Volume 60, Issue 3, Pages (September 2001)

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Volume 60, Issue 3, Pages 882-889 (September 2001) Abnormalities of Gq-mediated cell signaling in Bartter and Gitelman syndromes1  Lorenzo Calò, Giulio Ceolotto, Monia Milani, Elisa Pagnin, Lambertus P. Van Den Heuvel, Michelangelo Sartori, Paul A. Davis, Rodolfo Costa, Andrea Semplicini  Kidney International  Volume 60, Issue 3, Pages 882-889 (September 2001) DOI: 10.1046/j.1523-1755.2001.060003882.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Basal and fMLP (3 × 10-7 mol/L)-stimulated membrane protein kinase C (PKC) activity in neutrophils from patients with Bartter syndrome (BS) and Gitelman syndrome (GS), and from healthy controls. *P < 0.05; **P < 0.01. BS/GS, Bartter and Gitelman patients. Kidney International 2001 60, 882-889DOI: (10.1046/j.1523-1755.2001.060003882.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Representative immunoblot of the PKCα isoform in the membrane fraction of monocytes from one patient (P) and one control (C) subject in the presence and in the absence of fMLP (3 × 10-7 mol/L; A). (B and C) The densitometric analysis of PKCα at baseline and after fMLP in controls and BS/GS patients is shown. There was no significant difference between the two groups at baseline, but the increase after fMPL was blunted in BS/GS patients in comparison to controls (P < 0.01, right lower panel). Abbreviations are: fMLP, formylmethionyl-leucyl-phenylalanina. Other abbreviations are in the legend to Figure 1. Kidney International 2001 60, 882-889DOI: (10.1046/j.1523-1755.2001.060003882.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Representative experiments of an endothelial isoform of nitric oxide synthase (ecNOS) gene expression in monocytes at baseline and after exposure to the PKC stimulator PMA at the indicated concentrations. (A) PMA dose-dependently reduces ecNOS gene expression in a patient with BS. (B) Comparison of one control subject and one BS patient. At baseline, ecNOS gene expression is higher in the patient than in the control subject, and PMA reduces ecNOS gene expression to the same extent in the patient and in the control subject. Abbreviation is: Mwm, DNA molecular weight marker IX (ФX174 Hae III). The other abbreviations are as in Figure 1. Kidney International 2001 60, 882-889DOI: (10.1046/j.1523-1755.2001.060003882.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 ecNOS gene expression at baseline and after exposure to PKC inhibitor GF109203X (1.2 μmol/L) in monocytes from one control subject and from one GS patient. The PKC inhibitor GF109203X increases ecNOS gene expression in the patient and in the control subject to the same magnitude. GFX is GF109203X; the other abbreviations are the same as in Figure 2. Kidney International 2001 60, 882-889DOI: (10.1046/j.1523-1755.2001.060003882.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 Representative assay for Gαq RT-PCR amplified oligonucleotides from patients with BS (lane 5) and GS (lanes 6 and 7) and from controls (lanes 2, 3, and 4). The DNA molecular weight marker IX (ФX174 HaeIII) is reported in lane 1. It can be seen that the level of Gαq gene expression is decreased in the patients in comparison with the control subjects. Kidney International 2001 60, 882-889DOI: (10.1046/j.1523-1755.2001.060003882.x) Copyright © 2001 International Society of Nephrology Terms and Conditions