Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]_i, nitric oxide, and gap formation in intact venules

Abstract

Zhou X, He P. Temporal and spatial correlation of plateletactivating factor-induced increases in endothelial [Ca 2ϩ ]i, nitric oxide, and gap formation in intact venules. Am J Physiol Heart Circ Physiol 301: H1788 -H1797, 2011. First published August 26, 2011 doi:10.1152/ajpheart.00599.2011We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca 2ϩ ]i, NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca 2ϩ ]i was measured on fura-2-or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca 2ϩ ]i, NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca 2ϩ ]i preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca 2ϩ ]i in each cell, suggesting that the initial levels of endothelial [Ca 2ϩ ]i determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca 2ϩ response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation. microvessel permeability; endothelial Ca 2ϩ imaging; nitric oxide imaging; endothelial junctions; intracellular Ca 2ϩ concentration INCREASED MICROVESSEL PERMEABILITY to fluid and macromolecules is a hallmark of inflammation, resulting in edema formation and organ dysfunctions. A better understanding of the mechanisms that regulate microvessel permeability will benefit the development of potential therapeutic strategies for inflammation-related diseases. Gap formation between endothelial cells has been indicated as the main transport pathway responsible for the increased permeability to fluid and macromolecules during inflammation (3,6,15,17,18,22,24,29). Our previous study (17) demonstrated that the dynamic changes in endothelial gaps upon platelet-activating factor (PAF) stimulation closely correlated with the time course of increases in microvessel hydraul...

Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function

“…However, we cannot obviously rule out subtle regulatory roles for cytosolic Ca 2ϩ and MLCK (or other Ca 2ϩ -activated enzymes) during the signaling processes impacting on cytoskeletal rearrangements such as endothelial permeability or migration. In most in vivo studies, Ca 2ϩ signals and endothelial permeability to agonists, measured for instance on mesenteric venules of anesthetized rats, are merely correlative and are based on the use of pharmacological compounds with questioned specificity (57)(58)(59). However, a regulatory role for cytosolic Ca 2ϩ rise and subcellular activation of Ca 2ϩ -activated enzymes in endothelial barrier function might be more significant under in vivo conditions that are more complex and where subtle rearrangements of the barrier at specific sites are sufficient to allow passage of cells and solutes.…”

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

“…For many of our recent studies we have used male Sprague-Dawley rats which are acclimated in our animal facility at least one week before use at about age 3-4 months at which time we find they generally have available long, straight mesenteric microvessels. By contrast, our colleagues have successfully used female Sprague-Dawley rats of 2-3 months age 13 .…”

“…While early investigations used frog microvessels in mesentery and thin cutaneous pectoris muscle 7,8 , by far the most commonly used preparation in mammalian models is the rat mesentery [9][10][11][12][13][14][15] . Most investigations have focused on acute changes in vascular permeability studied over periods of 1-4 hr, but more recent investigations have been extended to measurements on individual vessels 24-72 hr after an initial perfusion [18][19][20][21][22][23][24][25][26] The fundamental aspect of the technique is the measurement of volume flow (J v ) across a defined surface area (S) of the microvessel wall.…”

“…Measurement of transvascular flows may also be combined with more detailed investigations using a sophisticated fluorescence microscope with appropriate filters such as rigs used for measurements of solute permeability, fluorescent ratio monitoring of cytoplasmic calcium or other cellular mechanisms, and confocal imaging 6,12,13,27 . A key advantage of all microperfusion approaches is the ability to make repeated measures, on the same vessel, under controlled change of driving force such as hydrostatic and oncotic pressures, or induced change in vessel responses to inflammatory conditions.…”

Delineation of Platelet Activation Pathway of Scutellarein Revealed Its Intracellular Target as Protein Kinase C

“…At last, ] i ) was determined as described previously. [27][28][29][30][31] Briefly, washed platelets were resuspended at concentrations of 10 8 /mL in modified Tyrode buffer (136.5 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO 3 , 0.42 mM NaH 2 PO 4 , 1 mM MgCl 2 , 5 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (Hepes), 1.11 mM dextrose, 0.35% BSA, pH 7.4) with 10 U/mL heparin, 0.2 U/mL Apyrase and 1 µM prostaglandin I 2 (PGI 2 ), and then incubated for 8 min. After centrifugation, about 10 8 platelets were incubated in 300 µL of modified Tyrode buffer with 2.5 mM probenecid, 0.02% pluronic, 10 µM Fluo-3, 0.5 µM PGI 2 and 0.02 U/mL apyrase for 40 min at 37°C and washed, while the test drug was added in the last 10 min.…”