Abnormalities of Gq-mediated cell signaling in Bartter's and Gitelman's syndome

The constitutive endothelial isoform of nitric oxide synthase (ecNOS) and nitric oxide (NO) production are increased in patients with Bartter syndrome (BS) and Gitelman (GS) syndrome and may reduce vascular tone. Moreover, these patients present an abnormal cell signaling [reduced stimulated intracellular calcium ([Ca(2+)](i)) and inositol-1,4,5,triphosphate ([IP(3)](i)) in neutrophils], suggesting the presence of a generalized reduction of protein kinase C (PKC) and cell reactivity. Since PKC regulates ecNOS gene expression, we evaluated the signal transduction system involving Gq protein, PKC, and ecNOS in circulating nucleated cells from patients with BS/GS. METHODS: Nucleated blood cells from 2 BS and 7 GS and from 10 controls (C) were used. PKC activity was evaluated in neutrophils by radioenzymatic assay; PKC alpha concentration was evaluated in monocytes by Western blot analysis. ecNOS and G alpha q mRNA production was evaluated in monocytes by reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers and quantitated by PCR-based semiquantitative analysis of ecNOS and G alpha q mRNA expression. RESULTS: Cytosol and membrane basal PKC activity were similar in neutrophils from BS/GS and C (70 +/- 3 vs. 80 +/- 2; 37 +/- 3 vs. 46 +/- 2 pmol/min/mg protein, respectively), while fMLP-stimulated membrane PKC activity increased to a lower extent in BS/GS (from 43 +/- 2 to 53 +/- 3 vs. 38 +/- 2 to 66 +/- 3 pmol/min/mg protein, P < 0.05 for the difference). Membrane PKC alpha expression was similar in basal conditions (8.5 +/- 1.5 vs. 12.4 +/- 4.0 densitometric units), but increased after fMLP was reduced in BS/GS (4.5 +/- 1.4 vs. 9.5 +/- 2.1, P < 0.01). In BS/GS, PKC stimulation with PMA dose dependently reduced ecNOS gene expression (from 0.80 +/- 0.05 to 0.78 +/- 0.03 densitometric units; PMA 50 nmol/L, P = NS; to 0.55 +/- 0.07, PMA 100 nmol/L, P < 0.001) to an undetectable expression (PMA 200 nmol/L). Qualitatively similar effects were seen in monocytes from control subjects. Incubation of monocytes from patients and controls with the PKC inhibitor GF109203X increased ecNOS mRNA, with no difference between patients and controls. G alpha q mRNA was reduced in BS/GS versus controls (0.87 +/- 0.013 vs. 0.98 +/- 0.005 densitometric units, P < 0.0004). CONCLUSION: An abnormal G alpha q expression blunts cell signaling and PKC production in BS/GS. A reduced PKC up-regulated NO system may contribute to the vascular hyporeactivity of BS/GS.