Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells.
The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca(2+)](i). [Ca(2+)](i) and membrane potentials were strongly correlated. In whole cell clamped cells, BK(Ca) currents were activated by increasing [Ca(2+)](i) via cell dialysis with pipette solution, and the activated BK(Ca) currents were further enhanced by S1P. When [Ca(2+)](i) was buffered at 1 microM, the S1P concentration required to evoke half-maximal activation was 403 +/- 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BK(Ca) channel activity in a reversible manner and shifted the relationship between Ca(2+) concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; 1 mM) using a patch pipette, GDPbetaS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK(Ca) current and channel activation. These results suggest that S1P enhances BK(Ca) channel activity by increasing Ca(2+) sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca(2+) influx through Ca(2+) entry channels. Inasmuch as S1P activates BK(Ca) channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca(2+) mobilization in human endothelial cells.
Kim
Moon Young
MY
Department of Physiology, College of Medicine, Ewha Woman's Univ., 911-1 Mok-6-dong, Yang Chun-gu, Seoul, Republic of Korea, 158-710.
Liang
Guo Hua
GH
Kim
Ji Aee
JA
Kim
Young Ju
YJ
Oh
Seikwan
S
Suh
Suk Hyo
SH
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism
D008246:Lysophospholipids; D024681:Potassium Channels, Calcium-Activated; D043562:Receptors, G-Protein-Coupled; C060506:sphingosine 1-phosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D013110:Sphingosine; D002118:Calcium