Related Experiment Videos
Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular
Chinnaswamy Tiruppathi1, Marc Freichel, Stephen M Vogel
1Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Ill 60612, USA.
Abstract:
We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4(-/-) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4(-/-) LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 micromol/L). In TRPC4(-/-) LECs, thrombin or TFLLRNPNDK-NH2 produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4(-/-) endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (K(f,c)), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4(-/-); La3+ (1 micromol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4(-/-). These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.
Insights
The TRPC4 channel is crucial for store-operated calcium entry, which increases microvascular endothelial permeability. TRPC4 knockout mice show reduced permeability responses to thrombin, highlighting TRPC4
Area of Science:
- Physiology
- Molecular Biology
- Cell Biology
Background:
- Microvascular endothelial permeability is regulated by intracellular calcium signaling.
- Store-operated calcium (SOC) entry channels, including the TRPC gene family, are implicated in modulating cellular responses.
- The specific role of TRPC channels in endothelial permeability remains to be fully elucidated.
Purpose of the Study:
- To investigate the contribution of the TRPC gene family, specifically TRPC4, to increased microvascular endothelial permeability.
- To determine if TRPC4 mediates the prolonged intracellular calcium signaling observed in response to agonists that increase endothelial permeability.
Main Methods:
- Utilized wild-type (wt) and TRPC4 knockout (TRPC4(-/-)) mice and isolated lung vascular endothelial cells (LECs).
- Assessed TRPC gene expression via RT-PCR.
- Stimulated LECs and perfused lungs with thrombin or a Proteinase-Activated Receptor-1 (PAR-1) agonist peptide.
- Measured intracellular calcium (Ca2+) transients, Ca2+ influx, actin-stress fiber formation, endothelial cell retraction, and microvessel filtration coefficient (K(f,c)).
Main Results:
- TRPC4 mRNA was expressed in wt LECs but absent in TRPC4(-/-) LECs.
- Thrombin or PAR-1 agonist induced prolonged Ca2+ influx in wt LECs, but this was significantly reduced in TRPC4(-/-) LECs.
- TRPC4 deficiency impaired thrombin-induced actin-stress fiber formation and endothelial cell retraction.
- In isolated lungs, PAR-1 agonist increased vascular permeability (K(f,c)) in wt mice, an effect significantly blunted in TRPC4(-/-) mice.
Conclusions:
- TRPC4-dependent calcium entry is a key regulator of agonist-induced microvascular endothelial permeability.
- TRPC4 channels play a critical role in mediating the increase in endothelial permeability by facilitating calcium influx.
- Targeting TRPC4 may offer a therapeutic strategy for conditions involving excessive vascular permeability.