Regulatory role of tyrosine phosphorylation in the swelling-activated chloride current in isolated rabbit articular chondrocytes.
Articular chondrocytes are exposed in vivo to the continually changing osmotic environment and thus require volume regulatory mechanisms. The present study was designed to investigate (i) the functional role of the swelling-activated Cl(-) current (I(Cl,swell)) in the regulatory volume decrease (RVD) and (ii) the regulatory role of tyrosine phosphorylation in I(Cl,swell), in isolated rabbit articular chondrocytes. Whole-cell membrane currents were recorded from chondrocytes in isosmotic, hyposmotic and hyperosmotic external solutions under conditions where Na(+), K(+) and Ca(2+) currents were minimized. The cell surface area was also measured using microscope images from a separate set of chondrocytes and was used as an index of cell volume. The isolated chondrocytes exhibited a RVD during sustained exposure to hyposmotic solution, which was mostly inhibited by the I(Cl,swell) blocker 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl)oxobutyric acid (DCPIB) at 20 microM. Exposure to a hyposmotic solution activated I(Cl,swell), which was also largely inhibited by 20 microM DCPIB. I(Cl,swell) in rabbit articular chondrocytes had a relative taurine permeability (P(tau)/P(Cl)) of 0.21. Activation of I(Cl,swell) was significantly reduced by the protein tyrosine kinase (PTK) inhibitor genistein (30 microM) but was only weakly affected by its inactive analogue daidzein (30 microM). Intracellular application of protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (250 and 500 microM) resulted in a gradual activation of a Cl(-) current even in isosmotic solutions. This Cl(-) current was almost completely inhibited by 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS, 500 microM) and was also largely suppressed by exposure to hyperosmotic solution, thus indicating a close similarity to I(Cl,swell). Pretreatment of chondrocytes with genistein significantly prevented the activation of the Cl(-) current by sodium orthovanadate, suggesting that the basal activity of endogenous PTK is required for the activation of this Cl(-) current. Our results provide evidence to indicate that activation of I(Cl,swell) is involved in RVD in isolated rabbit articular chondrocytes and is facilitated by tyrosine phosphorylation.
Okumura
Noriaki
N
Department of Physiology, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan.
Imai
Shinji
S
Toyoda
Futoshi
F
Isoya
Eiji
E
Kumagai
Kousuke
K
Matsuura
Hiroshi
H
Matsusue
Yoshitaka
Y
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D048429:Cell Size; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007189:Indans / Q000494:pharmacology; D008297:Male; D009995:Osmosis / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
C443139:4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-one-5-yl)oxybutyric acid; D018118:Chloride Channels; D003517:Cyclopentanes; D007189:Indans; D047428:Protein Kinase Inhibitors; D014638:Vanadates; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid