Contractility of mesangial cells (MC) is tightly controlled by [Ca(2+)](i). Ca(2+) influx across the plasma membrane constitutes a major component of mesangial responses to vasoconstrictors. Canonical transient receptor potential 1 (TRPC1) is a Ca(2+)-permeable cation channel in a variety of cell types. This study was performed to investigate whether TRPC1 takes part in vasoconstrictor-induced mesangial contraction by mediating Ca(2+) entry. It was found that angiotensin II (AngII) evoked remarkable contraction of the cultured MC. Downregulation of TRPC1 using RNA interference significantly attenuated the contractile response. Infusion of AngII or endothelin-1 in rats caused a decrease in GFR. The GFR decline was significantly reduced by infusion of TRPC1 antibody that targets an extracellular domain in the pore region of TRPC1 channel. However, the treatment of TRPC1 antibody did not affect the AngII-induced vasopressing effect. Electrophysiologic experiments revealed that functional or biologic inhibition of TRPC1 significantly depressed AngII-induced channel activation. Fura-2 fluorescence-indicated that Ca(2+) entry in response to AngII stimulation was also dramatically inhibited by TRPC1 antibody and TRPC1-specific RNA interference. These results suggest that TRPC1 plays an important role in controlling contractile function of MC. Mediation of Ca(2+) entry might be the underlying mechanism for the TRPC1-associated MC contraction.
[Show abstract][Hide abstract] ABSTRACT: The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca(2+) signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca(2+) responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca(2+) influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca(2+) entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca(2+) signaling of MCs seen in diabetes.
American journal of physiology. Renal physiology 11/2007; 293(4):F1381-90. DOI:10.1152/ajprenal.00185.2007 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular Ca(2+) concentration [Ca(2+)](i) to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 microM SNP, an NO donor, or 100 microM 8-Br-cGMP. Addition of DT-3 (2.5 microM), a specific PKG-1alpha inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 microM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1alpha transcript and protein in HMC. Immunocytochemical analysis localized PKG-1alpha to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca(2+) entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG-1alpha-mediated inhibition of the TRPC4-SOC response in HMC.
American journal of physiology. Renal physiology 01/2008; 293(6):F1768-76. DOI:10.1152/ajprenal.00365.2007 · 3.25 Impact Factor
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