Major odorants released as urinary volatiles by urinary incontinent patients.

In this study, volatile urinary components were collected using three different types of samples from patients suffering from urinary incontinence (UI): (1) urine (A); (2) urine + non-used pad (B); and (3) urine + used pad (C). In addition, urine + non-used pad (D) samples from non-patients were also collected as a reference. The collection of urinary volatiles was conducted with the aid of a glass impinger-based mini-chamber method. Each of the four sample types (A through D) was placed in a glass impinger and incubated for 4 hours at 37 °C. Ultra pure air was then passed through the chamber, and volatile urine gas components were collected into Tedlar bags at the other end. These bag samples were then analyzed for a wide range of VOCs and major offensive odorants (e.g., reduced sulfur compounds (RSCs), carbonyls, trimethylamine (TMA), ammonia, etc.). Among the various odorants, sulfur compounds (methanethiol and hydrogen sulfide) and aldehydes (acetaldehyde, butylaldehyde, and isovaleraldehyde) were detected above odor threshold and predicted to contribute most effectively to odor intensity of urine incontinence.

Identification of subdomains in NADPH oxidase-4 critical for the oxygen-dependent regulation of TASK-1 K+ channels.

Hypoxic inhibition of K+ current is a critical O2-sensing mechanism. Previously, it was demonstrated that the cooperative action of TASK-1 and NADPH oxidase-4 (NOX4) mediated the O2-sensitive K+ current response. Here we addressed the O2-sensing mechanism of NOX4 in terms of TASK-1 regulation. In TASK-1 and NOX4-coexpressing human embryonic kidney 293 cells, hypoxia (5% O2) decreased the amplitude of TASK-1 current (hypoxia-DeltaI(TASK-1)). To examine whether reactive oxygen species (ROS) mediate the hypoxia-DeltaI(TASK-1), we treated the cells with carbon monoxide (CO) which is known to reduce ROS generation from the heme-containing NOX4. Unexpectedly, CO failed to mimic hypoxia in TASK-1 regulation, rather blocked the hypoxia-DeltaI(TASK-1). Moreover, the hypoxia-DeltaI(TASK-1) was neither recovered by H2O2 treatment nor prevented by antioxidant such as ascorbic acid. However, the hypoxia-DeltaI(TASK-1) was noticeably attenuated by succinyl acetone, a heme synthase inhibitor. To further evaluate the role of heme, we constructed and expressed various NOX4 mutants, such as HBD(-) lacking the heme binding domain, NBD(-) lacking the NADPH binding domain, FBD(-) lacking the FAD binding domain, and HFBD(-) lacking both heme and FAD domains. The hypoxia-DeltaI(TASK-1) was significantly reduced in HBD(-)-, FBD(-)-, or HFBD(-)-expressing cells, versus wild-type NOX4-expressing cells. However, NBD(-) did not affect the TASK-1 response to hypoxia. We also found that p22 is required for the NOX4-dependent TASK-1 regulation. These results suggest that O2 binding with NOX4 per se controls TASK-1 activity. In this process, the heme moiety and FBD seem to be responsible for the NOX4 regulation of TASK-1, and p22 might support the NOX4-TASK-1 interaction.

ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBPalpha and STAT-3.

Hepcidin, a liver peptide, systemically inhibits iron utilization and is downregulated under hypoxic conditions. However, little is known about the mechanism underlying the hypoxic suppression of hepcidin. Here, we tested the possibility that HIF-1 and ROS are involved in hepcidin regulation. Hepcidin mRNA, pre-mRNA, and protein levels were reduced in mouse livers and in HepG2 cells after hypoxic incubation, and HIF-1 overexpression and knock-down studies showed that hepcidin regulation is independent of HIF-1. On the other hand, ROS levels were significantly elevated in hypoxic HepG2 cells, and anti-oxidants prevented the hypoxic down-regulation of hepcidin. Conversely, a prooxidant, H(2)O(2), suppressed hepcidin expression in these cells even in normoxia. Of the various transcription factors examined, C/EBPalpha and STAT-3 were found to dissociate from hepcidin promoter under hypoxia, but to become fully engaged after anti-oxidant treatment. These results suggest that ROS repress the hepcidin gene by preventing C/EBPalpha and STAT-3 binding to hepcidin promoter during hypoxia.