Girardi AC, Fukuda LE, Rossoni LV, Malnic G, Rebouças NA. Dipeptidyl peptidase IV inhibition downregulates Na ϩ -H ϩ exchanger NHE3 in rat renal proximal tubule.

Abstract-We previously demonstrated a differential activation of the endothelin-1 (ET-1) pathway in male and female deoxycorticosterone (DOCA)-salt hypertensive rats, with the male rats exhibiting marked alterations in vascular and pressor responses to ET-1 and Suc-[Glu, 9 Ala 11,15 ]-ET-1(8-21) (IRL-1620), an ET B agonist. Mechanisms underlying these gender differences are unclear, and we hypothesized that the ovarian hormones attenuate vascular ET B responses in female DOCA-salt rats. Female Wistar rats were randomized in 3 groups: sham-operated, ovariectomized (OVX), and OVX plus hormone replacement with estradiol (E) or estradiol/progesterone (EP). Two weeks later, rats were uninephrectomized and further randomized in DOCA-salt (subcutaneous injections of desoxycorticosterone and drinking water containing NaCl/KCl) and control normotensive (subcutaneous injections of vehicle and tap water). Blood pressure was evaluated both by direct and standard tail-cuff methods. Responses to IRL-1620 were evaluated in vivo/in situ in the mesenteric microcirculation. mRNA expression of ET-1 and ET A/B receptors was evaluated in mesenteric arteries by reverse transcription-polymerase chain reaction and expressed relative to GAPDH. OVX-DOCA rats developed a more severe form of hypertension than did DOCA rats. Treatment with E or EP restored blood pressure to levels observed in DOCA rats. In the mesentery, IRL-1620 induced vasodilatation in control rats, a mild vasoconstriction in DOCA rats, and marked vasoconstriction in OVX-DOCA rats. Both E and EP decreased IRL-1620 -induced vasoconstriction in the DOCA group. In the normotensive group, OVX did not change blood pressure or IRL-1620 -induced vasodilation. Removal of the ovaries increased ET-1 mRNA in arteries from DOCA and control rats, although treatment with E or EP reversed these changes. Vascular ET B receptor mRNA levels were greatly enhanced in OVX-DOCA but not OVX-control rats. Hormone replacement with E or EP restored ET B receptor expression in the DOCA group. A greater blood pressure-lowering effect of bosentan (ET A /ET B blocker) was observed in OVX-DOCA rats. The observation that OVX worsens hypertension as well as the altered ET B receptor-mediated responses and the effects of bosentan in female DOCA rats supports our suggestion that the ovarian hormones modulate ET-1/ET B receptor vascular responses/expression in DOCA-salt hypertension. Key Words: deoxycorticosterone Ⅲ vasoconstriction Ⅲ endothelin Ⅲ estrogen Ⅲ receptors, endothelin I n deoxycorticosterone (DOCA)-salt hypertension and other experimental models of hypertension, male rats develop an earlier and more severe form of hypertension than do female rats. 1-2 We have recently suggested that differential activation of endothelin-1 (ET-1) pathways, expressed by a functional upregulation of ET B receptors, may play a role in the higher blood pressure (BP) levels observed in male DOCA-salt hypertensive rats. [3][4] We observed that mesenteric arterioles from male but not female DOCA-salt rats display incre...

Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+ salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide and stimulated by GTP and diazoxide. These results suggest the existence of ATP-sensitive K+ channels in these mitochondria, similar to those previously described in heart, liver, and brain. Renal mitochondrial ATP-sensitive K+ uptake rates are ∼140 nmol·min–1·mg protein–1. This K+ transport results in a slight increase in respiration and decrease in the inner membrane potential. In addition, the activation of ATP-inhibited K+ uptake using diazoxide leads to a decrease of ATP hydrolysis through the reverse activity of the F0F1 ATP synthase when respiration is inhibited. In conclusion, we characterize an ATP-sensitive K+ transport pathway in kidney mitochondria that affects volume, respiration, and membrane potential and may have a role in the prevention of mitochondrial ATP hydrolysis.

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (JH) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 microM, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of JH by the NHE3 inhibitor S3226 (1 microM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 microM EIPA abolished JH entirely, but NHE4 was not inhibited at 10 microM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of JH. Cl--dependent NHE was not detected: in 0 Cl- solutions JH was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside -83/-62 and -134/-113 bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the -134/-113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation.