Multiple acid-base and electrolyte disturbances upregulate NBCn1, NBCn2, IRBIT and L-IRBIT in the mTAL.

KEY POINTS: The roles of the Na+ /HCO3- cotransporters NBCn1 and NBCn2 as well as their activators IRBIT and L-IRBIT in the regulation of the mTAL transport of NH4+ , HCO3- , and NaCl are investigated. Dietary challenges of NH4 Cl, NaHCO3 or NaCl all increase the abundance of NBCn1 and NBCn2 in the outer medulla. The three challenges generally produce parallel increases in the abundance of IRBIT and L-IRBIT in the outer medulla. Both IRBIT and L-IRBIT powerfully stimulate the activities of the mTAL isoforms of NBCn1 and NBCn2 as expressed in Xenopus oocytes. Our findings support the hypothesis that NBCn1, NBCn2, IRBIT and L-IRBIT appropriately promote NH4+ shunting but oppose HCO3- and NaCl reabsorption in the mTAL, and thus are at the nexus of the regulation pathways for multiple renal transport processes. ABSTRACT: The medullary thick ascending limb (mTAL) plays a key role in urinary acid and NaCl excretion. NBCn1 and NBCn2 are present in the basolateral mTAL, where NBCn1 promotes NH4+ shunting. IRBIT and L-IRBIT (the IRBITs) are two powerful activators of certain acid-base transporters. Here we use western blotting and immunofluorescence to examine the effects of multiple acid-base and electrolyte disturbances on expression of NBCn1, NBCn2 and the IRBITs in rat kidney. We also use electrophysiology to examine the functional effects of IRBITs on NBCn1 and NBCn2 in Xenopus oocytes. NH4 Cl-induced metabolic acidosis (MAc) substantially increases protein expression of NBCn1 and NBCn2 in the outer medulla (OM) of rat kidney. Surprisingly, NaHCO3 -induced metabolic alkalosis (MAlk) and high-salt diet (HSD) also increase expression of NBCn1 and NBCn2 (effect of NaHCO3  > HSD). Moreover, all three challenges generally increase OM expression of the IRBITs. In Xenopus oocytes, the IRBITs substantially increase the activities of NBCn1 and NBCn2. We propose that upregulation of basolateral NBCn1 and NBCn2 plus the IRBITs in the mTAL: (1) promotes NH4+ shunting by increasing basolateral HCO3- uptake to neutralize apical NH4+ uptake during MAc; (2) inhibits HCO3- reabsorption during MAlk by opposing HCO3- efflux via the basolateral anion exchanger AE2; and (3) inhibits NaCl reabsorption by mediating (with AE2) net NaCl backflux into the mTAL cell during HSD. Thus, NBCn1, NBCn2 and the IRBITs are at the nexus of the regulatory pathways for multiple renal transport processes.

Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins.

Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys - hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 - were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes.

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes.

The α-carbonic anhydrases (CAs) are zinc-containing enzymes that catalyze the interconversion of CO2 and HCO3 (-). Here, we focus on human CA II (CA II), a ubiquitous cytoplasmic enzyme. In the second paper in this series, we examine CA IV at the extracellular surface. After microinjecting recombinant CA II in a Tris solution (or just Tris) into oocytes, we expose oocytes to 1.5% CO2/10 mM HCO3 (-)/pH 7.50 while using microelectrodes to monitor intracellular pH (pHi) and surface pH (pHS). CO2 influx causes the familiar sustained pHi fall as well as a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA II increases the magnitudes of the maximal rate of pHi change, (dpHi/dt)max, and the maximal change in pHS, ΔpHS. Preincubating oocytes with the inhibitor ethoxzolamide eliminates the effects of CA II. Compared with pHS, pHi begins to change only after a delay of ~9 s and its relaxation has a larger (i.e., slower) time constant (τpHi > τpHS ). Simultaneous measurements with two pHi electrodes, one superficial and one deep, suggest that impalement depth contributes to pHi delay and higher τpHi . Using higher CO2/HCO3 (-) levels, i.e., 5%/33 mM HCO3 (-) or 10%/66 mM HCO3 (-), increases (dpHi/dt)max and ΔpHS, though not in proportion to the increase in [CO2]. A reaction-diffusion mathematical model (described in the third paper in this series) accounts for the above general features and supports the conclusion that cytosolic CA-consuming entering CO2 or replenishing exiting CO2-increases CO2 fluxes across the cell membrane.

Evidence from mathematical modeling that carbonic anhydrase II and IV enhance CO2 fluxes across Xenopus oocyte plasma membranes.

Exposing an oocyte to CO2/HCO3 (-) causes intracellular pH (pHi) to decline and extracellular-surface pH (pHS) to rise to a peak and decay. The two companion papers showed that oocytes injected with cytosolic carbonic anhydrase II (CA II) or expressing surface CA IV exhibit increased maximal rate of pHi change (dpHi/dt)max, increased maximal pHS changes (ΔpHS), and decreased time constants for pHi decline and pHS decay. Here we investigate these results using refinements of an earlier mathematical model of CO2 influx into a spherical cell. Refinements include 1) reduced cytosolic water content, 2) reduced cytosolic diffusion constants, 3) refined CA II activity, 4) layer of intracellular vesicles, 5) reduced membrane CO2 permeability, 6) microvilli, 7) refined CA IV activity, 8) a vitelline membrane, and 9) a new simulation protocol for delivering and removing the bulk extracellular CO2/HCO3 (-) solution. We show how these features affect the simulated pHi and pHS transients and use the refined model with the experimental data for 1.5% CO2/10 mM HCO3 (-) (pHo = 7.5) to find parameter values that approximate ΔpHS, the time to peak pHS, the time delay to the start of the pHi change, (dpHi/dt)max, and the change in steady-state pHi. We validate the revised model against data collected as we vary levels of CO2/HCO3 (-) or of extracellular HEPES buffer. The model confirms the hypothesis that CA II and CA IV enhance transmembrane CO2 fluxes by maximizing CO2 gradients across the plasma membrane, and it predicts that the pH effects of simultaneously implementing intracellular and extracellular-surface CA are supra-additive.

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes.

Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3 (-) hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3 (-)/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pHi changes (τpHi ) and pHS relaxations (τpHS ). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3 (-) buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τpHS , indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane.

Relative CO2/NH3 permeabilities of human RhAG, RhBG and RhCG.

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (DpHS) caused by exposing the same oocyte to 5 % CO2/33 mM HCO3⁻ (an increase) or 0.5 mM NH3/NH4⁺ (a decrease). Subtracting the respective values for day-matched, H2O-injected control oocytes yielded channel-specific values (*). (ΔpH*(S))(CO2) and (-ΔpH*(S))(NH3) were each significantly >0 for all channels, indicating that RhBG and RhCG--like RhAG--can carry CO2 and NH3. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH3 transport. However, surface biotinylation experiments indicate the mutants RhBG(D178N) and RhCG(D177N) have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG--like RhAG--have significant CO2 permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH3 permeability. However, as evidenced by (ΔpH*(S))(CO2)/ (-ΔpH*(S))(NH3) values, we could not distinguish among the CO2/ NH3 permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH3 but also CO2 across the membranes of CD cells.

Movement of NH3 through the human urea transporter B: a new gas channel.

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [¹4C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (ΔpHS) caused by exposing oocytes to 5% CO2/33 mM HCO3⁻ (pHS increase) or 0.5 mM NH3/NH4⁺ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ΔpHS but doubled the NH3-induced ΔpHS. Phloretin reduced UT-B-dependent urea uptake (Jurea*) by 45%, Pf* by 50%, and (- ΔpHS*)NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea* by 25%, Pf* by 30%, and (ΔpHS*)NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9.

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel.

The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)(*)) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (ΔpH(S)) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (ΔpH(S)(*))((CO)2)/P(f)(*) is about half that of human AQP1, and the ratio (ΔpH(S)(*))(NH3)/P(f)(*) is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes.

The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (approximately 1mm diameter), (b) it has an established capacity to produce-from microinjected mRNAs or cRNAs-exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion transporters as green fluorescent protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl(-), H(+) (and hence base equivalents like OH(-) and HCO(3)(-)), K(+), and Na(+) is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates.

Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG.

The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO(2) and NH(3). Here, we use changes in surface pH (pH(S)) to assess relative CO(2) vs. NH(3) permeability of Xenopus oocytes expressing members of the AQP or Rh family. Exposed to CO(2) or NH(3), AQP1 oocytes exhibit a greater maximal magnitude of pH(S) change (DeltapH(S)) compared with day-matched controls injected with H(2)O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO(2), AQP1 oocytes also have faster time constants for pH(S) relaxation (tau(pHs)). Thus, AQP1, but not the other proteins, conduct CO(2) and NH(3). Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater DeltapH(S)(CO(2)) and faster tau(pHs) compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater DeltapH(S)(NH(3)) values. Only AQPs exhibited significant osmotic water permeability (P(f)). We computed channel-dependent (*) DeltapH(S) or P(f) by subtracting values for H(2)O oocytes from those of channel-expressing oocytes. For the ratio DeltapH(S)(CO(2))*/P(f)*, the sequence was AQP5 > AQP1 congruent with AQP4. For DeltapH(S)(CO(2))*/DeltapH(S)(NH(3))*, the sequence was AQP4 congruent with AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO(2) vs. NH(3) permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.

Concentration-dependent effects on intracellular and surface pH of exposing Xenopus oocytes to solutions containing NH3/NH4(+).

Others have shown that exposing oocytes to high levels of NH(3)/NH(4)(+) (10-20 mM) causes a paradoxical fall in intracellular pH (pH(i)), whereas low levels (e.g., 0.5 mM) cause little pH(i) change. Here we monitored pH(i) and extracellular surface pH (pH(S)) while exposing oocytes to 5 or 0.5 mM NH(3)/NH(4)(+). We confirm that 5 mM NH(3)/NH(4)(+) causes a paradoxical pH(i) fall (-DeltapH(i) approximately equal 0.2), but also observe an abrupt pH(S) fall (-DeltapH(S) approximately equal 0.2)-indicative of NH(3) influx-followed by a slow decay. Reducing [NH(3)/NH(4)(+)] to 0.5 mM minimizes pH(i) changes but maintains pH(S) changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates -DeltapH(S) at both NH(3)/NH(4)(+) levels. During removal of 0.5 or 5 mM NH(3)/NH(4)(+), failure of pH(S) to markedly overshoot bulk extracellular pH implies little NH(3) efflux and, thus, little free cytosolic NH(3)/NH(4)(+). A new analysis of the effects of NH(3) vs. NH(4)(+) fluxes on pH(S) and pH(i) indicates that (a) NH(3) rather than NH(4)(+) fluxes dominate pH(i) and pH(S) changes and (b) oocytes dispose of most incoming NH(3). NMR studies of oocytes exposed to (15)N-labeled NH(3)/NH(4)(+) show no significant formation of glutamine but substantial NH(3)/NH(4)(+) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH(i) and pH(S) demonstrate that NH(3) flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane-AmtB-enhances the flux of a gas across a biological membrane.

Characterization of human SLC4A10 as an electroneutral Na/HCO3 cotransporter (NBCn2) with Cl- self-exchange activity.

The SLC4A10 gene product, commonly known as NCBE, is highly expressed in rodent brain and has been characterized by others as a Na(+)-driven Cl-HCO(3) exchanger. However, some of the earlier data are not consistent with Na(+)-driven Cl-HCO(3) exchange activity. In the present study, northern blot analysis showed that, also in humans, NCBE transcripts are predominantly expressed in brain. In some human NCBE transcripts, splice cassettes A and/or B, originally reported in rats and mice, are spliced out. In brain cDNA, we found evidence of a unique partial splice of cassette B that is predicted to produce an NCBE protein with a novel C terminus containing a protein kinase C phosphorylation site. We used pH-sensitive microelectrodes to study the molecular physiology of human NCBE expressed in Xenopus oocytes. In agreement with others we found that NCBE mediates the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent transport of HCO(3)(-). For the first time, we demonstrated that this transport process is electroneutral. Using Cl(-)-sensitive microelectrodes positioned at the oocyte surface, we found that, unlike both human and squid Na(+)-driven Cl-HCO(3) exchangers, human NCBE does not normally couple the net influx of HCO(3)(-) to a net efflux of Cl(-). Moreover we found that that the (36)Cl efflux from NCBE-expressing oocytes, interpreted by others to be coupled to the influx of Na(+) and HCO(3)(-), actually represents a CO(2)/HCO(3)(-)-stimulated Cl(-) self-exchange not coupled to either Na(+) or net HCO(3)(-) transport. We propose to rename NCBE as the second electroneutral Na/HCO(3) cotransporter, NBCn2.

Effect of uroguanylin on potassium and bicarbonate transport in rat renal tubules.

The effect of uroguanylin (UGN) on K+ and H+ secretion in the renal tubules of the rat kidney was studied using in vivo stationary microperfusion. For the study of K+ secretion, a tubule was punctured to inject a column of FDC-green-colored Ringer's solution with 0.5 mmol KCl/L+/-10(-6) mol UGN/L, and oil was used to block fluid flow. K+ activity and transepithelial potential differences (PD) were measured with double microelectrodes (K+ ion-selective resin vs. reference) in the distal tubules of the same nephron. During perfusion, K+ activity rose exponentially, from 0.5 mmol/L to stationary concentration, allowing for the calculation of K+ secretion (JK). JK increased from 0.63+/-0.06 nmol.cm-2.s-1 in the control group to 0.85+/-0.06 in the UGN group (p<0.01). PD was -51.0+/-5.3 mV in the control group and -50.3+/-4.98 mV in the UGN group. In the presence of 10(-7) mol iberiotoxin/L, the UGN effect was abolished: JK was 0.37+/-0.038 nmol.cm-2.s-1 in the absence of, and 0.38+/-0.025 in the presence of, UGN, indicating its action on maxi-K channels. In another series of experiments, renal tubule acidification was studied, using a similar method: proximal and distal tubules were perfused with solutions containing 25 mmol NaHCO3/L. Acidification half-time was increased both in proximal and distal segments and, as a consequence, bicarbonate reabsorption decreased in the presence of UGN (in proximal tubules, from 2.40+/-0.26 to 1.56+/-0.21 nmol.cm-2.s-1). When the Na+/H+ exchanger was inhibited by 10(-4) mol hexamethylene amiloride (HMA)/L, the control and UGN groups were not significantly different. In the late distal tubule, after HMA, UGN significantly reduced JHCO3-, indicating an effect of UGN on H+-ATPase. These data show that UGN stimulated JK+ by acting on maxi-K channels, and decreased JHCO3- by acting on NHE3 in proximal and H+-ATPase in distal tubules.

Action of ANG II and ANP on colon epithelial cells.

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH(i)) and calcium ([Ca2+](i)) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH(i) recovery rate mediated by the Na(+)/H+ exchanger (NHE) was examined following an NH4Cl pulse. Under control conditions pH(i) recovered at 0.114+/-0.005 pH units/min (n=35). ANG II (10(-12) or 10(-9) M) increased this value, whilst ANG II (10(-7) M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 microM or 25 microM HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH(i) recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca2+]i concentration-dependently. ANP (10(-6) M) or dimethyl-BAPTA/AM (50 microM) blocked the effects of ANG II on [Ca2+]i and on the rate of pH(i) recovery. Thapsigargin (10(-5) M) enhanced the effect of ANG II on [Ca2+]i and reversed its stimulatory effect on the rate of pH(i) recovery to an inhibitory one. External Ca(2+)-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca2+]i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na(+)/H+ exchanger, one stimulating Na(+)/H+ activity by increases of [Ca2+]i in the lower range (at 10(-12) or 10(-9) M ANG II) and the other inhibiting this activity at high [Ca2+]i levels (at 10(-7) M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca2+]i, block both the stimulatory and inhibitory effects of ANG II.

Signaling path of the action of AVP on distal K+ secretion.

BACKGROUND: Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (JK) via V1 receptors (Am J Physiol 278:F809-F816, 2000). In the present work, we investigate the cell signaling mechanism of this process. METHODS: In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K+ was measured using double K+ resin/reference microelectrodes. RESULTS: In control conditions, JK was 0.71 +/- 0.05 nmol.cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, JK increased to 0.88 +/- 0.08 nmol.cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, JK was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca(2+)-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated JK by 36.4%, and AVP-independent JK (after blocking V1 receptors) by only 16%. CONCLUSION: The results suggest that the luminal V1-receptor effect of AVP on JK was mediated by the phospholipase C (PLC)/Ca2+/PKC signaling path and not byadenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

Arginine vasopressin stimulates H+-ATPase in MDCK cells via V1 (cell Ca2+) and V2 (cAMP) receptors.

The effect of arginine vasopressin (AVP) and/or atrial natriuretic peptide (ANP) on the regulation of intracellular pH (pHi) via H+-ATPase and of cytosolic calcium ([Ca2+]i) was investigated in Madin-Darby canine kidney (MDCK) cells by the fluorescent probes BCECF-AM and fluo-4-AM, respectively. The pHi recovery rate was examined after intracellular acidification following an NH4Cl pulse, in the presence of zero Na+ plus Schering 28080 (a specific inhibitor of H+-K+-ATPase). AVP (10-12-10-6 M) increased the rate of pHi recovery and [Ca2+]i in a dose-dependent manner. V1- or V2-receptor antagonists impaired the effect of AVP on both processes, and DDAVP (10-12-10-6 M; a V2-selective agonist) caused a dose-dependent stimulation of them. [Ca2+]i or cAMP (as increased by 10-5 M thapsigargin or 8-BrcAMP, respectively) alone had no effect on H+-ATPase, but their synergic action was necessary to stimulate H+-ATPase. In agreement with these findings, ANP (10-6 M) or dimethyl-BAPTA-AM (5 x 10-5 M), impairing the increase of [Ca2+]i in response to AVP, blocks the stimulatory effect of AVP on H+-ATPase.

Peritubular AVP regulates bicarbonate reabsorption in cortical distal tubule via V(1) and V(2) receptors.

Peritubular arginine vasopressin (AVP) regulates bicarbonate reabsorption in the cortical distal tubule via V(1) and V(2) receptors. The dose-dependent effects of peritubular AVP on net bicarbonate reabsorption (J(HCO)) were evaluated by stationary microperfusion of in vivo early (ED; distal convoluted tubule) and late distal (LD; connecting tubule and initial collecting duct) segments of rat kidney, using double-barreled H(+)-sensitive, ion-exchange resin/reference (1 M KCl) microelectrodes. AVP (10(-11) M) perfused into peritubular capillaries increased J(HCO), compared with basal levels during intact capillary perfusion with blood, in ED and LD segments. AVP (10(-9) M) also increased J(HCO) in both segments, but the effect of AVP (10(-11) M) was significantly higher. A specificV(1)-receptor antagonist alone or with AVP (10(-11) or 10(-9) M) reduced J(HCO) below basal levels. A specific V(2)-receptor antagonist alone or plus AVP (10(-11) M) did not affect J(HCO) but increased AVP (10(-9) M)-mediated stimulation. 8-Bromoadenosine 3',5'-cyclic monophosphate alone reduced J(HCO) below basal levels and also reduced AVP (10(-11) M)-mediated stimulation. (Deamino-Cys(1), D-Arg(8)) vasopressin (a V(2)-selective agonist) also reduced J(HCO) below basal levels. These results show that peritubular AVP stimulates J(HCO) in ED and LD segments via basolateral V(1) receptors and that basolateral V(2) receptors have a dose-dependent inhibitory effect mediated by cAMP. The data also indicate that endogenous AVP stimulates distal J(HCO) via basolateral V(1) receptors.