Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells

Xiaoxia Wang, Jennifer Pluznick, Deann C. Settles, Steven C. Sansom

Research output: Contribution to journalArticle

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 Ca2+ 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 μM SNP, an NO donor, or 100 μM 8-Br-cGMP. Addition of DT-3 (2.5 μM), a specific PKG-1α inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 μM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1α transcript and protein in HMC. Immunocytochemical analysis localized PKG-1α to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca2+ 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-1α-mediated inhibition of the TRPC4-SOC response in HMC.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume293
Issue number6
DOIs
StatePublished - Dec 2007
Externally publishedYes

Fingerprint

Mesangial Cells
Cations
Calcium
Serine
Western Blotting
Phosphorylation
Nitroprusside
Fura-2
vasodilator-stimulated phosphoprotein
Cytoplasm
Immunohistochemistry
Cell Membrane
Polymerase Chain Reaction
Growth

Keywords

  • cGMP
  • Nitric oxide
  • SNP
  • SOC

ASJC Scopus subject areas

  • Physiology

Cite this

Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells. / Wang, Xiaoxia; Pluznick, Jennifer; Settles, Deann C.; Sansom, Steven C.

In: American Journal of Physiology - Renal Physiology, Vol. 293, No. 6, 12.2007.

Research output: Contribution to journalArticle

@article{af8a57c2fafa4d97ace9d703edaa963f,
title = "Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells",
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 Ca2+ 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 μM SNP, an NO donor, or 100 μM 8-Br-cGMP. Addition of DT-3 (2.5 μM), a specific PKG-1α inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 μM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1α transcript and protein in HMC. Immunocytochemical analysis localized PKG-1α to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca2+ 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-1α-mediated inhibition of the TRPC4-SOC response in HMC.",
keywords = "cGMP, Nitric oxide, SNP, SOC",
author = "Xiaoxia Wang and Jennifer Pluznick and Settles, {Deann C.} and Sansom, {Steven C.}",
year = "2007",
month = "12",
doi = "10.1152/ajprenal.00365.2007",
language = "English (US)",
volume = "293",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "6",

}

TY - JOUR

T1 - Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells

AU - Wang, Xiaoxia

AU - Pluznick, Jennifer

AU - Settles, Deann C.

AU - Sansom, Steven C.

PY - 2007/12

Y1 - 2007/12

N2 - 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 Ca2+ 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 μM SNP, an NO donor, or 100 μM 8-Br-cGMP. Addition of DT-3 (2.5 μM), a specific PKG-1α inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 μM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1α transcript and protein in HMC. Immunocytochemical analysis localized PKG-1α to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca2+ 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-1α-mediated inhibition of the TRPC4-SOC response in HMC.

AB - 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 Ca2+ 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 μM SNP, an NO donor, or 100 μM 8-Br-cGMP. Addition of DT-3 (2.5 μM), a specific PKG-1α inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 μM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1α transcript and protein in HMC. Immunocytochemical analysis localized PKG-1α to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca2+ 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-1α-mediated inhibition of the TRPC4-SOC response in HMC.

KW - cGMP

KW - Nitric oxide

KW - SNP

KW - SOC

UR - http://www.scopus.com/inward/record.url?scp=36549046451&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36549046451&partnerID=8YFLogxK

U2 - 10.1152/ajprenal.00365.2007

DO - 10.1152/ajprenal.00365.2007

M3 - Article

VL - 293

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 6

ER -