Role of F-actin in the activation of Na+-K+-Cl? cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells

Wu, Mai Szu; Bens, Marcelle; Cluzeaud, Françoise; Vandewalle, Alain

doi:10.1007/bf00233439

Cited by 44 publications

(20 citation statements)

References 41 publications

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“…Both PKA and PKG inhibit RhoA by phosphorylation at Ser-188 (18,49), a process that has been proposed for the regulation of several membrane transporters (55,62,63,69). Thus cAMP-or cGMP-dependent inhibition of RhoA and ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation and subsequent increase in V-ATPase-dependent proton secretion in clear cells.…”

Section: Discussionmentioning

confidence: 97%

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

Shum

Silva

Belleannée

et al. 2011

American Journal of Physiology-Cell Physiology

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ϩ -ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10 -30 M) and HA1077 (30 M) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 g/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 M) or HA1077 (30 M) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.actin; proton pump; luminal acidification; proton ATPase; epididymis SEVERAL SPECIALIZED biological tissues, such as kidney tubules (8 -10, 66), the male reproductive tract (1,6,32,33,43,57), the inner ear (27, 59) salivary glands (51, 65), and osteoclasts (30, 52) establish an acidic extracellular local environment. These tissues all have in common a high expression of the proton pumping V-ATPase, which is localized in the plasma membrane of specialized acidifying cells. In the male reproductive tract, luminal acidification is a critical process for sperm maturation and for their storage in a quiescent state. We have shown that the V-ATPase localizes in the apical membrane of clear cells in the epididymis (2,3,6,42,43,57) and that these cells are important players in luminal acidification, especially in the distal region of the epididymis, where they are present in high numbers. Impairment of the acidification process in the epididymis, either by genetic mutations or environmental factors, such as the heavy metal cadmium, reduces male fertility (4,5,11,22,64).We have shown previously that luminal bicarbonate via bicarbonate-regulated adenylyl cyclase (sAC)-dependent intracellular cAMP production (42), or luminal angiotensin II via the nitric oxide (NO)/soluble guanylate cyclase (sGC)-dependent pathway (57), induce the apical membrane ...

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Section: Discussionmentioning

confidence: 97%

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

Shum

Silva

Belleannée

et al. 2011

American Journal of Physiology-Cell Physiology

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“…Further experiments should determine whether the interaction of NKCC2 with aldolase B indeed involves SNX9 or other intermediary regulatory proteins. In this regard, the affinity of aldolase for actin (24,76) and the involvement of actin cytoskeleton in the regulation of Na-K-Cl co-transport in TAL cells (58) opens the possibility for a key role of actin in aldolase effect on NKCC2 surface expression.…”

Section: Discussionmentioning

confidence: 99%

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

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Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

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“…None of the subunits of the V-ATPase have been shown to be phosphorylated by protein kinase A (49,50), which indicates that an indirect mechanism might be involved in the cAMPinduced apical accumulation of the pump. Interestingly, modulation of the actin cytoskeleton by cAMP has been proposed for the regulation of several membrane transporters (81)(82)(83)(84)(85)(86), and phosphorylation of RhoA by protein kinase A, leading to actin disassembly, was invoked in their regulation. In this study, depolymerization of the actin cytoskeleton by cAMP, leading to inhibition of V-ATPase endocytosis, would be compatible with the apical accumulation of V-ATPase induced by cAMP.…”

Section: Discussionmentioning

confidence: 99%

Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling

Beaulieu

Silva

Pastor-Soler

et al. 2005

Journal of Biological Chemistry

124

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The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar

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Role of F-actin in the activation of Na+-K+-Cl? cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells

Cited by 44 publications

References 41 publications

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

NKCC2 Surface Expression in Mammalian Cells

Modulation of the Actin Cytoskeleton via Gelsolin Regulates Vacuolar H+-ATPase Recycling

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