Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) and Nacetyl-S-farnesyl-l-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-␣. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway. INTRODUCTIONVascular injury has been implicated in the pathogenesis of disorders such as sepsis and acute respiratory distress syndrome (ARDS). Endothelial cell apoptosis, or programmed cell death, may be important in vascular injury and repair. Apoptotic cells have been identified in increased quantities in the lungs of patients with ARDS, indicating that apoptosis occurs in this syndrome (Polunovsky et al., 1993). Apoptosis can be triggered by disruption of cell-extracellular matrix communication (anoikis) (Frisch and Francis, 1994) or by extracellular factors, such as lipopolysaccharide (Han and Wyche, 1994;Hoyt et al., 1995;Mebmer et al., 1999), tumor necrosis factor (TNF)-␣ (Polunovsky et al., 1994), or UV light (Chatterjee and Wu, 2001). We have previously demonstrated that increased extracellular ATP causes endothelial cell apoptosis after conversion to adenosine and uptake into cells. Moreover, apoptosis caused by intracellular adenosine was enhanced by homocysteine (Dawicki et al., 1997). In subsequent work, we found that increased concentrations of adenosine and homocysteine or inhibition of Sadenosylhomocysteine hydrolase resulted in enhanced levels of S-adenosylhomocysteine (SAH) (Rounds et al., 1998). Because enhanced intracellular concentrations of SAH may result in product inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases (Perna et al., 1997) (Figure 1), we postulated that methyltransferase activity is important in the modulation of endothelial cell apoptosis.Among the methyltransferases is isoprenylcysteine-O-carboxyl methyltransferase (ICMT), the substrates ...

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the K-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters. Graphical abstract ᅟ.

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