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Pathological lung overdistention associated with mechanical ventilation at high tidal volumes (ventilator-induced lung injury; VILI) compromises endothelial cell (EC) barrier leading to development of pulmonary edema and increased morbidity and mortality. We have previously shown involvement of microtubule (MT)-associated Rho-specific guanine nucleotide exchange factor GEF-H1 in the agonist-induced regulation of EC permeability. Using an in vitro model of human pulmonary EC exposed to VILI-relevant magnitude of cyclic stretch (18% CS) we tested a hypothesis that CS-induced alterations in MT dynamics contribute to the activation of Rho-dependent signaling via GEF-H1 and mediate early EC response to pathological mechanical stretch. Acute CS (30 rain) induced disassembly of MT network, cell reorientation, and activation of Rho pathway, which was prevented by MT stabilizer taxol, siRNA-based GEF-H1 knockdown suppressed CS-induced disassembly of MT network, abolished Rho signaling, and attenuated CS-induced stress fiber formation and EC realignment compared with nonspecific RNA controls. Depletion of GEF-H1 in the murine two-hit model of VILI attenuated vascular leak induced by lung ventilation at high tidal volume and thrombin-derived peptide TRAP6. These data show for the first time the critical involvement of microtubules and microtubule-associated GEF-H1 in lung vascular endothelial barrier dysfunction induced by pathological mechanical strain. cyclic stretch; actin; microtubules; endothelium; Rho GEF; mechanical forces doi: 10.1152/ajplung.00263.2009.

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purineselective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with [K.sub.m] values of 2.5 [+ or -] 0.7 [micro]M for [[sup.3]H]adenine, 6.4 [+ or -] 0.5 [micro]M for [[sup.3]H]guanine, 1.1 [+ or -] 0.1 mM for [[sup.3]H]guanosine, and 4.2 [+ or -] 0.5 mM [[sup.3]H]adenosine. The uptake of [[sup.3]H]guanine (0.05 [micro]M) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [[sup.3]H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [[sup.3]H]guanine uptake with ICso values of 68 [+ or -] 5 and 99 [+ or -] 10 [micro]M, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-[beta]-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 ([IC.sub.50] of ~1 [micro]M), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells. [[sup.3]H]guanine; adenine; cladribine; adenosine