Platelet-conditioned medium increases endothelial electrical resistance independently of cAMP/PKA and cGMP/PKG

Abstract

Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensitization protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharmacological inhibitors, we determined that the activity of PCM is independent of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented the increase in endothelial electrical resistance. Because lysophosphatidic acid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the G(i) protein, inhibitors of the G protein pertussis toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmannin were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay in the characteristic rise in electrical resistance induced by PCM. Inhibition of phosphorylation of extracellular signal-regulated kinase with the mitogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols and inhibition of G(i) and PI3K were obtained for 1-oleoyl-LPA. These results demonstrate that PCM increases endothelial electrical resistance in vitro via a novel, signal transduction pathway independent of cAMP/PKA and cGMP/PKG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosine phosphorylation, the G(i) protein, and PI3K.

Cellular Electrical Micro-Impedance Parameter Artifacts Produced by Passive and Active Current Regulation

“…At each frequency, the statistics of 64 voltage measurements sampled at a rate of 32 Hz for 2 s were evaluated using Eqs. (9)(10)(11). The electrode circuit model frequency-dependent average voltages are in qualitative agreement with those of the gold electrode.…”

“…One of the most common microimpedance sensors currently available consists of a gold micro-electrode with a large counter electrode. 12,13 Although such sensors have been increasingly used to probe and study time-and frequency-dependent cellular impedances, 1,4,[6][7][8]11,18,[20][21][22][23]25,[27][28][29] several forms of random and systematic errors may potentially corrupt these impedance measurements. Despite the importance of this technology, few studies have attempted to identify and reduce these errors and their propagation into cellular barrier function parameter estimates based on cell-cell, cell-matrix, and membrane impedance components.…”

Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products

“…These observations clearly establish that the platelet, via soluble platelet-derived factors, is essential in maintaining normal vascular homeostasis. We and others have reported on vasoactive plateletderived lipids that activate endothelium, including our first report that S1P activated EC phospholipase D (7,10,23,33,48). Subsequent studies have since demonstrated that platelet-derived phospholipids modulate the barrier function of the vascular endothelium via both endothelial barrier-perturbing lipid products such as phosphatidate (6) and barrier-stabilizing/enhancing lipid products such as lysophosphatidic acid (LPA) (8,32) and S1P (11).…”

“…S1P is a normal constituent in plasma with increased (micromolar) levels in serum that are consistent with S1P release during whole blood coagulation (51). S1P exerts diverse biological effects on cells and is now recognized as an important courier of cellular information (7,10,23,33,48). S1P stimulates proliferation, calcium mobilization, adhesion molecule expression, and suppression of caspasedependent apoptosis (7,20,40,46) via ligation of G protein-coupled receptors of the EDG family (26).…”

“…Preliminary characterization indicates that the active barrier-promoting factor is heat stable and resistant to inactivation by exposure to freeze-thaw cycles; therefore, it is likely not a peptide (1,18,32). Additionally, the barrier-enhancing effect of platelet-conditioned media was shown to involve signaling via the small GTPase G i (10). A variety of platelet-released products including adenosine, adenine nucleotides, serotonin, norepinephrine, and arachidonic acid metabolites have been excluded as possible factors involved in the plateletinduced decrease in endothelial permeability (1,18,37,45).…”