Role of cytoskeleton in shear stress-induced endothelial nitric oxide production.
To study the role of the cytoskeleton in mechanochemical transduction, human umbilical vein endothelial cells were exposed to cytoskeleton-disrupting or -stabilizing agents, and the flow-induced production of nitric oxide (NO) as monitored by intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) was examined. A shear stress of 20 dyn/cm2 elevated cGMP levels approximately twofold relative to basal (stationary) levels (1.9 +/- 0.1 pmol cGMP in stationary controls; P < 0.01). Treatment with 1 microM phalloidin and 0.5 microM cytochalasin D did not significantly affect the flow-induced response (1.77 +/- 0.23 and 2.89 +/- 0.18 pmol cGMP in stationary controls, respectively), whereas disruption of microtubules with 0.5 microM colchicine significantly elevated the response (3.64 +/- 0.18 pmol cGMP in stationary controls; P < 0.01). The NO synthase inhibitor NG-amino-L-arginine abrogated all flow-induced elevations of cGMP, indicating that increased cGMP levels were mediated by NO. Cytoskeletal disruption with 0.2 microM cytochalasin D or 0.5 microM colchicine did not alter cGMP levels in response to 10 nM bradykinin. The role of the plasma membrane in mechanochemical transduction was examined by treatment with cholesteryl hemisuccinate, which attenuated the flow-induced response in a dose-dependent manner. In conclusion, the pathways of flow- and bradykinin-mediated NO production in endothelial cells did not require actin filament turnover or intact actin or microtubule cytoskeletons, and cholesterol, possibly by stiffening the plasma membrane, attenuated the flow response.
Knudsen
H L
HL
Department of Bioengineering, University of California, San Diego 92093-0412, USA.
Frangos
J A
JA
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D000199:Actins / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D001920:Bradykinin / Q000494:pharmacology; D002478:Cells, Cultured; D002788:Cholesterol Esters / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009569:Nitric Oxide / Q000096:biosynthesis*; D010590:Phalloidine / Q000494:pharmacology; D013314:Stress, Mechanical; D014471:Umbilical Veins
D000199:Actins; D002788:Cholesterol Esters; D010590:Phalloidine; D015638:Cytochalasin D; D009569:Nitric Oxide; D006152:Cyclic GMP; D001920:Bradykinin; D003078:Colchicine; C013440:cholesteryl succinate