Figure 4.

TRPC1-mediated Ca²⁺ entry suppresses SPHK1 activity and S1P generation of S1P. Intracellular Ca²⁺ in response to thapsigargin in HPAECs transduced with dominant negative TRPC1 or control vector following incubation in Ca²⁺-free medium or 2 mM extracellular Ca²⁺. A) Plot shows means ± sd of fold increase in steady-state Ca²⁺ after addition or without addition of extracellular Ca²⁺ in presence of thapsigargin. *Significant increase in intracellular Ca²⁺, P < 0.05. B) Plot showing means ± sd of fold increase in SPHK1 and SPHK2 mRNA expression relative to GAPDH in HPAECs transducing indicated mutants; n = 3. C) SPHK1, SPHK2, and VE-cadherin levels in HPAECs transducing dominant negative TRPC1 or control vector. Actin was used as loading control. D) S1P levels in HPAECs expressing indicated mutants. *Values different from WT ECs; P < 0.05. E) SPHK1 promoter activity in HPAECs cotransfected along with dominant negative TRPC1 or control vector. *Values different from WT ECs; P < 0.05.