Biphasic Regulation of Renal Proximal Bicarbonate Absorption by Luminal AT1A Receptor
Yanan Zheng*,
Shoko Horita*,
Chiaki Hara*,
Motoei Kunimi*,
Hideomi Yamada*,
Takeshi Sugaya,
Atsuo Goto*,
Toshiro Fujita* and
George Seki*
*Department of Internal Medicine, Faculty of Medicine, Tokyo University, Tokyo, Japan; and Discovery Research Laboratory, Tanabe Seiyaku Co, Ltd, Osaka, Japan.
Correspondence to Dr. George Seki, Department of Internal Medicine, Faculty of Medicine, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-3815-5411 ext. 33004; Fax: 81-3-5800-8806;
ABSTRACT. Angiotensin II (AngII) regulates renal proximal transportin a biphasic way. It has been recently shown that the basolateraltype 1A receptor (AT1A) mediates the biphasic regulation ofNa+-HCO3- cotransporter (NBC) by AngII. However, the receptorsubtype(s) responsible for the luminal AngII actions remainedto be established. To clarify this issue, the luminal AngIIeffects in isolated proximal tubules from wild-type (WT) andAT1A-deficient mice (AT1A KO) were compared. In WT, the rateof bicarbonate absorption (JHCO3-), analyzed with a stop-flowmicrospectrofluorometric method, was stimulated by 10-10 mol/Lluminal AngII but was inhibited by 10-6 mol/L luminal AngII.Both stimulatory and inhibitory effects of AngII were completelyblocked by valsartan (AT1 antagonist) but unaffected by PD 123,319(AT2 antagonist). In AT1A KO, in contrast, luminal AngII (10-10- 10-6 mol/L) did not change JHCO3-. In WT, 10-6 mol/L luminalAngII increased cell Ca2+ concentrations ([Ca2+]i), which wasagain blocked by valsartan but not by PD 123,319. However, luminalAngII did not increase [Ca2+]i in AT1A KO. On the other hand,the addition of arachidonic acid similarly inhibited JHCO3-in WT and AT1A KO. Furthermore, the acute activation of proteinkinase C by phorbol 12-myristate 13-acetate similarly stimulatedJHCO3- in WT and AT1A KO, indicating that the inhibitory andstimulatory pathways necessary for the AngII actions were preservedin AT1A KO. These results indicate that the luminal AT1A mediatesthe biphasic regulation of bicarbonate absorption by luminalAngII, while no evidence was obtained for a role of AT2. E-mail:georgeseki-tky@uminac.jp
Angiotensin II (AngII) receptors are divided into two distinctreceptor subtypes, type 1 (AT1) and type 2 (AT2) receptors,and AT1 receptors are further divided into AT1A and AT1B inrodents (1,2). Most of the physiologic effects of AngII arethought to be mediated by AT1, while the role of AT2 remainsunclear (1–3). In addition to the effects on renal hemodynamics,AngII has direct effects on renal tubular functions. In particular,AngII acts on sodium and bicarbonate reabsorption from proximaltubules, and this process is thought to have significant impacton body fluid and sodium homeostasis (4–7). Interestingly,AngII is known to regulate proximal transport in a biphasicway: stimulation by low (picomolar to nanomolar) concentrationsof AII, and inhibition by high (nanomolar to micromolar) concentrationsof AII (4,5). Previous studies have consistently shown thatAT1 mediates the stimulatory effect of AngII. However, conflictingresults have been reported regarding the receptor subtype mediatingthe inhibitory effect of AngII. Thus, several studies supporteda view that AT1 mediates both stimulatory and inhibitory effectsof AngII (8,9), whereas Haithcock et al. (10) presented theevidence that AT2 mediates the inhibitory effect of AngII. Methodologicaldifferences may not fully account for these conflicting results,and the receptor subtype responsible for the inhibitory effectof AngII remained to be definitely determined. To clarify thisissue, we have recently compared the effects of AngII on Na+-HCO3-cotransporter (NBC) activity in isolated proximal tubules fromwild-type (WT) and AT1A-deficient mice (AT1A KO). The resultsclearly showed that the basolateral AT1A mediates both stimulationand inhibition of NBC by AngII (11). It could be still possible,however, that the apical AT2 may mediate inhibition by AngII,because the study by Haithcock et al. (10) was conducted oncultured proximal tubular cells that might express the apicalAT2, whereas our study was restricted on the basolateral receptors(11). Previous studies have indeed shown that luminal AngIIhas also biphasic effects on proximal transport (12,13). Inaddition, a recent finding of remarkably high (nanomolar) proximalintratubular concentrations of AngII suggests an important roleof luminal AII actions (13). The purpose of the present studyis therefore to compare the roles of luminal AT1A and AT2 inmediating the stimulatory and inhibitory effects of AngII usinga combination of gene-targeted mice and specific receptor antagonists.To accomplish this task, we compared the effects of luminalAngII on the rate of bicarbonate absorption (JHCO3-) in isolatedproximal tubules from WT and AT1A KO. This study was facilitatedby a convenient microspectrofluorometric method, which we haverecently developed for the determination of JHCO3- in isolatedrabbit proximal tubules (14,15).
Animals
Male AT1A KO mice (16) and WT mice (Discovery Research Laboratory,Tanabe Seiyaku), 5- to 8-wk-old, from the same genetic backgroundwere used in the present study. They were provided with standardfood and water ad libitum. All animal procedures were in accordancewith local institutional guidelines.
Microperfusion Technique
Mice were anesthetized with pentobarbital sodium, and the thinsections from the left kidney were obtained and stored in ice-cold,gas-equilibrated (5% CO2/95% O2) Ringer solution containing:144 mmol/L Na+, 5 mmol/L K+, 1.5 mmol/L Ca2+, 1 mmol/L Ma2+,125 mmol/L Cl-, 25 mmol/L HCO3-, 2 mmol/L H2PO4-, 1 mmol/L SO42-,and 5.5 mmol/L D-glucose. Proximal tubules (S2 segment) weremicrodissected without collagenase treatment and then microperfusedaccording to the method described by Burg et al. (17) with amodified version of the perfusion and sampling capillary system(14,15). The tubular lumen was perfused with the same gas-equilibratedRinger solution; however, D-glucose was omitted and 20 mmol/LNaCl was replaced by 40 mmol/L raffinose. We have previouslyshown that tissue culture media such as Dulbecco modified Eaglemedium (DMEM) are essential to maintain a better functionalstate of isolated proximal tubules (18–20). In pilot experiments,we indeed confirmed that JHCO3- in isolated mouse proximal tubuleswas kept at high rates for more than 60 min in DMEM solution,but it deteriorated within 60 min in Ringer solution. In thepresent study, DMEM was therefore used as the bath perfusate.The experimental chamber was continuously perfused at a rateof approximately 10 ml/min with prewarmed (38°C) and gas-equilibrated(5% CO2/95% O2) DMEM solution for 30 min, and the JHCO3- measurementswere started.
Determination of JHCO3-
We used the recently described stop-flow microperfusion technique(14,15). In brief, isolated tubules were mounted on the stageof an inverted epifluorescence microscopy (IMT-2, Olympus).After background fluorescence was measured, a pH-sensitive fluorescencedye 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein (BCECF)was added to the luminal perfusate. Luminal pH (pHL) was monitoredby a microspectrofluorometer system (OSP-10, Olympus), whichalternately illuminates the preparation with light of 440 and490 nm and measures emission at the 530 nm wavelength. To obtaina calibration curve, lumen was perfused at high rates (>80nl/min) with NaCl solutions buffered to pH 6.5 to 7.5 with 4-(2-hydroxyethyl)-1-piperazineethanesulphonicacid. To determine JHCO3-, the rapid (approximately 80 nl/min)luminal perfusion was abruptly stopped by suddenly reducingthe perfusion pressure from approximately 18 to 0 cmH2O. Afterstop-flow, pHL fell from 7.4 to values near 6.8 within 30 s,where it remained virtually constant. This decrease in pHL reflectsthe gradual absorption of HCO3- and the attainment of a steady-statezero net volume flux that develops because of the presence ofpoorly absorbable raffinose in the luminal perfusate as described(14,15). The decay in luminal HCO3- concentration ([HCO3-]L)was calculated from the changes in pHL, and JHCO3- was calculatedfrom the following equation:
(1)
Where r is the luminal radius before stop-flow, [HCO3-]0, and[HCO3-] are [HCO3-]L before stop-flow and in the steady-state,respectively, and k is the rate constant of [HCO3-]L decline.During rapid luminal perfusion, the perfusion pressure willact to dilate the tubular lumen because the tubule itself aswell as the collection pipette offers some resistance to flow.When the perfusion pressure is suddenly reduced to zero uponstop-flow, the tubular lumen tends to partially collapse. Thecorrection for this volume loss into the pipettes was achievedby using the decaying 440 nm (pH-insensitive) fluorescence signalsas a marker of the residual luminal volume. The validity ofthis correction strategy was confirmed by comparison with microphotographicand video measurements as described previously (14).
Measurements of Cell Ca2+ Concentrations ([Ca2+]i)
[Ca2+]i was measured as described previously (11). Briefly,after autofluorescence was measured, the tubules were incubatedwith 30 µmol/L Fura-2/AM for 60 min in DMEM under 5% CO2/95%O2 gas at 37°C. Thereafter, the tubule was transferred intothe perfusion chamber, microperfused as described above, and[Ca2+]i was monitored with the OSP-10 system. The calibrationcurves were obtained at the end of each experiment, and [Ca2+]iwere calculated according to the method by Grynkiewicz et al.(21).
Materials and Statistics
BCECF and Fura-2/AM were obtained from Dojindo, valsartan wasfrom Novartis, 5-ile-AngII, PD 123319, arachidonic acid, (PMA),acetazolamide, and ouabain were from Sigma, and all the otherchemicals were from Wako. The data were represented as meanvalues ± SEM. Significant differences were determinedby applying the paired or unpaired t test as appropriate.
The Validity of Stop-Flow Microspectrofluorometric Method
To test whether the stop-flow microspectrofluorometric method,which we have recently developed to measure bicarbonate absorptionfrom rabbit proximal tubules (14,15), can also apply to mouseproximal tubules, we performed a series of experiments on isolatedproximal tubules from WT. Bicarbonate absorption from proximaltubules has been shown to largely depend on the activities ofcarbonic anhydrases as well as on the Na+ gradient (22). Totest for the role of carbonic anhydrases, we examined the effectof acetazolamide. The addition of acetazolamide (1 mmol/L) toboth luminal and basolateral sides for 10 min substantiallyreduced JHCO3- from 13.0 ± 1.1 to 4.1 ± 0.5 pmol/cms (68 ± 3% inhibition; n = 6; P < 0.005). To testfor Na+-dependency, we examined the effect of ouabain. The additionof ouabain (1 mmol/L) to the basolateral side for 10 min markedlyreduced JHCO3- from 12.3 ± 0.8 to 3.0 ± 0.3 pmol/cms (76 ± 2% inhibition; n = 7; P < 0.005). We alsotested for Na+-dependency more directly by removing Na+ fromthe ambient solutions. Similarly to all the other experiments,tubules were first perfused for 30 min using Ringer solutionand DMEM as the luminal and the basolateral perfusate, respectively.After the control JHCO3- value was obtained, however, both perfusateswere switched to Na+-free solution, in which all the Na+ inRinger solution was replaced by N-methely-D-glucamine. The bilateralNa+-removal by this procedure for 10 min also markedly reducedJHCO3- from 11.7 ± 0.9 to 2.4 ± 0.2 pmol/cm s(78 ± 3% inhibition; n = 7; P < 0.005). In contrast,JHCO3- did not change (n = 5) when Ringer solution, insteadof Na+-free solution, was used for the bilateral perfusates,indicating that the reduction in JHCO3- after the Na+-removalwas not due to the omission of DMEM. These results are consistentwith the characteristics of bicarbonate absorption from proximaltubules (22), indicating that the stop-flow microspectrofluorometricmethod can successfully apply to measure bicarbonate absorptionfrom isolated mouse proximal tubules.
Effects of Luminal AngII in WT
To examine the roles of luminal AngII receptors in the regulationof proximal bicarbonate absorption, we first examined the effectsof luminal AngII in isolated proximal tubules from WT. As canbe seen in Figure 1, 10-min perfusion with the luminal solutioncontaining 10-10 mol/L AngII increased JHCO3- by 42 ±7% (n = 8; P < 0.005). On the other hand, 10-8 mol/L AngIIhad no effects (n = 8), and 10-6 mol/L AngII rather decreasedJHCO3- by 34 ± 4% (n = 7; P < 0.05). Time controlexperiments without AngII confirmed that JHCO3- did not changeduring repeated stop-flow (n = 6). These results indicate thatluminal AngII has biphasic effects on JHCO3- in isolated mouseproximal tubules as previously shown in isolated rabbit proximaltubules (12).
Figure 1. Effects of luminal angiotensin II (AngII) on rate of bicarbonate absorption (JHCO3-) in wild-type mice (WT). Open bars indicate control values; closed bars indicate values after AngII addition. n = 8, 8, and 7 for 10-10, 10-8, and 10-6 mol/L AngII, respectively. *P < 0.005 versus control responses.
To examine the receptor subtype(s) mediating these AngII effects,we tested an AT1 antagonist, valsartan. The addition of 2 x10-7 mol/L valsartan into the luminal perfusate did not changeJHCO3- (11.8 ± 2.0 versus 12.0 ± 1.9 pmol/cm s;n = 6; NS), but it completely blocked the effects of luminalAngII. Thus, in the presence of valsartan, JHCO3- was not changedby 10-10 mol/L AngII (12.8 ± 1.4 versus 12.2 ±1.5 pmol/cm s; n = 7; NS) and by 10-6 mol/L AngII (11.3 ±1.2 versus 11.3 ± 1.1 pmol/cm s; n = 7; NS). The additionof higher concentrations (2 x 10-6 mol/L) of valsartan intothe luminal perfusate also did not change JHCO3- (n = 6). Totest for a role of AT2, we examine the effects of an AT2 antagonistPD 123,319. The addition of 10-5 mol/L PD 123,319 into the luminalperfusate did not change JHCO3- (11.9 ± 1.8 versus 11.6± 2.2 pmol/cm s; n = 6; NS) and did not modify the effectsof luminal AngII. Thus, in the presence of PD 123,319, 10-10mol/L AngII increased JHCO3- by 39 ± 8% (n = 7; P <0.005), and 10-6 mol/L AngII decreased JHCO3- by 32 ±3% (n = 7, P < 0.005). These results indicate that the luminalAT1 mediates both stimulation and inhibition of JHCO3- by AngII.
A previous study on isolated rabbit proximal tubules (12) suggestedthat the presence of physiologic concentrations of AngII inthe basolateral side could counteract the inhibition by highconcentrations of luminal AngII. We therefore examined the effectof luminal 10-6 mol/L AngII in the presence of basolateral 10-10mol/L AngII. The addition of 10-10 mol/L AngII to the basolateralside alone for 10 min increased JHCO3- from 10.7 ± 0.7to 14.1 ± 0.8 pmol/cm s (33 ± 5% stimulation;n = 6; P < 0.01), as expected. On the other hand, the simultaneousaddition of basolateral 10-10 mol/L AngII and luminal 10-6 mol/LAngII for 10 min significantly reduced JHCO3- from 11.7 ±0.6 to 7.8 ± 0.5 pmol/cm s (34 ± 2% inhibition;n = 7; P < 0.005). The degree of inhibition by luminal 10-6mol/L AngII in the presence of basolateral 10-10 mol/L AngIIwas not statistically different from that by luminal 10-6 mol/LAngII alone.
Effects of AngII in AT1A KO
We next examined the effects of luminal AngII in AT1A KO. Thecontrol JHCO3- in AT1A KO (11.9 ± 0.8 pmol/cm s; n =24) was very similar to that in WT (11.7 ± 0.5 pmol/cms; n = 26). As shown in Figure 2, the addition of 10-10, 10-8,and 10-6 mol/L AngII all failed to change JHCO3- in AT1A KO.Time control experiments without AngII confirmed that JHCO3-did not change during repeated stop-flow (n = 5). These resultsare consistent with a view that the biphasic regulation of JHCO3-in WT was mediated by the luminal AT1A.
Figure 2. Effects of luminal AngII on JHCO3- in type 1A receptor (AT1A)–deficient mice (AT1A KO). Open bars indicate control values; closed bars indicate values after AngII addition. n = 8 for each concentration of AngII.
Effects of AngII on [Ca2+]i
The increase in [Ca2+]i could be an important factor in AngII-mediatedsignaling pathways (8,9,23,24); we therefore also examined the[Ca2+]i responses to luminal AngII. In WT, the addition of 10-10mol/L AngII did not induce a significant increase in [Ca2+]i(n = 6), and 10-8 mol/L induced only a marginal increase inthree of seven tubules. However, 10-6 mol/L AngII consistentlyinduced a spikelike increase in [Ca2+]i as shown in Figure 3.To examine the receptor subtype mediating the [Ca2+]i responseto AngII, we tested valsartan and PD 123,319. In three tubules,sequential additions of 10-6 mol/L AngII in the same tubule,separated for more than 4 min, induced comparable [Ca2+]i responses.As also shown in Figure 3, however, the addition of 2 x 10-7mol/L valsartan into the luminal perfusate almost completelyinhibited the [Ca2+]i increase by 10-6 mol/L AngII (n = 4).On the other hand, the addition of 10-5 mol/L PD 123,319 intothe luminal perfusate did not inhibit the [Ca2+]i increase by10-6 mol/L AngII (n = 4). To test whether the luminal AngIIactions are mediated solely by the apical receptor(s), we examinedthe effect of basolateral valsartan on the [Ca2+]i responseto luminal AngII. In three tubules, the [Ca2+]i increase bythe luminal addition of 10-6 mol/L AngII was not inhibited byvalsartan added into the basolateral perfusate. These resultsindicate that the [Ca2+]i response to luminal AngII is solelymediated by the luminal AT1.
Figure 3. Effects of luminal AngII on concentrations of Ca2+ ([Ca2+]I) in the absence and presence of luminal valsartan. Note the [Ca2+]i increase by 10-6 mol/L AngII was almost completely inhibited by 2 x 10-7 mol/L valsartan.
We also examined the effects of luminal AngII in AT1A KO. However,up to 10-5 mol/L AngII did not induce a significant [Ca2+]iincrease in AT1A KO.
Effects of Arachidonic Acid and PMA on JHCO3-
Previous studies have shown that the activation of phospholipaseA2 (PLA2) and the subsequent release of arachidonic acid couldbe responsible for the inhibition by high concentrations ofAngII (9,25,26). We therefore compared the effects of arachidonicacid on JHCO3- in WT and AT1A KO. As shown in Figure 4, theaddition of 10-8 mol/L arachidonic acid to the bath perfusatesimilarly reduced JHCO3- in WT (-27 ± 2%; n = 7) andAT1A KO (-32 ± 1%; n = 7).
Figure 4. Effects of arachidonic acid on JHCO3- in WT and AT1A KO. Open bars indicate control values; shaded bars indicate values obtained 5 min after arachidonic acid (A.A) addition. n = 7 for each mice. *P < 0.005 versus control responses.
On the other hand, activation of protein kinase C (PKC) couldbe involved in the stimulation by low concentrations of AngII(24,27). Therefore we finally compared the effects of acutePKC activation in WT and AT1A KO. As shown in Figure 5, theaddition of 5 x 10-7 mol/L PMA similarly increased JHCO3- inWT (33 ± 4%; n = 5) and AT1A KO (36 ± 6%; n =5).
Figure 5. Effects of phorbol 12-myristate 13-acetate (PMA) on JHCO3- in WT and AT1A KO. Open bars indicate control values; shaded bars indicate values obtained 5 min after PMA addition. n = 5 for each mice. *P < 0.005 versus control responses.
In 1977, Harris and Young (4) reported that AngII added intothe peritubular fluid regulates volume absorption from rat proximaltubules in the biphasic way. This finding has been confirmedin various experimental conditions (8,9,23,28,29), and otherinvestigators have shown that AngII added into the luminal fluidcan also exert the similar biphasic effects (12,13). Regardingthe signal transduction pathways, the stimulation by AngII isgenerally thought to be mediated by the activation of PKC and/orthe decrease in the level of cAMP in the cell (7,23,24,27),though a recent study suggests that the activation of tyrosinekinases could be also involved (30). On the other hand, theactivation of PLA2 and the subsequent release of arachidonicacid seem to be involved in the inhibition by AngII. In particular,a P-450–dependent arachidonic acid metabolite, 5,6-epoxyeicosatrienoicacid, could be the final mediator of the inhibition by AngII(9,25,26). Although the stimulation by AngII is certainly mediatedby AT1, conflicting results have been reported as to the receptorsubtype responsible for the inhibition by AngII (8,9,10). Bycomparing the effects of AngII in WT and AT1A KO, we have recentlyshown that the biphasic regulation of NBC by AngII is mediatedby AT1A in the basolateral membranes (11). The study was, however,limited on the basolateral receptors; we could not thereforeexclude a possibility that the luminal AT2 may mediate the inhibitionby AngII. To clarify this issue, we compared the effects ofluminal AngII in WT and AT1A KO in the present study. To measurebicarbonate absorption, we used the stop-flow microspectrofluorometricmethod, which we have recently developed for isolated rabbitproximal tubules (14,15). The validity of this method was confirmedby series of experiments with acetazolamide, ouabain, and Na+-freesolution on isolated proximal tubules from WT, which reproducedthe previously characterized properties of proximal bicarbonatetransport (22).
In WT, the biphasic regulation of JHCO3- by AngII was completelyinhibited by the highly selective AT1 antagonist, valsartan(31). However, the AT2 antagonist PD 123,319 did not modifythe effects of AngII. In AT1A KO, the effects of luminal AngIIon JHCO3- were completely lost. On the other hand, the stimulationby PMA as well as the inhibition by arachidonic acid was verysimilar in WT and AT1A KO, indicating that the stimulatory andinhibitory signaling pathways required for AngII actions areintact in AT1A KO as we previously reported (11). These resultsare consistent with a view that the luminal AT1A mediates thebiphasic regulation of bicarbonate absorption from proximaltubules.
Because of remarkably high proximal intratubular concentrationsof AngII and the presence of angiotensinogen and its mRNA inproximal tubules, AngII is thought be directly secreted intoproximal tubular lumen by the epithelial cells (13). Baum etal. (32) reported that this endogenously secreted AngII wasable to modify bicarbonate absorption from isolated proximaltubules. In the present study, however, the addition of sufficientconcentrations of valsartan did not change JHCO3- in WT, whichdid not support a role of endogenous AngII. One possible explanationfor these apparently conflicting results could be that we usedthe very high luminal perfusion rates (>80 nl/min) to performstop-flow measurements, whereas Baum et al. (32) used the muchlower (approximately 10 nl/min) perfusion rates to measure JHCO3-by the conventional picapnotherm method. On the other hand,a previous study on isolated rabbit proximal tubules (12) provideddata suggesting that the inhibition by high concentration ofluminal AngII could be blunted by the presence of physiologicconcentrations of basolateral AngII. In contrast, we showedthat the inhibition by 10-6 mol/L luminal AngII in WT was preservedeven in the presence of basolateral 10-10 mol/L AngII. The reasonfor these discrepant results is not apparent, but species differencecould be involved.
In the previous study, we observed that 10-6 mol/L basolateralAngII slightly stimulated the NBC activity in AT1A KO, whichwas completely inhibited by valsartan (11). In the present study,however, 10-6 mol/L luminal AngII had no effects on JHCO3- inAT1A KO. The pharmacologic properties of AT1A and AT1B are knownto be very similar (1,2); therefore, the most likely explanationfor these observations would be that AT1B is expressed at alow level in the basolateral membranes but not in the apicalmembranes of proximal tubules. A previous study has confirmedthe low-level expression of AT1B in proximal tubules (33), butwhether it is expressed in the basolateral or the apical membraneshas not been determined. The future studies on AT1B-deficientmice would help clarify this issue.
When high concentrations of AngII were added into the luminalfluid, some of the peptide could diffuse out of the lumen throughthe paracellular pathways and might act also on the basolateralreceptors. However, the [Ca2+]i increase by 10-6 mol/L luminalAngII was completely inhibited by the luminal valsartan butunaffected at all by the basolateral valsartan in WT. In addition,up to 10-5 mol/L luminal AngII did not increase [Ca2+]i in AT1AKO. These observations indicate that the effects of luminalAngII are really mediated by the luminal AT1A, but not by thebasolateral receptor. Regarding the concentration dependencyof [Ca2+]i responses to AngII in WT, 10-10 mol/L luminal AngIIdid not increase [Ca2+]i and only 10-6 mol/L luminal AngII induceda typical spike-like [Ca2+]i increase. Previously Nagami (34)reported, however, that much lower concentrations of luminalAngII can increase [Ca2+]i in isolated mouse proximal tubules.At present the reason for these discrepant results is unknown,but the difference in metabolic status of isolated tubules couldbe responsible.
Taken together with the results of our previous study aboutthe NBC regulation by AngII (11), we can now conclude that AT1A,whether expressed in the basolateral or the apical membranes,mediates the biphasic regulation of proximal transport. In contrastto our conclusion, Haithcock et al. (10), using cultured proximaltubular cells, presented the evidence that AT2 mediates theinhibition by high concentrations of AngII. Other studies oncultured proximal tubular cells also suggested a role of AT2in the acute inhibitory effect of AngII (35,36). While the reasonfor the discrepant results is not apparent, it has been shownthat the expression of AT2 in kidney is very high in the developingfetus but declines soon after birth (37,38). It could be possiblethat cultured proximal tubular cells, depending on the cultureconditions, might express a significant amount of AT2. On theother hand, we cannot exclude a possibility that AT2 may playsome physiologic roles in adult kidney. For example, AngII hasbeen shown to activate NF-B in proximal tubules through bothAT1 and AT2 (39), and AT2 could mediate long-term effects ofAngII in vivo through such a mechanism. Nevertheless, the resultsfrom our studies on isolated proximal tubules strongly argueagainst a significant role of AT2 in the acute regulation ofproximal transport by AngII.
Acknowledgments
This study was in part supported by grant 14571013 from theMinistry of Education, Science and Culture of Japan.
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Received for publication November 20, 2002.
Accepted for publication February 10, 2003.
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Top
Abstract
Introduction
are [HCO3-]L before stop-flow and in the steady-state, respectively, and k is the rate constant of [HCO3-]L decline. During rapid luminal perfusion, the perfusion pressure will act to dilate the tubular lumen because the tubule itself as well as the collection pipette offers some resistance to flow. When the perfusion pressure is suddenly reduced to zero upon stop-flow, the tubular lumen tends to partially collapse. The correction for this volume loss into the pipettes was achieved by using the decaying 440 nm (pH-insensitive) fluorescence signals as a marker of the residual luminal volume. The validity of this correction strategy was confirmed by comparison with microphotographic and video measurements as described previously (14). 
B in proximal tubules through both AT1 and AT2 (39), and AT2 could mediate long-term effects of AngII in vivo through such a mechanism. Nevertheless, the results from our studies on isolated proximal tubules strongly argue against a significant role of AT2 in the acute regulation of proximal transport by AngII. 
