Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability.
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.
Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Ill 60612, USA.
Chinnaswamy
C
Tiruppathi
Marc
M
Freichel
Stephen M
SM
Vogel
Biman C
BC
Paria
Dolly
D
Mehta
Veit
V
Flockerzi
Asrar B
AB
Malik
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005838:Genotype; D006801:Humans; D007811:Lanthanum / Q000494:pharmacology; D008168:Lung / Q000098:blood supply* / Q000166:cytology / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D009842:Oligopeptides / Q000494:pharmacology; D011652:Pulmonary Circulation / Q000187:drug effects / Q000502:physiology; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000819:agonists; D050052:TRPC Cation Channels; D013917:Thrombin / Q000494:pharmacology
D015220:Calcium Channels; D009842:Oligopeptides; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D050052:TRPC Cation Channels; D007811:Lanthanum; D013917:Thrombin; D002118:Calcium