cAMP increases surface expression of NKCC2 in rat thick ascending limbs: role of VAMP

2006 ◽  
Vol 290 (3) ◽  
pp. F608-F616 ◽  
Author(s):  
Pablo A. Ortiz
Keyword(s):  
Confocal Microscopy ◽  
Membrane Protein ◽  
Tetanus Toxin ◽  
Apical Membrane ◽  
Surface Expression ◽  
Thick Ascending Limb ◽  
Dibutyryl Camp ◽  
Nacl Absorption ◽  
Stimulation Of

NaCl absorption by the thick ascending limb of Henle’s loop (TAL) is mediated by the apical Na-K-2Cl cotransporter NKCC2. cAMP increases NaCl absorption in the TAL by stimulating NKCC2. In oocytes, cAMP increases NKCC2 activity by regulating its trafficking. However, the mechanism by which cAMP stimulates NKCC2 in TALs is not clear. We hypothesized that cAMP increases surface expression of NKCC2 and NaCl absorption in TALs and that vesicle-associated membrane protein (VAMP) is involved in this mechanism. We used surface biotinylation of rat medullary TALs (mTAL) to examine surface and total NKCC2 levels. When mTAL suspensions were treated with dibutyryl cAMP (db-cAMP) or forskolin plus IBMX for 20 min, surface NKCC2 expression increased by 126 ± 23 and 92 ± 17% above basal, respectively ( P < 0.03). No changes in total NKCC2 expression were observed, suggesting that cAMP increased translocation of NKCC2. We studied the role of VAMP in NKCC2 translocation and found that incubating mTALs with tetanus toxin (30 nM), which inhibits vesicle trafficking by inactivating VAMP-2 and -3, completely blocked the stimulatory effect of db-cAMP on surface NKCC2 expression (tetanus toxin = 100% vs. tetanus toxin + db-cAMP = 102 ± 21% of control; not significant). We studied VAMP-2 and -3 expression and localization in isolated perfused TALs by confocal microscopy and found that both of them were located in the subapical space of the TAL. Finally, in isolated perfused mTALs, db-cAMP increased net Cl absorption by 95.0 ± 34.8% ( P < 0.03), and pretreatment of TALs with tetanus toxin blocked the stimulation of Cl absorption (from 110.9 ± 15.9 to 109.7 ± 15.6 pmol·min−1·mm−1; not significant). We concluded that cAMP increases NKCC2 surface expression by a mechanism involving VAMP and that NKCC2 trafficking to the apical membrane is involved in the stimulation of TAL NaCl absorption by cAMP.

scholarly journals Constitutive endocytosis and recycling of NKCC2 in rat thick ascending limbs

2010 ◽  
Vol 299 (5) ◽  
pp. F1193-F1202 ◽  
Author(s):  
Gustavo R. Ares ◽  
Pablo A. Ortiz
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Acute Treatment ◽  
Apical Membrane ◽  
Surface Expression ◽  
Membrane Cholesterol ◽  
Thick Ascending Limb ◽  
Apical Surface ◽  
Total Pool ◽  
Stimulation Of

The Na-K-2Cl cotransporter (NKCC2) mediates NaCl absorption by the thick ascending limb of Henle's loop (THAL). Exocytosis and endocytosis regulates surface expression of most transporters. However, little is known about the mechanism of NKCC2 trafficking in the absence of stimulating hormones and whether this mechanism contributes to regulation of steady-state surface expression of apical NKCC2 in the THAL. We tested whether NKCC2 undergoes constitutive endocytosis that regulates steady-state surface NKCC2 and NaCl reabsorption in THALs. We measured steady-state surface NKCC2 levels and the rate of NKCC2 endocytosis by surface biotinylation and Western blot and confocal microscopy of isolated perfused rat THALs. We observed constitutive NKCC2 endocytosis over 30 min that averaged 21.5 ± 2.7% of the surface pool. We then tested whether methyl-β-cyclodextrin (MβCD), a compound that inhibits endocytosis by chelating membrane cholesterol, blocked NKCC2 endocytic retrieval. We found that 30-min treatment with MβCD (5 mM) blocked NKCC2 endocytosis by 81% ( P < 0.01). Blockade of endocytosis by MβCD induced accumulation of NKCC2 at the apical membrane as demonstrated by a 60 ± 16% ( P < 0.05) increase in steady-state surface expression and enhanced apical surface NKCC2 immunostaining in isolated, perfused THALs. Acute treatment with MβCD did not change the total pool of NKCC2. MβCD did not affect NKCC2 trafficking when it was complexed with cholesterol before treatment. Inhibition endocytosis with MβCD enhanced NKCC2-dependent NaCl entry by 57 ± 16% ( P < 0.05). Finally, we observed that a fraction of retrieved NKCC2 recycles back to the plasma membrane (36 ± 7%) over 30 min. We concluded that constitutive NKCC2 trafficking maintains steady-state surface NKCC2 and regulates NaCl reabsorption in THALs. These are the first data showing an increase in apical membrane NKCC2 in THALs by altering the rates of constitutive NKCC2 trafficking, rather than by stimulation of hormone-dependent signaling.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

scholarly journals Renal Na+-K+-Cl− cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent

2008 ◽  
Vol 295 (3) ◽  
pp. F789-F802 ◽  
Author(s):  
Pia Welker ◽  
Alexandra Böhlick ◽  
Kerim Mutig ◽  
Michele Salanova ◽  
Thomas Kahl ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
Cholesterol Depletion ◽  
Xenopus Laevis Oocytes ◽  
Thick Ascending Limb ◽  
Short Term ◽  
Nacl Transport ◽  
Triton X

Apical bumetanide-sensitive Na+-K+-2Cl− cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40–70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na+-Cl− cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-β-cyclodextrin (MβCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MβCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

Stimulation of Na+/K+-ATPase activity by polyamines in the rat renal medullary cells of the thick ascending limb of Henle's loop

1990 ◽  
Vol 127 (3) ◽  
pp. 377-382 ◽  
Author(s):  
J. A. Charlton ◽  
P. H. Baylis
Keyword(s):  
Atpase Activity ◽  
Arginine Vasopressin ◽  
Thick Ascending Limb ◽  
Stimulus Response ◽  
Spermine Synthase ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Specific Inhibitors ◽  
Stimulation Of

ABSTRACT Previous studies have indicated that ornithine decarboxylase (ODC) may be involved in the stimulation of Na+/K+-ATPase activity by arginine vasopressin (AVP) in the rat renal medullary thick ascending limb of Henle's loop. The present study was aimed at establishing the role of the polyamines, the conversion products of ODC activity, in the stimulation of Na+/K+-ATPase by AVP. Using cytochemical methods, we have demonstrated an increase in Na+/K+-ATPase activity after stimulation with putrescine, spermidine and spermine (each 1 mmol/l) for 2·5,2 and 1·5 min respectively. The specific inhibitors of spermidine and spermine synthase, bis-cyclohexylammonium sulphate and N-alkylated-1,3-diaminopropane respectively, inhibited the stimulation of Na+/K+-ATPase by AVP, this inhibition being reversed by spermine. These findings suggest that polyamines are involved in the stimulus-response coupling of the hormone-mediated response. Journal of Endocrinology (1990) 127, 377–382

scholarly journals Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct

2010 ◽  
Vol 298 (4) ◽  
pp. F988-F996 ◽  
Author(s):  
Monu Goel ◽  
Cheng-Di Zuo ◽  
William P. Schilling
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Surface Membrane ◽  
Transient Receptor Potential Channels ◽  
Signaling Cascade ◽  
Dibutyryl Camp ◽  
Principal Cells ◽  
V2 Receptor ◽  
Stimulation Of

Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys1, d-Arg8]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl1, O-Et-d-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 ± 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

scholarly journals Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption.

1986 ◽  
Vol 87 (4) ◽  
pp. 567-590 ◽  
Author(s):  
S C Hebert ◽  
T E Andreoli
Keyword(s):  
Apical Membrane ◽  
Electrical Conductance ◽  
Transport Processes ◽  
Diffusion Resistance ◽  
Osmotic Gradient ◽  
Thick Ascending Limb ◽  
Nacl Absorption ◽  
Medullary Thick Ascending Limb ◽  
Paired Measurement

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.

Effects of apical membrane Cl(-)-formate exchange on cell volume in rabbit proximal tubule

1990 ◽  
Vol 258 (3) ◽  
pp. F530-F536 ◽  
Author(s):  
L. Schild ◽  
P. S. Aronson ◽  
G. Giebisch
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Volume Changes ◽  
Proximal Tubule Cells ◽  
Volume Increase ◽  
Tubule Lumen ◽  
Stimulation Of

We used real-time recordings of cell volume changes to test for the role of the Cl(-)-formate exchanger in mediating NaCl entry across the apical membrane of rabbit proximal tubule cells. In the absence of extracellular Cl-, 0.5 and 5 mM formate in the tubule lumen induced an increase in cell volume of 1 and 9%, respectively. Formate-induced cell swelling was reduced by alkalinizing the tubule lumen or by addition of luminal amiloride (2 mM), indicating that the increase in cell volume results from the intracellular accumulation of Na-formate via nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. The cell volume increase induced by 0.5 mM formate was potentiated (from 1 to 4%) by Cl-, as expected for a formate-mediated stimulation of NaCl uptake via parallel Cl(-)-formate exchange and Na(+)-H+ exchange across the apical membrane. By contrast, the cell volume increase induced by 5 mM formate was attenuated (from 9 to 4%) by Cl-. The attenuating effect of Cl- on formate-induced cell swelling required the operation of the apical membrane Cl(-)-formate exchanger. The effect of 1:1 Cl(-)-formate exchange to attenuate formate-induced cell swelling can be explained if the cell possesses a volume-activated anion exit pathway, most likely at the basolateral cell membrane, that is capable of mediating the efflux of Cl- but not formate from the cell.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470 ◽  
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

Abstract This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

scholarly journals Regulation of ROMK (Kir1.1) channels: new mechanisms and aspects

2006 ◽  
Vol 290 (1) ◽  
pp. F14-F19 ◽  
Author(s):  
Wen-Hui Wang
Keyword(s):  
Protein Tyrosine Kinase ◽  
Surface Expression ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Superoxide Anions ◽  
Channel Activity ◽  
Recent Developments ◽  
Low K ◽  
Small Conductance ◽  
Stimulation Of

This brief review attempts to provide an overview regarding recent developments in the regulation of ROMK channels. Studies performed in ROMK null mice suggest that ROMK cannot only form hometetramers such as the small-conductance (30-pS) K channels but also construct heterotetramers such as the 70-pS K channel in the thick ascending limb (TAL). The expression of ROMK channels in the plasma membrane is regulated by protein tyrosine kinase (PTK), serum and glucorticoid-induced kinase (SGK), and with-no-lysine-kinase 4. PTK is involved in mediating the effect of low K intake on ROMK channel activity. Increases in superoxide anions induced by low dietary K intake are responsible for the stimulation of PTK expression and tyrosine phosphorylation of ROMK channels. Finally, a recent study indicated that ROMK channels can be monoubiquitinated and monoubiquitination regulates the surface expression of ROMK channels.

scholarly journals Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

2000 ◽  
Vol 11 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Eric Féraille ◽  
Pascal Béguin ◽  
Maria-Luisa Carranza ◽  
Sandrine Gonin ◽  
Martine Rousselot ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Phorbol Ester ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Wild Type ◽  
Α1 Subunit ◽  
Stimulation Of

The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

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