Electrogenic Sodium Transport Research Articles

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

MCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells.

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCDcl1 parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCDcl1 cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCDcl1 cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCDcl1 cells being precursor cells.

Additive Effect of Chronic Hypoxia and Aldosterone on Electrogenic Sodium Transport in Rat Rectal Epithelium

Background Both chronic hypoxia and mineralocorticoids independently stimulate electrogenic sodium absorption in rat distal colon It is hitherto unknown whether there is any interaction between these two stimuli The aim of this work was to assess the interaction of these stimuli on rat rectal epithelium in vitro Methods Isolated rectal mucosa samples were obtained from adult rats submitted to hypobaric hypoxia for days or breathing at normal pressure Tissues were mounted in Ussing chambers which allow simultaneous measurement of short circuit current ISC and oxygen consumption rate QO Both variables were measured at baseline after h incubation with aldosterone nM and after addition of the epithelial sodium channel amiloride Statistical analysis was performed with Student rsquo s t tests and linear regression Results At baseline samples from hypoxic rats had higher ISC p but its QO did not differ from the control group p Incubation with aldosterone caused a progressive increase of ISC in both groups but the difference in ISC between them was maintained throughout the incubation period At the end of the incubation period both groups showed significant increases in ISC and QO Amiloride reduced ISC and QO to the same extent in both groups QO and ISC were linearly related to each other both at the baseline and at the end of the incubation period Conclusion While the rectal epithelium of chronically hypoxic rats had a higher ISC its response of incubation with aldosterone at nanomolar concentration was preserved and followed the same time course than that of the rectal epithelium of non hypoxic rats Chronic hypoxia and aldosterone showed an additivity of effects in the rectal epithelium The increase in ISC and QO caused by hypoxia and aldosterone was abolished by amiloride suggesting an ENaC dependent response to both stimuli

The effects of amiloride and age on oxygen consumption coupled to electrogenic sodium transport in the human sigmoid colon.

Background/Aim:Aerobic metabolism is necessary for ion transport in many transporting epithelia, including the human colonic epithelium. We assessed the effects of the epithelial sodium channel blocker, amiloride, on oxygen consumption and short-circuit current of the human sigmoid epithelium to determine whether these effects were influenced by the age of the subject.Materials and Methods:Segments of the sigmoid colon were obtained from the safety margin of resections performed in patients of 62–77 years of age. Isolated mucosa preparations were obtained and mounted in airtight Ussing chambers, fit for simultaneous measurement of short-circuit current and oxygen concentration, before and after blocking epithelial sodium channels with amiloride (0.1 mmol/L). Regression analyses were performed to assess the associations between short-circuit current, oxygen consumption, and age of the subject as well as to define the relationship between the decreases in short-circuit current and oxygen consumption after blockade.Results:Epithelial sodium channel blockade caused an 80% reduction in short-circuit current and a 26% reduction in oxygen consumption. Regression analysis indicated that both changes were significantly related (r = 0.884; P = 0.0007). Oxygen consumption decreased by 1 μmol/h/cm2 for each 25 μA/cm2 decrease in short-circuit current. Neither short-circuit current nor oxygen consumption had any significant relationship with the age of the subjects.Conclusion:The decrease in epithelial oxygen consumption caused by amiloride is proportional to the decrease in short-circuit current and independent of the age of the subject.

Ion transport and barrier function are disturbed in microscopic colitis

In this paper, we identify mechanisms of watery diarrhea in microscopic colitis (MC). Biopsies from the sigmoid colon of patients with collagenous colitis and treated lymphocytic colitis were analyzed in miniaturized Ussing chambers for electrogenic sodium transport and barrier function with one-path impedance spectroscopy. Cytometric bead arrays (CBA) served to analyze cytokine profiles. In active MC, electrogenic sodium transport was diminished and epithelial resistance decreased. CBA revealed a Th1 cytokine profile featuring increased IFN-γ, TNF-α, and IL-1β levels. After four weeks of steroid treatment with budesonide, electrogenic sodium transport recovered while epithelial barrier defects remained. Diarrhea in MC results at least in part from a combination of impaired electrogenic sodium transport and barrier defects. From a therapeutic perspective it can be postulated that the functional importance of loss of ions may be higher than that caused by barrier impairment.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

Involvement of protons in the ion transport cycle of Na+,K+-ATPase

The effect of pH on electrogenic sodium transport by the Na+ ,K+ -ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid mem­ brane with adsorbed membrane fragments containing purified Na+ ,K+ -ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solu­ tions containing 150 m M Na+, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance incre­ ments at low frequencies decreased with the increasing pH. In the absence ofNaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCI. These results may be explained by involvement of protons in the Na+, K+ -ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.

Identification of functional transport in non lactating breast epithelium and its relationship to parity and menstrual cycle.

Abstract Abstract #4084 Background: The electrophysiological properties of breast epithelia have not been extensively studied in non-lactating women. Furthermore, it is known that parity is protective against breast cancer, but it is unknown how parity may influence the functional characteristics of breast epithelia and whether there is a carry over effect after childbirth.&amp;#x2028; Patients and Methods: With IRB and patient consent, electrical contact with ductal epithelium was established non-invasively, using a specially designed nipple sensor in 15 normal women under the age of 40 with no risk factors for breast cancer. The transepithelial potential (TEP) and resistance of the skin were abolished with sonopheresis. TEP was measured across the breast epithelium using an isolated millivoltmeter. In a separate study isolated mammary epithelial cells were obtained from 14 women undergoing surgery to identify the origins of transepithelial potential in non-lactating epithelia. Cultures were established in media permissive for epithelial cells, which were immunomagnetically purified and mounted on porous substrates. Ion transport was measured in an Ussing chamber coupled to a voltage clamp apparatus. Data were analyzed using t-test, Mann-Whitney test, or ANOVA as appropriate.&amp;#x2028; Results: Nulliparous women (n=8) had an open circuit potential of -56 ± 2 mV (mean ± SEM), -55 ± 1 mV, -56 ± 2 mV and -57 ± 2 mV during weeks 1-4 for 64 observations weekly through 2 complete menstrual cycles. Parous women (n=7) had open circuit potentials which were hyperpolarized compared to nulliparous women with values of -64 ± 1 mV (p &amp;lt; 0.001), -65 ± 1 mV (p &amp;lt; 0.001), -60 ± 2 mV (p n.s.) and -63 ± 2 mV (p&amp;lt;0.005) during weeks 1-4 for 56 observations weekly through 2 menstrual cycles. Therefore the transepithelial potential was 6-10 mV hyperpolarized in parous women compared to nulliparous women except in the 3rd week of menstrual cycle. Primary cultures of human mammary epithelial cells, which thrived in culture and matured successfully into competent (&amp;gt; 200 Ω.cm2) monolayers (50%), upregulated transepithelial ion movement in response to adrenergic stimulus (10uM norepinephrine). These currents were sensitive to pharmacologic inhibitors of ENaC (electrogenic sodium transport), NKCC (Na,K,2Cl) and Na/K ATPase. These findings suggest electrogenic sodium and chloride transport are responsible for the TEP observed in vivo. Post partum modulation of the activity of one of these functioning elements identified in non-cancerous monolayers may be responsible for the observed differences in TEP observed in this study.&amp;#x2028; Discussion: Parity appears to make the epithelium functionally competent so that it maintains a higher transepithelial potential, and is “primed for lactation”. Depolarization of the epithelium throughout cycle (as in the nulliparous group) may make it more susceptible to proliferative signals. Epithelial depolarization may represent a biomarker of breast cancer risk, the ionic basis of which remains to be elucidated. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4084.

A New Drug Delivery System Targeting Ileal Epithelial Cells Induced Electrogenic Sodium Absorption: Possible Promotion of Intestinal Adaptation

We previously demonstrated the induction of the epithelial sodium channel, prostasin, and 11β-hydroxysteroid dehydrogenase type 2 and activation of sodium transport mediated by those molecules in the remnant ileum after total proctocolectomy. The aims of the present study were to develop a new drug delivery system that targets ileal epithelial cells and to enhance local mineralocorticoid action without systemic effects. Orally administered d-aldosterone-containing d,l-lactide/glycolide acid copolymer microspheres are absorbed in the rat terminal ileum and released aldosterone. Blood and terminal ileal tissues were collected 2 weeks after the administration of the microspheres, and the aldosterone concentrations, mRNA, and protein expressions of the above molecules and sodium transport were evaluated. Significantly high levels of tissue aldosterone in the absence of elevated plasma levels were detected in the microspheres-treated rats. Epithelial mRNA and protein expression of the above molecules increased significantly in the microspheres-treated animals. Electrogenic sodium transport in the ileum was enhanced in the microspheres-treated rats. Aldosterone-containing microspheres successfully induced the expression of the above molecules and activated sodium transport in the ileal mucosa, both of which are essential for intestinal adaptation. Pre- and/or postoperative treatment with this drug may compensate for the excessive loss of sodium and water following proctocolectomy.

Effect of chaotropic anions on the sodium transport by the Na,K-ATPase

The effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na(+) transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, tau (2). Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E(1)/E(2). Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer.

Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis

Background & Aims : The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). Methods : Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na + transport (J Na) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC α-, β-, and γ-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of β- and γ-ENaC were analyzed in reporter gene assays. Results : In controls, aldosterone stimulated J Na and induced ENaC β- and γ-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC α-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as β- and γ-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor α and interferon γ. Promoter analysis revealed that down-regulation of β- and γ-ENaC gene expression was primarily induced by tumor necrosis factor α. Conclusions : We conclude that, in UC, elevated proinflammatory cytokines selectively impair β- and γ-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.

The Serum and Glucocorticoid Kinase sgk Increases the Abundance of Epithelial Sodium Channels in the Plasma Membrane of Xenopus Oocytes

The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of beta and gamma subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Molecular diversity of electrogenic sodium transport

Molecular diversity of electrogenic sodium transport

Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.

The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (R(T) approximately 4700 ohm x cm2) and potential difference (P(D) approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 microA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10(-10) to 10(-6) M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5x10(-7) M)-induced Isc (fourfold) was associated with a three- to fivefold increase in alpha-ENaC mRNA (but not in those for beta- or gamma-ENaC) and three- to 10-fold increases in alpha-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Electrogenic Sodium Transport Mediated by an Amiloride-sensitive Conductance in a Porcine Trophectoderm Cell Line

This study examined the electrical properties and Na+transport function of a porcine trophectoderm cell line, TE1, which forms a polarized epithelium in culture. Specifically, the capacity of TE1 cells to generate a transepithelial potential difference, and to modify selectively the Na+, K+and Cl−ionic composition of medium in the apical and basolateral compartments, was examined over a 48-h period using monolayers cultured on permeable tissue culture supports. TE1-cell monolayers formed ‘tight epithelia’ in that significant transepithelial electrical resistances (RT; median value 5.30k|gQ/cm2, range 2.26–9.18k|gQ/cm2, n=72), and electrical potential differences (VT; maximum mean value at 24h, 42.9 mV, sem 7.14; n=6) were generated. It is concluded that: (1) the VTwas generated by the amiloride-sensitive, Na+absorptive function; (2) K+transport across the monolayers was related to the electrogenic Na+transport function; and (3) the three ions traverse the epithelium by active transport or co-transport, rather than simple diffusion. These data substantiate the proposed role for porcine trophectoderm of generating electrical and chemical potentials in vivo, and for regulating the environment of the blastocoel cavity in the pre-implantation porcine embryo.

Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium.

1. The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon. 2. Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice. 3. Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons. 4. The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes. 5. The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate-free solutions and acetazolamide reduced the frusemide-resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide-resistant current. 6. It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response.

Peptide YY inhibits vasopressin-stimulated chloride secretion in inner medullary collecting duct cells.

mIMCD-k2 cells are derived from the inner medullary collecting duct of a mouse and exhibit electrogenic sodium absorption and cAMP- and vasopressin (AVP)-stimulated electrogenic chloride secretion [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; and N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The purpose of the present study was to determine how peptide YY (PYY) affects electrogenic Na+ and Cl- current in mIMCD-k2 cells. Short-circuit currents (Isc) were measured across monolayers of mIMCD-k2 cells mounted in Ussing-type chambers. PYY did not alter baseline Isc, nor did it alter Isc in chloride-free conditions, indicating no effect on electrogenic sodium transport. Baseline chloride current in these cells is low; therefore, chloride short-circuit current (IClsc) was stimulated with AVP (10 nM) added to the basolateral surface and 10 microM amiloride added to the apical surface. Although apical applications of PYY had no effect, basolateral application of PYY caused attenuation of IClsc, with the maximal inhibitory dose (100 nM) causing 52 +/- 1.3% inhibition (IC50 = 0.11 nM). Inhibition by PYY of IClsc is mediated through the Y2 receptor subtype, as PYY-(3-36) was the only PYY analog tested that caused inhibition and was equipotent to PYY. Inhibition by PYY of IClsc was abolished following incubation with pertussis toxin. We also show that PYY inhibits AVP-stimulated cAMP accumulation, with a maximal inhibitory dose (100 nM) causing a 38% +/- 6% inhibition (IC50 = 0.16 nM), comparable to inhibition by PYY of IClsc. We conclude that PYY acts through either Gi or Go to inhibit adenylate cyclase activity, leading to a decrease in AVP-stimulated chloride current.

Vasopressin-induced activation of protein kinase C in renal epithelial cells

Recent studies indicate that the actions of arginine vasopressin (AVP) and other agonists that stimulate electrogenic sodium transport in renal epithelial A6 cells are linked to a Ca 2+-mobilizing signal transduction mechanism that involves generation of inositol trisphosphate. Since diacylglycerol is the other product in this pathway, studies were performed to determine the possible role of PKC in the stimulation of sodium transport. AVP induced a biphasic increase in diacylglycerol generation, characterized by an initial rapid rise and then a sustained elevation, and PKC activation, reflected by phosphorylation of a specific 80 kDa myristoylated alanine-rich PKC substrate (MARCKS). To determine the PKC isoform(s) involved in this process, immunoblot analysis was performed using antisera that recognize both classical PKC isoforms, XPKC-I and XPCK-II, cloned from Xenopus oocytes. The transcripts of both isoforms were expressed in the A6 cell. Since protein recognized by antisera was translocated from cytosol to the particulate fraction after exposure to AVP, one or both isoforms were activated in the A6 cell. Further studies showed that cyclohexyladenosine and insulin, additional agonists of sodium transport in A6 cells, also stimulated phosphorylation of MARCKS. These results argue that Ca 2+-dependent PKC is involved in the action of AVP, and that of other agonists, which stimulate sodium transport.

Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

Segmental Difference of Water and Electrolyte Transport in Rat Colon in vivo

The segmental difference of water and electrolyte transport in the rat colon was studied in vivo. The proximal and distal colon segments were perfused separately but simultaneously at a constant rate with physiological solution, and net movements of water, sodium and chloride were determined. The effects of osmolality and sodium concentration of perfusate were assessed. The effect of a sodium channel blocker on the net transport of water and electrolytes was also studied in each colon segment. The net absorption of water, sodium and chloride correlated with the sodium concentration and osmolality of the perfusion solution in both colon segments and were dominant in the distal colon segment in each condition, compared with that in the proximal colon segment. The concentrations of three electrolytes in the collected fluid were almost the same as those of the perfusion solutions in both segments and these results indicated that water was transported isotonically through the colon lumen. Benzamil, a specific sodium ion channel blocker, inhibited net water and sodium absorption by 58.8% and 63.1% in the proximal colon segment and by 52.0% and 43.6% in the distal colon segment, respectively. These results suggest the existence of an electrogenic sodium transport mechanism and a paracellular pathway in normal (i.e., not treated with corticosteroids or sodium-depleted food) rats which has not been detected in in vitro studies with both apical and basolateral membrane vesicles.