrolysable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) into perfused bovine chromaffin cells were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence [Ca2+]i measurements. 2. GTP gamma S (5-300 microM) induced a slowly developing transient current (inwardly directed at the holding potential -60 to -70 mV) and [Ca2+]i oscillations. The current activated with a 10-50 s delay after the start of whole-cell dialysis, peaked at 70-120 s and decayed almost to its initial level during the next 150-300 s. Calcium oscillations were observed within the first 100-150 s of cell perfusion. 3. GTP competitively lowered the probability of current activation by GTP gamma S. At low GTP gamma S/GTP ratio (5 and 300 microM, respectively) activation of the current was observed only rarely. 4. The activation of the current was accompanied by an increase in conductance but not by changes in the current reversal potential. The changes in the conductance did not depend on the membrane potential; no time-dependent relaxation of the current was induced by steps in the membrane voltage. 5. The current reversal potential was close to the Cl- equilibrium potential; changes in the extracellular Cl- concentration induced corresponding changes in the current amplitude and shifted its reversal potential. The permeability to larger anions--aspartate, glutamate and isethionate--was about one-tenth of that for chloride. 6. Single-channel conductance, estimated from the ratio of the mean current and its variance, was about 1-2 pS. 7. The current could be reversibly blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 10 microM), chlorpromazine (5 microM) and tolbutamide (0.5-5 mM). 8. It is suggested that the GTP gamma S-induced increase in the permeability to Cl- ions is due to a G protein-mediated production of an as yet unidentified second messenger.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Second messengers mediating activation of chloride current by intracellular GTP gamma S in bovine chromaffin cells.

1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP gamma S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP gamma S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300 microM; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37 degrees C) did not affect the current activation by GTP gamma S nor did it cause current activation in the absence of GTP gamma S. 6. Activation of the current by GTP gamma S could be prevented by incubating the cells for 10-15 min with 2.5 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP gamma S. 7. Activation of the current by GTP gamma S could also be prevented by incubating the cells with 1 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), an inhibitor of cyclo-oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP gamma S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism.

Adrenal Medulla↗