Nucleotides regulate NaCl transport in mIMCD-K2 cells  via P2X and P2Y purinergic receptors

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

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Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00297.2019 ◽

2020 ◽

Vol 318 (4) ◽

pp. F956-F970 ◽

Cited By ~ 4

Author(s):

Wei-Ling Wang ◽

Shih-Han Su ◽

Kit Yee Wong ◽

Chan-Wei Yang ◽

Chin-Fu Liu ◽

...

Keyword(s):

Plasma Membrane ◽

Apical Membrane ◽

Collecting Duct ◽

Water Channel ◽

Small Gtpase ◽

Apical Plasma Membrane ◽

Water Reabsorption ◽

Recycling Endosomes ◽

Early Endosomes ◽

Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

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Antidiuretic hormone moves membranes

AJP Renal Physiology ◽

10.1152/ajprenal.1988.255.3.f375 ◽

1988 ◽

Vol 255 (3) ◽

pp. F375-F382 ◽

Cited By ~ 3

Author(s):

J. S. Handler

Keyword(s):

Plasma Membrane ◽

Antidiuretic Hormone ◽

Water Permeability ◽

Apical Membrane ◽

Collecting Duct ◽

Toad Bladder ◽

Cell Swelling ◽

Apical Plasma Membrane ◽

Principal Cells ◽

Particle Aggregates

This review focuses on events at the apical plasma membrane of toad urinary bladder and mammalian collecting duct as their permeability to water changes in response to antidiuretic hormone (ADH) and to its withdrawal. The major marker of the permeability change is observed in freeze-fracture electron microscopy of the apical plasma membrane and consists of a dramatic increase in membrane particle aggregates and, in toad bladder but not in collecting duct, in fused vesicles (aggrephores) that contain particle aggregates in their limiting membranes. Withdrawal of ADH is accompanied by endocytosis at the apical membrane, reflecting retrieval of water-permeable, particle aggregate-containing membrane. Covalent labeling of the external surface of the apical membrane of toad bladder identifies specific proteins that are present in the apical membrane only during the response to ADH. Proteins of the same molecular weights are also present in the retrieved membrane when ADH is withdrawn. Several controversial areas are considered, including the extent of cell swelling as water flows across the epithelium from dilute apical solution to isotonic basal solution, whether only principal cells or principal cells and intercalated cells participate in the water permeability response of the collecting duct, the role of the cytoskeleton in the water permeability response, and the proposed second water permeability barrier that is affected by ADH, but not by adenosine 3',5'-cyclic monophosphate.

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CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts

AJP Renal Physiology ◽

10.1152/ajprenal.2001.281.3.f434 ◽

2001 ◽

Vol 281 (3) ◽

pp. F434-F442 ◽

Cited By ~ 19

Author(s):

Marcelle Bens ◽

Jean-Paul Duong Van Huyen ◽

Françoise Cluzeaud ◽

Jacques Teulon ◽

Alain Vandewalle

Keyword(s):

Collecting Duct ◽

Short Circuit ◽

Primary Cultures ◽

Short Circuit Current ◽

Similar Degree ◽

Transmembrane Conductance Regulator ◽

Cortical Collecting Duct ◽

Transmembrane Conductance ◽

Nacl Transport ◽

Cystic Fibrosis Transmembrane

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current ( I sc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I sc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I sc in BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl− in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl− flux in CFTR(−/−) CCDs but not the basal-to-apical 36Cl− flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl− in mouse CCD.

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Expression of the unconventional myosin Myo1c alters sodium transport in M1 collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.00569.2003 ◽

2005 ◽

Vol 289 (1) ◽

pp. C120-C129 ◽

Cited By ~ 18

Author(s):

Mark C. Wagner ◽

Bonnie L. Blazer-Yost ◽

Judy Boyd-White ◽

Anjaiah Srirangam ◽

Janice Pennington ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Collecting Duct ◽

Short Circuit ◽

Direct Interaction ◽

Short Circuit Current ◽

Duct Cell ◽

Unconventional Myosin ◽

Myosin I ◽

Green Fluorescent

Epithelial cells rely on proper targeting of cellular components to perform their physiological function. This dynamic process utilizes the cytoskeleton and involves movement of vesicles to and from the plasma membrane, thus traversing the actin cortical cytoskeleton. Studies support both direct interaction of actin with channels and an indirect mechanism whereby actin may serve as a track in the final delivery of the channel to the plasma membrane. Actin-dependent processes are often mediated via a member of the myosin family of proteins. Myosin I family members have been implicated in multiple cellular events occurring at the plasma membrane. In these studies, we investigated the function of the unconventional myosin I Myo1c in the M1 mouse collecting duct cell line. Myo1c was observed to be concentrated at or near the plasma membrane, often in discrete membrane domains. To address the possible role of Myo1c in channel regulation, we expressed a truncated Myo1c, lacking ATP and actin domains, in M1 cells and compared electrophysiological responses to control M1 cells, M1 cells expressing the empty vector, and M1 cells expressing the full-length Myo1c construct. Interestingly, cells expressing the Myo1c constructs had modulated antidiuretic hormone (ADH)-stimulated short-circuit current and showed little inhibition of short-circuit current with amiloride addition. Evaluation of enhanced green fluorescent protein-Myo1c constructs supports the importance of the IQ region in targeting the Myo1c to its respective cellular domain. These data are consistent with Myo1c participating in the regulation of the Na+ channel after ADH stimulation.

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Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

AJP Renal Physiology ◽

10.1152/ajprenal.00253.2011 ◽

2012 ◽

Vol 302 (7) ◽

pp. F801-F808 ◽

Cited By ~ 23

Author(s):

Takamitsu Saigusa ◽

Ryan Reichert ◽

Jennifer Guare ◽

Brian J. Siroky ◽

Monika Gooz ◽

...

Keyword(s):

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Renal Cysts ◽

Short Circuit Current ◽

Polycystic Kidney ◽

Collecting Duct Cells ◽

And Function ◽

Apical Membranes ◽

Apical Localization

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (−), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/ Ift88 gene). Interestingly, only cilia (−) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (−) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (−) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current ( Isc) in cilia (−) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of Isc in cilia (−) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

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Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

The Journal of General Physiology ◽

10.1085/jgp.200409124 ◽

2004 ◽

Vol 125 (1) ◽

pp. 81-101 ◽

Cited By ~ 130

Author(s):

Michael B. Butterworth ◽

Robert S. Edinger ◽

John P. Johnson ◽

Raymond A. Frizzell

Keyword(s):

Hormonal Regulation ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Membrane Capacitance ◽

Apical Surface ◽

Cortical Collecting Duct ◽

Ussing Chambers ◽

Camp Stimulation

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

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Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

AJP Renal Physiology ◽

10.1152/ajprenal.00197.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. F1308-F1313 ◽

Cited By ~ 64

Author(s):

Mitsi A. Blount ◽

Janet D. Klein ◽

Christopher F. Martin ◽

Dmitry Tchapyjnikov ◽

Jeff M. Sands

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Apical Membrane ◽

Collecting Duct ◽

Rat Kidney ◽

Basolateral Membrane ◽

Apical Plasma Membrane ◽

Membrane Staining ◽

Protein Protein Interaction ◽

Medullary Collecting Duct

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

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Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v4111855 ◽

1994 ◽

Vol 4 (11) ◽

pp. 1855-1860

Author(s):

B A Stoos ◽

O A Carretero ◽

J L Garvin

Keyword(s):

Nitric Oxide ◽

Maximum Rate ◽

Apical Membrane ◽

Collecting Duct ◽

Short Circuit ◽

Short Circuit Current ◽

Sodium Absorption ◽

Cortical Collecting Duct ◽

Duct Cells ◽

Collecting Duct Cells

Previously, it has been shown that the addition of bradykinin (Bk) to M-1 cortical collecting duct cells in the presence of endothelial cells decreased short-circuit current (Isc), a measure of net active transport. This effect is presumably due to the release of endothelium-derived nitric oxide (EDNO), because the decrease in Isc could be blocked with Nw-nitro-L-arginine. To show that the inhibition of Isc was due to EDNO rather than prostaglandins, the ability of a cyclooxygenase inhibitor to block the inhibition was examined. When Bk was added to cocultures in the presence of meclofenamate (10(-5) M), Isc decreased from 62 +/- 12 to 44.5 +/- 7 muA/cm2, not significantly different from that in the absence of meclofenamate. To determine if the effect was due to an alteration of sodium absorption, Bk (10(-9) M) was added to cocultures, resulting in a decrease in Na flux from 28 +/- 3.1 to 20 +/- 2.2 nEq/min (P < 0.05), with Isc decreasing from 25 +/- 2.4 to 20 +/- 3.6 nEq/min (P < 0.05). To examine if the inhibition was due to blockade at the apical membrane sodium channel or the basolateral Na+/K+ ATPase, the cation-selective ionophore nystatin was used. Nystatin reversed the effect of EDNO on Isc. The effects of EDNO on Na+/K+ ATPase were also measured directly. Under maximum rate conditions, the Na+/K+ ATPase activity of control and Bk-treated cocultures was 5.2 +/- 0.3 and 6.8 +/- 1.0 nmol/min per square centimeter, respectively (not significantly different).(ABSTRACT TRUNCATED AT 250 WORDS)

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Hydrogen peroxide stimulates chloride secretion in primary inner medullary collecting duct cells via mPGES-1-derived PGE2

AJP Renal Physiology ◽

10.1152/ajprenal.00132.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. F1571-F1576 ◽

Cited By ~ 20

Author(s):

Sunhapas Soodvilai ◽

Zhanjun Jia ◽

Tianxin Yang

Keyword(s):

Collecting Duct ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Chloride Secretion ◽

Dependent Manner ◽

Semipermeable Membranes ◽

Nacl Transport ◽

Inner Medullary Collecting Duct ◽

Medullary Collecting Duct

We investigated the role and mechanism of H2O2 in regulation of NaCl transport in primary inner medullary collecting duct (IMCD) cells. IMCD cells were isolated from wild-type mice and grown onto semipermeable membranes, and short-circuit current ( Isc) was determined by Ussing chamber. Exposure of IMCD cells to H2O2 at a range of 100–300 μM caused a rapid increase in Isc in a time- and dose-dependent manner. This increase was almost abolished by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel inhibitors diphenylamine-2-carboxylic acid (DPC) and CFTRinhibitor-172. In contrast, the magnitude of stimulation was unaffected by the epithelial Na+ channel (ENaC) inhibitor amiloride. The H2O2-induced Cl− secretion was significantly inhibited by indomethacin, as well as by microsomal PGE synthase-1 (mPGES-1) deficiency. Like H2O2, PGE2 treatment induced a twofold increase in Isc that was reduced by the protein kinase A (PKA) inhibitors H-89 and KT5720. These data suggest that H2O2 stimulates CFTR Cl− channel-mediated Cl− secretion through cyclooxygenase- and mPGES-1-dependent release of PGE2 and subsequent activation of PKA.

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Cyclic AMP-induced mucin exocytosis is independent of Cl− movements in human colonic epithelial cells (HT29-Cl.16E)

Biochemical Journal ◽

10.1042/bj3040675 ◽

1994 ◽

Vol 304 (3) ◽

pp. 675-678 ◽

Cited By ~ 18

Author(s):

A Jarry ◽

D Merlin ◽

U Hopfer ◽

C L Laboisse

Keyword(s):

Plasma Membrane ◽

Cyclic Amp ◽

Cell Line ◽

Short Circuit ◽

Short Circuit Current ◽

Apical Plasma Membrane ◽

Colonic Epithelial Cells ◽

Mucin Release ◽

Fold Stimulation ◽

Stimulation Of

The human colonic epithelial goblet cell line HT29-Cl.16E was used to test whether stimulated Cl- transport is involved in the mucin exocytotic response to an increase in intracellular cyclic AMP by measuring in parallel the short-circuit current (Isc) and mucin exocytosis. Addition of 50 microM forskolin to HT29-Cl.16E cells resulted in a 2-fold stimulation of mucin release and an increase in Isc by 20 microA/cm2. To evaluate the requirement for cosecretion of Cl-, the Cl- flux was altered by three different manipulations: (1) Cl- in the medium was replaced by the poorly transported anion gluconate; (2) basolateral Cl- influx through the Na(+)-K(+)-2Cl- cotransporter was inhibited by bumetanide; and (3) an inward Cl- flux through the apical plasma membrane was generated by reversing the Cl- gradient. These manipulations did not change the forskolin-stimulated mucin release and thereby provide evidence that Cl- movements are not required for fusion of mucin granules with the plasma membrane.

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