Bradykinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation

Abstract

We determined whether activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and a subsequent increase in cytosolic calcium concentration ([Ca2+]i) was an obligatory signaling event mediating the increase in transendothelial permeability induced by bradykinin (BK) and alpha-thrombin (alpha-T). Both BK and alpha-T (each at a concentration range of 0.01-1 microM) caused dose-dependent increases in transendothelial 125I-albumin permeability in cultured bovine pulmonary artery endothelial cell monolayers. Both agonists also produced a rise in inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] by 10 sec that was followed by a prolonged increase in [Ca2+]i. Pretreatment of endothelial cells with the PLC inhibitor, 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dion [(U73122) at 10 microM for 15 min], prevented the increases in Ins(1,4,5)P3 and [Ca2+]i induced by both BK and alpha-T. However, inhibition of PLC with U73122 or another PLC inhibitor, neomycin, did not prevent the increase in endothelial permeability induced by either agonist. In contrast, depletion of cellular protein kinase C (PKC) with phorbol-12-myristate 13-acetate (0.01 microM for 20 hr) increased both BK- and alpha-T-induced phosphoinositide turnover but inhibited the agonist-induced increase in permeability. A PKC inhibitor, staurosporine (5 microM) likewise inhibited the BK-induced increase in endothelial cell permeability to albumin. We conclude that increases in endothelial permeability induced by the inflammatory mediators, BK and thrombin, can occur independently of PLC activation and increased [Ca2+]i but that a PKC-dependent pathway is required for the permeability response.

Anandamide initiates Ca2+ signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells

“…The intracellular Ca 2 þ release in response to bradykinin is known to be mediated by phospholipase C-mediated activation of inositol 1,4,5-trisphosphate (Graier et al, 1991). In our experiments, the phospholipase C inhibitor U73122 (Aschner et al, 1997) was found to prevent anandamide-induced Ca 2 þ signaling with the same potency as bradykinin. Furthermore, the inositol 1,4,5-trisphosphate inhibitor 2-APB (Koganezawa & Shimada, 2001) prevented anandamide-and bradykinininduced Ca 2 þ signaling in the presence and absence (data not shown) of extracellular Ca 2 þ .…”

Cerebral Microvessel Responses to Focal Ischemia

“…2) (Gotoh et al, 1985;Olesen, 1986;Dietrich et al, 1993;Okada et al, 1994a;Risau et al, 1998). There is evidence that loss of the blood-brain barrier and the microvascular ECM results from the actions of bradykinin (Kamiya et al, 1993;Aschner et al, 1997), VEGF Zhang et al, 2000), thrombin (Okada et al, 1994a;Aschner et al, 1997), active matrix metalloproteinases (MMPs) , proteases released by activated leukocytes (Hasty et al, 1990;Garcia et al, 1994;Armao et al, 1997;Opdenakker et al, 2001), and other protease activities (Hosomi et al, 2001). Blockade of bradykinin receptors has been associated with reduced injury and edema formation (Relton et al, 1997).…”

“…Blockade of bradykinin receptors has been associated with reduced injury and edema formation (Relton et al, 1997). Thrombin can increase edema formation by direct action on the cerebral microvascular endothelium (Aschner et al, 1997;Lee et al, 1997;Kubo et al, 2000). VEGF disrupts the interendothelial cell tight-junctions, which involve the gap junction complex containing connexin 43 (Suarez and Ballmer-Hofer, 2001).…”

Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury

“…Throughout the last five decades, investigation into the pathogenesis of the increased endothelial permeability associated with RDS has indicated a role for many mediators, such as cytokines (e.g., IL-1, TNF-␣) (52,93,146), growth factors [e.g., vascular endothelial growth factor (VEGF)] (93), peptides (substance P, bradykinin) (7,150,183), proteases (e.g., elastase) (19), complement activation (e.g., C5a) (125,188), intravascular coagulation (e.g., thrombin) (59,102,103,110,170), reactive oxygen and nitrogen species (e.g., (20,80,89,164,165), and lung sequestration of neutrophils (95) (Fig. 1).…”