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Platelet-activating factor increases endothelial [Ca²⁺]_i and NO production in individually perfused intact microvessels

Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, West Virginia

Submitted 22 October 2004 ; accepted in final form 13 January 2005

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195–H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the initiating event and Ca²⁺-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca²⁺]_i and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca²⁺]_i increased from 69 ± 8 nM to a peak value of 374 ± 26 nM within 3 min and then declined to a sustained level at 190 ± 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca²⁺]_i. PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 ± 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 ± 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with L-NMMA and attenuated in the absence of extracellular Ca²⁺. These results provide direct evidence from intact microvessels that increased endothelial [Ca²⁺]_i is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.

nitric oxide; permeability; fluorescence imaging; 4,5-diaminofluorescein diacetate; endothelial cytoplasmic Ca²⁺ concentration

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