The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (QO 2 ) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced QO 2 in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL QO 2 . To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212-2 reduced QO 2 in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on QO 2 . In contrast, the CB2-selective agonist JHW-133 had no effect on QO 2 , while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on Q O 2 . To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor N G -nitro-L-arginine methyl ester blocked the anandamide effects on Q O 2 . We conclude that anandamide inhibits TAL Na transport-related Q O 2 via activation of CB1 receptor and NOS.

Suspended particulate matter trigger the production of reactive oxygen species. However, most of the studies dealing with oxidative damage of airborne particles focus on the effects of individual compounds and not real mixtures. In order to study the enzymatic superoxide production resulting from the exposition to a complex mixture, we derived organic extracts from airborne particles collected daily in an urban area and exposed kidney, liver, and heart mammal tissues. After that, we measured DNA damage employing the comet assay. We observed that in every tissue, NADPH oxidase and xanthine oxidase were involved in O2 (-) production when they were exposed to the organic extracts, as the lucigenin's chemiluminescence decays when enzymes were inhibited. The same trend was observed with the percentage of cells with comets, since DNA damage was higher when they were exposed to same experimental conditions. Our data allow us to hypothesize that these enzymes play an important role in the oxidative stress produced by PAHs and that there is a mechanism involving them in the O2 (-)generation.

Background: Sodium reabsorption is increased in the thick ascending limb (TAL) of Henle in several hypertensive models. In this segment, while transport is increased by ADH via cAMP, sodium reabsorption results from Ang II-induced superoxide (O₂^-) production. Surprisingly, it is unknown whether these mechanisms overlap in hypertension. We hypothesized that Ang II and ADH have accumulative effects on TAL's transport during hypertension. Methods: The effect of ADH/Ang II in TALs from spontaneously hypertensive rats (SHR) on oxygen consumption (QO₂), cAMP and O₂^- was measured. Results: Basal QO₂ was 113.3 ± 14.2 nmol O₂/min/mg protein. Addition of ADH (1 nM) increased QO₂ by 198%. In the presence of ADH, Ang II (1 nM) elicited a QO₂ transient response and then rose to 321.5 ± 28.3 (p = 0.003 vs. ADH). These accumulative effects could be due to nitric oxide synthase (NOS) uncoupling, lower Ang II ability to decrease cAMP or increased O₂^-. We first measured QO₂ using a NOS inhibitor. Pretreatment with L-NAME did not block the observed interaction (p = 0.001 Ang II vs. ADH). Also, Ang II blocked the ADH-stimulated cAMP accumulation in TAL of SHRs. In the presence of ADH, Ang II increased O₂^- production in TALs from SHR by 309% (p = 0.015 vs. basal). The O₂^- scavenger tempol blocked the Ang II effects on QO₂. In the presence of the NADPH oxidase inhibitor apocynin, the accumulative effects of ADH and Ang II were abolished. We conclude that (1) in SHR, Ang II has accumulative effects on ADH-stimulated transport; (2) this effect is mediated by AT1 receptors, and increased O₂^- production.