Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization.
Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.
Camiña
Jesus P
JP
Department of Medicine, Research Area, Molecular Endocrinology Laboratory, Complejo Hospitalario Universitario de Santiago (CHUS) and University of Santiago de Compostela, Spain.
Diaz-Rodriguez
Esther
E
Harks
Erik G A
EG
Theuvenet
Alexander P R
AP
Ypey
Dirk L
DL
Casanueva
Felipe F
FF
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002450:Cell Communication / Q000187:drug effects; D002999:Clone Cells; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D010712:Phosphatidic Acids / Q000378:metabolism; D010743:Phospholipids / Q000302:isolation & purification / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014822:Vitreous Body / Q000737:chemistry*
D005456:Fluorescent Dyes; D007476:Ionophores; D010712:Phosphatidic Acids; D010743:Phospholipids; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; C472625:bovine vitreous lipid factor; D015544:Inositol 1,4,5-Trisphosphate; D011188:Potassium; D002118:Calcium