Effect of isolated removal of either basolateral or basolateral CO2 on reabsor - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 24 下一页

返回列表

查看: 454153\|回复: 237	go Effect of isolated removal of either basolateral or basolateral CO2 on reabsor [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:40 |只看该作者 |倒序浏览 |打印

作者：Jinhua Zhao, Yuehan Zhou,  Walter F. Boron作者单位：Department of Cellular and Molecular Physiology, Yale University Schoolof Medicine, New Haven, Connecticut 06520 $ @" @' S2 |2 o  f! r


      " }$ O- q7 q2 i' T) m# w" D% Z- t8 @

         . X# D3 Z+ O8 D. ?) k% s) Y

         . k- n) {4 H# i$ P

                  2 V/ p, W" h0 W: o" n: u( i



      【摘要】
   The equilibrium had made it impossible to determine how isolated changes in basolateralCO 2 ([CO 2 ]) or concentration(), at a fixed basolateral pH,modulate renal or reabsorption. Inthe present study, we have begun to address this issue by measuring reabsorption( J HCO 3 ) and intracellular pH (pH i )in isolated perfused rabbit S2 proximal tubules exposed to three differentbasolateral (bath) solutions: 1 ) equilibrated 5% CO 2 /22 mM 7.40, 2 ) an out-of-equilibrium (OOE) solution containing 5% CO 2 /pH 7.40 butminimal ("pureCO 2 "), and 3 ) an OOE solution containing 22 mM 7.40 but minimalCO 2 ("pure "). Tubule lumens wereconstantly perfused with equilibrated 5% CO 2 /22 mM. Compared with the equilibratedbath solution ( J HCO 3 = 76.5 ± 7.7pmol·min - 1 ·mm - 1,pH i = 7.09 ± 0.04), the pure CO 2 bath solutionincreased J HCO 3 by 25% but decreasedpH i by 0.19. In contrast, the pure bath solution decreased J HCO 3 by 37% but increased pH i by0.24. Our data are consistent with two competing hypotheses: 1 ) theisolated removal of basolateral (or CO 2 ) causes a pH i decrease (increase) that in turnraises (lowers) J HCO 3; and 2 ) removal raises J HCO 3 by reducing inhibition of basolateral Na/HCO 3 cotransport and/or reducing backleak, whereas CO 2 removal lowers J HCO 3 by reducing stimulation of a CO 2 sensor. ) `& ~' b4 V& a4 D! x) ^
      【关键词】 bicarbonate carbon dioxide intracellular pH acidbase volume reabsorption outofequilibrium solutions
   THE KIDNEYS, ALONG WITH THE lungs, are one of the two major organ systems that regulate the acid-base balance of the extracellular fluid.A half-century ago, Brazeau and Gilman( 6 ) as well as Dorman et al.( 12 ) showed that acuterespiratory acidosis (i.e., an increase in P CO 2 thatcauses a decrease in pH) raises renal reabsorption in whole dogs. Bothgroups found that isohydric hypercapnia {i.e., a proportional increase inP CO 2 and concentration (), with no changein pH} raises absolute reabsorption to the same extent as a respiratory acidosis in whichP CO 2 is elevated to the same degree. They concluded thatan increase in CO 2, and not the accompanying decrease in blood pH,is the stimulus that elevates reabsorption. However, increasing plasma in isohydric hypercapnia alsoincreased the filtered load of andmay have had other unintended effects as well. If one expresses reabsorption as the fractionalreabsorption of the filtered load,the conclusions of the above studies are quite different: isohydrichypercapnia inhibits reabsorption.3 d! `% T5 G7 l1 O

P CO 2 also influences reabsorption in the proximaltubule, which is responsible for reabsorbing 80% of the filtered. Cogan( 11 ) found that acute respiratory alkalosis (lowering plasma P CO 2 by 20 mmHg)decreased reabsorption( J HCO 3 ) by 25% in free-flow micropunctureexperiments in rats. Moreover, Sasaki et al. ( 23 ) found that although acutemetabolic alkalosis (i.e., an increase in basolateral and pH with no change inP CO 2 ) inhibits reabsorption, raisingP CO 2 sufficiently to normalize basolateral pH (i.e., producing isohydric hypercapnia) reverses the inhibition.

The above-mentioned studies clearly demonstrate that an acute increase inP CO 2 and/or the accompanying decrease in basolateral pHleads to an increase in J HCO 3. Conversely, anacute increase in basolateral and/or the accompanying increase in basolateral pH leads to a decrease inproximal reabsorption. However,the equilibrium that interrelates CO 2,, and H had previously made it impossible to determine the importance of basolateral CO 2 per se and of per se,independently of basolateral pH, in controlling proximal reabsorption. In 1995, ourlaboratory introduced the concept of exploiting the relatively slowequilibrium CO 2 H 2 O H 2 CO 3 to generate out-of-equilibrium (OOE) solutions ( 29 ). The approachis to use a rapid-mixing technique to combine two solutions with different characteristics. With a judicious choice of the above parameters, it ispossible to generate OOE solutions with virtually any combination of [CO 2 ], , and pH, atleast within a range of several pH units that encompasses all pH values ofpathophysiological interest. In the present study, we exploit this OOEapproach to determine the extent to which reabsorption by the S2 segment ofthe rabbit proximal tubule is affected by the isolated removal of from the basolateral or"bath" solution (keeping bath P CO 2 fixed at5% and bath pH fixed at 7.4), or by the isolated removal of CO 2 (keeping bath fixed at 22 mM andbath pH fixed at 7.4). Our results, which are the first to examine an isolated change in CO 2 or on anyphysiological parameter in a vertebrate cell, show that the isolated removalof basolateral stimulates reabsorption, whereas the isolatedremoval of basolateral CO 2 inhibits reabsorption.1 ?- K; m( {2 ^1 I
  H: c4 b5 A- y
METHODS. @' v# y  V; a

Biological Preparation% x. q0 {& f# N( Y1 _0 g

The Yale Animal Care and Use Committee approved the following procedures.Pathogen-free, female New Zealand White rabbits, obtained from Covance(Denver, PA) and weighing 1.4-2.0 kg, were euthanized by a singleoverdose of 3 ml ( 20 mg) of intravenous pentobarbital sodium. The kidneyswere then removed, transferred to a dissection dish, sliced into transverse 1- to 1.50-mm-thick sections, and further dissected by hand to yield individualmidcortical 1 S2segments 2 1.5-1.7 mm in length. We perfused these tubules in a manner similar tothat originally described by Burg and co-workers( 7 ), using severalmodifications described by Baum et al.( 1 ). In particular, we doublycannulated the tubule (i.e., using holding, perfusion, and exchange pipettes)at the perfusion end of the tubule and singly cannulated the tubule (i.e.,using a holding pipette as well as a calibrated collection pipette with avolume of 55 nl) at the collection end. The collection pipette was coated with cured Sylgard (Sylgard 184 Silicone Elastomer Kit, Dow Corning, Midland,MI) and filled with liquid Sylgard (the silicone elastomer base) at the distal 50 µm of the pipette. About 80% of the tubules (depending on how theylanded on being transferred to the chamber) were cannulated and perfusedorthograde. For these, typically 0.1-0.2 mm of the proximal part of the proximal convoluted tubule was held in the perfusion pipette, and typically0.2-0.3 mm of the proximal straight tubule was held in the collectionpipette. The mean length of perfused tubules in our J V / J HCO 3 experiments (where J V is the fluid reabsorption rate), as measured with aneyepiece micrometer, was 1.33 ± 0.06 mm ( n = 16 tubules),representing the distal end of the proximal convoluted tubule. The meanluminal perfusion rate in our J V / J HCO 3 experiments was13.0 ± 0.6 nl/min. We superfused the basolateral (i.e., bath) side ofthe tubule with a solution at 37°C and flowing at 7 ml/min.  R) \- Z9 X# I+ b- ^  @8 Y% w2 a
+ J# N; G) d4 p. G% _5 @
Solutions and Experimental Protocol

Dissection and luminal perfusion. The compositions of solutions 1-4 ( Table1 ) were similar to those described by Baum et al.( 1 ), except for changesrequired in the composition of solution 4 to make it compatible withthe OOE solutions ( solutions 5 and 6 ). The tubules weredissected in Hanks' solution ( solution 1 ) at 4°C. Followingtransfer of the tubule to the chamber filled with cold Hanks' solution, weperfused the tubule lumen with a solution at 37°C buffered to pH 7.40 with5% CO 2 /22 mM ( solution 2 ) and delivered by gravity. In experiments whereby wemeasured J HCO 3, this luminal solution alsocontained dialyzed [ 3 H]methoxyinulin ( 30 µCi/ml).

Table 1. Physiological solutions& T$ }( Z' K9 s4 ]5 Q/ y: `
0 k7 P0 D/ w& `1 R' U9 I! W
Warm-up period. After establishing luminal perfusion, we initiated flow of a 37°C bath "warm-up" solution ( solution 3 )that was buffered to pH 7.40 with 5% CO 2 /22 mM. After a 20- to 30-min warm-upperiod, we switched the bath solution from solution 3 to solution4 (which was identical to our OOE solutions except that it contained both5% CO 2 and 22 mM ), orto solution 5 or 6 (the OOE solutions). In any case,coincident with the switch to the bath solution for the first data-collectionperiod, we used the calibrated collection pipette to remove and discard the fluid that had accumulated in the holding pipette.

Data collection. We allowed the tubule to stabilize in the bath solution for the first data-collection period for 5-8 min. After thisperiod, we again used the calibrated collection pipette to remove and discardthe luminal fluid that had accumulated in the holding pipette and then began aseries of three (or four) timed and calibrated collections. The first two (orthree) were subsequently analyzed for [ 3 H]methoxyinulin for use in the calculation of J V, and the third was analyzed fortotal CO 2 for use in the calculation of J HCO 3. We then usually switched to a differentbath solution ( solution 4, 5, or 6 ) for the seconddata-collection period. We repeated the entire procedure of allowing thetubule to stabilize for 5-8 min in the new solution and then performingthree (or four) timed and calibrated collections.

OOE solutions: technical considerations. We generated OOE solutions using the approach outlined in a previous paper( 29 ). In brief, we used adual-syringe pump (model 55-2222, Harvard Apparatus, South Holliston,MA) to drive two 140-ml syringes (Monoject 140 ml, Sherwood MedicalIndustries, Ballymoney, UK), one containing solution 5A (or 6A ) and the other containing solution 5B (or 6B )( Table 1; see Fig. 1, A and B ). The A and B solutions each flowed at 3.5 ml/min, fora total flow of 7 ml/min. Separate lengths of Tygon tubing ( -in. outerdiameter x. innerdiameter, Norton Performance Plastics, Akron, OH) which has a relatively lowpermeability to CO 2, carried the outflow of each syringe to one oftwo inlets of paired computer-actuated five-way valves (Eagle P/NE4-1PP-00-000, Clippard Instrument Laboratory, Cincinnati, OH). Inone position, these two valves, which have zero dead space, sent one pair ofsolutions (e.g., solutions 5A and 5B ) toward the chamber andan alternate pair (e.g., solutions 6A and 6B ) to a waste recepticle; in the alternate position, the two valves reversed thedestinations of the two pairs of solutions. We actually used a series of foursuch valve pairs, connected to one another in daisy-chain fashion, so that wecould switch among up to five bath solutions. All bath solutions, includingthe ones that were not OOE, were delivered as described above by a pair ofsyringes driven by a separate dual-syringe pump.$ ^7 ?* [/ a5 h% _: C/ R8 f& k
9 V5 D0 F4 q* ?5 |8 T# Z! N
Fig. 1. Generating out-of-equilibrium (OOE) solutions. A : arrangement ofsyringes, valve assembly, stainless-steel tubule, mixing T, and mesh. Theportions of the tubing not indicated as stainless steel are made of Tygon. Thechemical reactions indicate the decay of the OOE solutions as they flow downthe tubing after mixing. B : side view of isolated perfused tubuleexposed to an OOE solution. C : concentrations of components. Left : compositions of the solutions inside the 4 syringes forgenerating solutions 5 ("pure CO 2 ") and 6 ("pure "). Middle : compositions of solutions 5 and 6 at theinstant of mixing. Right : compositions of solutions 5 and 6 after the solutions have passed the tubule, 200 ms aftermixing.
; j- ?! |$ w' d. J
The two outputs of the valve assembly, carrying solutions5A 5B or 6A 6B to generate OOE solutions, orcarrying two identical streams of either solution 4 or solution7, were carried by separate lengths of Tygon tubing to separate 22-cmlengths of 15-gauge stainless steel tubing, which, in turn, were enclosed within a jacket that was perfused with water by a circulating bath at 38°C. The downstream ends of the two lengths of stainless steeltubing were connected to 5-cm lengths of Tygon tubing, which carried thenow-warmed pair of solutions to the two arms of a polypropylene T( x x. Tee P/N6365-77, Cole-Parmer Instrument, Vernon Hills, IL). The base of the T was connected to a length of Tygon tubing stuffed with nylon mesh (35-µmnylon thread, Small Parts, Miami Lakes, FL) to generate turbulence and therebyensure adequate mixing of the two components of an OOE solution. The oppositeend of the tubing was situated at the entrance to the slot of the chamber (seebelow); the total volume of the system from the point of mixing at the middleof the T to the beginning of the chamber slot was 14 µl. From within thevalve assembly to the T, the two members of a pair of pieces of Tygon tubingwere of identical length. This precaution ensured that, following a switch inthe valve assembly, each member of the solution pair reached the T at the sameinstant.* r  x* t. ]5 N. _1 W
+ M/ o6 f, ~& i& {% W% w! j
OOE solutions: theoretical considerations. Our OOE approach exploits the slow, first equilibrium in the following reaction sequence' D3 V% R) b- S
5 G+ W$ I1 b" V. D9 y# U
Thus we have substantial and individual control over the CO 2 concentration ([CO 2 ]). However, because the reaction in Eq.2 is very rapid, we cannot independently control theH 2 CO 3 concnetration ([H 2 CO 3 ]),which at any instant should equal.Similarly, because the reaction in Eq. 3 is also very rapid, wecannot independently control concentration (), which at anyinstant should equal K 3.8 A! d- L1 V  H, x' Z# g
6 F; I3 X" `" J3 f5 p- |
Figure 1 C summarizes the concentrations of CO 2, H 2 CO 3,, and as computed from Eqs. 1-3. Take the pure CO 2 solution, for example. Asindicated by the top half of the left column on Fig. 1 C, solution5A contained 2,210 µM CO 2 (which corresponds to agas mixture of 10% CO 2 ) at a pH of 5.40 (2 pH units below thetarget pH of 7.40). Because the p K of the overall equilibrium is 6.1, the  in solution 5A was 441 µM. The top half of the middle column of Fig. 1 C shows, at the idealized instant of perfect mixing, theresult of combining solutions 5 and 6 in a 1:1 ratio. ThisOOE pure CO 2 solution contained 1,105 µM CO 2 (whichcorresponds to a gas mixture of 5% CO 2 ) and 223 µM; this is 1% of the value in anequilibrated 5% CO 2 /22 mM solution. In principle, we couldhave reduced the contaminating even further by lowering the pH of solution 5A below 5.40. The bottom half of Fig.1 C provides a similar analysis for the pure solution. Because solution6B contained 44 mM total at apH of 9.40 (2 pH units above the target pH of 7.40), the [CO 2 ] in solution 6B was 19.6 µM (which corresponds to a gas mixture of0.09% CO 2 ). Thus mixing this solution in a 1:1 ratio with solution 6A initially yielded an OOE pure solution containing nearly 22 mM and 9.8 µM CO 2 (which corresponds to a gas mixture of 0.04% CO 2 ); this [CO 2 ] is 2 /22 mM solution.Again, we could have reduced the contaminating [CO 2 ] even furtherby raising the pH of solution 6B above 9.40.* K2 @2 h7 g9 x! D

Our design criteria for the pure CO 2 OOE solution ( solution 5 ) were that solution 5B should be heavily buffered but have apH just high enough (i.e., 7.55) to drive the pH of the OOE solution to 7.40.This combination of design features yields an OOE solution, as measured with aP CO 2 electrode (MI 720, Microelectrodes, Londonderry,NH) at the end of the Tygon tubing that connects to the chamber, that isindistinguishable from 5%. In our experience, attempting to generate a pureCO 2 solution by using a solution B with an extremely highpH (e.g., pH 10) yielded an OOE solution with a [CO 2 ] markedlylower than the target value. We presume that rapid mixing of solutionsA and B creates short-lived microscopic pockets with a high[CO 2 ] (contributed by solution A ) and a high[OH - ] (contributed by solution B ) that allows theuncatalyzed reaction to consume CO 2 at a high rate.

Our design criteria for the pure solution ( solution 6 )were that solution 6A should be heavily buffered but have a pH justlow enough (i.e., 6.99) to drive the pH of the OOE solution to 7.40. Thisapproach yields an OOE solution that, when assayed as described in thepreceding paragraph, has a P CO 2 indistinguishable fromzero. Attempts to generate a pure solution by using a solution A with an extremely low pH (e.g., 5.00)yielded an OOE solution with a [CO 2 ] markedly higher than zero. Wepresume that short-lived microscopic pockets with a high (from solution B ) and alow pH/high [H ] (from solution A ) allowed the reaction to raise [H 2 CO 3 ] to such high levels that theuncatalyzed reaction H 2 CO 3 CO 2  H 2 O produces CO 2 at a high rate.
8 S2 I" w$ s8 d* l9 V; E
Because of the considerations discussed in the two previous paragraphs, wesuspect that it might be challenging to generate pure CO 2 solutionsat an extremely alkaline target pH or pure solutions at an extremely acidictarget pH.6 |6 r, }, E0 \3 r
+ Z+ Z3 U& Z  i' q8 E
Finally, it should be noted that we employed OOE solutions only in thebath. Although it is theoretically possible to generate OOE solutions for thelumen, the mixing of the A and B solutions would beextremely challenging, and the relatively slow transit of the fluid down thelumen would permit more extensive equilibration than we observe in thebath.

The chamber. The chamber (see Fig. 1 B ), which wasmachined out of polycarbonate, contained a channel 14-mm long x 2.5-mm wide by 2.5-mm high. The bottom of the slot was formed by a coverslip. Aboutone-fourth of the way (measured from the solution inflow) along this slot wasthe perfused tubule, with its long axis parallel to the axis of bath flow. Thechamber sat atop a heating plate (series 20 chamber and platform with integral TS70B thermistor, Warner Instruments, Hamden, CT) that was clamped to atemperature of 41°C. The combination of the prewarmed solution and theheating plate yielded a bath temperature of 37.5°C (confirmed with aminiature electronic temperature probe Digi-Sense with a type-K thermocouple,Cole Parmer). A length of Tygon tubing from the T (see above) entered the chamber through a port that was colinear with the long axis of the slot. As itterminated at the entrance to the slot, the Tygon tubing had an inner diameterof 1.6 mm, which was modestly less than the width of the chamber slot (2.5 mm)to promote laminar flow along the slot and minimize the formation of deadzones. Such dead zones would have permitted the equilibration of the bathsolution within these zones and, to some extent, thwarted our goal ofsurrounding the tubule with an OOE solution. At the opposite end of the slot,the bath solution was continuously sucked away. The height of the bathsolution within the slot was 1.2 mm.

We calculate that the time required for the newly mixed solution (7 ml/min)to travel from the middle of the T to the tubule (total volume = 14 µl forthe tubing 10 µl for 1/4 of the chamber slot = 24 µl) was 200 ms.Given an extracellular CO 2 concentration([CO 2 ] o ) of 1,105µM 3 for solution 5 at the instant of mixing (see middle column of Fig. 1 C, top portion) and a rate constant of 0.08 s - 1, thereaction CO 2 H 2 O H 2 CO 3 ( Eq. 1 ) would consume CO 2 and produceH 2 CO 3 at the rate of 88 µM/s. Thus after 200 ms(see right column of Fig.1 C, top portion), the preceding reaction wouldconsume 17.7 µM CO 2 or 1.6% of the original 1,105 µMCO 2. Simultaneously, the reaction ( Eq. 2 ) would produce 17.7 µM, which represents 0.08% of the present in an equilibrated 5%CO 2 /22 mM solution. Asa result, pH would fall by a trivial amount, [CO 2 ] would fall to1,087 µM CO 2 (which corresponds to a gas mixture of 4.92%CO 2 ), and  would riseby a trivial amount.

Conversely, given an extracellular concentration( ) of 22 mMfor solution 6 at the instant of mixing, the initial[H 2 CO 3 ] would be 2.765 µM (see middle columnof Fig. 1C, bottom portion). Because the reaction H 2 CO 3 CO 2 H 2 O ( Eq. 1 ) has a rate constant of 32s - 1, the preceding reaction would consumeH 2 CO 3 and produce CO 2 at the rate of 88µM s - 1. Thus, after 200 ms (see right column of Fig. 1 C, bottom portion), the preceding reaction would produce 17.7 µMCO 2, or 1.6% of that contained in an equilibrated 5%CO 2 /22 mM solution. Asa result, pH would rise by a trivial amount, would fall by a trivial amount,and [CO 2 ] would rise to 27.5 µM (which corresponds to a gasmixture of 0.12% CO 2 ). In a parallel series of experiments, we useda pair of liquid-membrane pH-sensitive electrodes to behave as adouble-barreled P CO 2 microelectrode( 28 ). We used this combinationto monitor P CO 2 in the chamber slot at room temperatureunder conditions mimicking those in the kidney-tubule experiments. Whilegenerating a pure solution, wefound that the [CO 2 ] in the chamber slot was indistinguishable fromzero (not shown). However, this gratifying result was achieved only afteremployment of the above considerations in chamber design.

Osmolality and pH of solutions. The osmolality of all solutions was measured using a vapor-pressure osmometer (model 5100C, Wescor, Logan,UT). The osmolality was adjusted, if necessary, to 300 ± 2mosmol/kgH 2 O by adding either water or NaCl. The pH was adjusted tothe value indicated in Table 1 using either HCl or NaOH. All solutions were made using water filtered througha Milli-Q biocel system (Millipore, Bedford, MA).# @  H, q2 J8 E* D- k; J

Measurement of J HCO 3 and J V
, C3 d" [6 G; v# ~  X
We measured J HCO 3 and J V using an approach similar to that used by McKinney and Burg( 16 ). We assayed totalCO 2 in aliquots of the perfusate and collected fluid using a WPINanoFlo device (World Precision Instruments, Sarasota, FL) to measure the -dependent decrease in thefluorescence at 340 nm of NADH as phospho enol pyruvate carboxylase(from "plant") and malate dehydrogenase (from porcine heart),respectively, catalyzed the following two reactions( 13, 26 )

We purchased the above reagents as a kit (Diagnostic Kit 132-A, Sigma-Aldrich,St. Louis, MO). Given the fluid collection rate (V i ), the tubule length ( L ), the concentration of total CO 2 in the luminalperfusate ([T CO 2 ] o ) and collected luminalfluid ([T CO 2 ] i ), and J V (see below), we used the following equation to compute J HCO 3+ r8 {& e, {2 M1 ~! {9 W
" J. I6 U& c  t$ x% d* \/ O9 H
We report J HCO 3 in picomoles divided by theproduct of minutes and millimeters tubule length.

We computed J V from V i, L, and theconcentration of [ 3 H]methoxyinulin in the perfusate (C o )and collected luminal fluid (C i )

We report J V in nanoliters divided by the product ofminutes and millimeters tubule length. We accepted the data only if J V was positive and if the computed concentration of reabsorbed NaHCO 3 (i.e., the ratio J HCO 3 / J V ) was less than 160 mM. 4

Measurement of Intracellular pH$ K) r2 G9 N5 b$ E) E  k
. P1 ?3 x  `  k* `/ R& u5 L
We calculated intracellular pH (pH i ) from the fluorescence excitation ratio of BCECF using the approach outlined previously forosteoclasts ( 21 ). We loadedperfused tubules with the dye by exposing them from the bath at roomtemperature for 5 min to solution 7 containing 10 µM ofBCECF-AM (B-1170, Molecular Probes, Eugene, OR). The microscope was a ZeissIM-35 inverted microscope, equipped with a x 40/numerical aperture 0.85objective and apparatus for epiillumination. The light source in thefluorescence experiments was a 100-W tungsten halogen lamp. Using a system ofdual filter wheels (Ludl Electronic Products, Hawthorne, NY), one wheelcarrying chromic filters and the other carrying neutral-density filters, wealternately excited the entire field with light in wavelengths of 495 ± 5 and 440 ± 5 nm (Thermo Oriel, Stratford, CT), using theneutral-density filters to equalize, as nearly as possible, the intensity ofincident light at the two wavelengths. The emitted fluorescent light, afterpassing through a 510-nm long-pass dichroic mirror and a 530-nm long-passfilter in the filter cube beneath the turret, was amplified by an imageintensifier (KS-1381 intensifier, Video Scope, Dulles, VA), before it was captured with a charge-coupled-device camera (CCD 72, Dage M.T.I., MichiganCity, IN).
/ u+ G  k/ I" r0 x
To limit both photobleaching of the dye and photodynamic damage of thetubule cells, we limited illumination to 370 ms for the 490-nm light(producing an I 490 image), followed immediately by 370 ms for the 440-nm light (producing an I 440 image). This pair of excitations was repeated at intervals varying from 6 to20 s; between the 490- and 440-nm excitations, we kept the tubule in the darkby closing the shutter on the filter wheel. Each cycle of excitations had thefollowing protocol.The filter wheel was rotated, under computer control, toput the 490-nm filter in the light path. With the shutter of the filter wheelstill closed, we snapped an image to be used for background subtraction andthen opened the shutter. After a delay of 100 ms to allow for stabilization ofthe shutter, we collected and averaged four successive video frames using animage-processing board (DT3155, Data Translation, Marlboro, MA). The shutter was then closed and, after storage of the averaged I 490 image, the 440-nm filter was rotated into position and the I 440 image was obtained in the same way. Finally, wesubtracted the average pixel intensity of the matched background image (seeabove) from the I 490 and I 440 images.Software developed in our laboratory, using the Optimas (Media Cybernetics,Silver Spring, MD) platform, controlled all aspects of data acquisition andreal-time storage to the hard disk of an Intel-based computer runningMicrosoft Windows 98SE. This software also allowed us to monitor I 490 / I 440 fluorescence-excitationratios for selected areas of interest during the experiments. After theexperiment, we transferred the data to a CD-ROM for later analysis usingOptimas-based software. For the analysis, we outlined two areas of interest, each of which typically represented 9% of the tubule length. In BCECF-loadedtubules, the mean I 490 signal was typically 3,000-fold greater than the I 490 signal in tubulesnot loaded with BCECF. The sum of the I 490 values of thepixels in the area of interest was divided by the sum of the corresponding I 440 values. The resulting ratio is strongly dependent onpH i but relatively insensitive to other factors, such as dyeconcentration.0 t% M6 o; V9 [" M
% R# M% Y2 s$ r7 X( j5 d
We converted the I 490 / I 440 ratiosto pH i values using the high-K /nigericin technique ofThomas et al. ( 27 ), asmodified by Boyarsky et al.( 4 ), to permit a one-pointcalibration at pH i 7.00 for each experiment. At the end of eachexperiment, we introduced into the bath a pH 7.00 high-K /nigericinsolution (100 mM KCl, 32.2 mM HEPES-free acid, 1 mM CaCl 2, 1.2 mMMgSO 4, titrated to pH 7.00 with 32.8 mM N -methyl- D -glucamine) that drives pH i toward7.00. Potential limitations of this approach are discussed elsewhere( 8 ). The I 490 / I 440 ratios of the entireexperiment were normalized by dividing them by the I 490 / I 440 ratio corresponding topH i 7.00, and we then calculated pH i using the following equation ( 4 )
; t$ ^3 n; d$ g& A6 T% c
The values of p K and b were determined from a separateseries of experiments in which we exposed the basolateral sides of isolatedperfused tubules to a series of solutions containing 10 µM nigericin at 10different pH values (pH 5.8-8.5 in increments of 0.3 pH units), alwaysincluding pH 7.00. The plot of( I 490 / I 440 )/( I 490 / I 440 ) pH = 7 vs. pH i (not shown), obtained from a total of 79 fluorescence measurements (1 measurement·pHvalue - 1 ·tubule - 1, not including measurements at pH i 7.00) on a total of 19 tubules, was a typical pH titration curve. The data were fitted by the above equation,which forces the best-fit curve to pass through unity at pH i =7.00, using a nonlinear least-squares method. The best-fit values, which weused in the above calibration equation for each experiment, were p K =7.08 ± (SD) 0.02 and b = 1.326 ± (SD) 0.029.

Because the nigericin used in the calibration can stick to the tubing andvalves and later dissociate and enter nominally nigericin-free solutions inlater experiments ( 2, 22 ), at the end of eachpH i experiment we routinely flushed ethanol (70% in water) and thenwater (filtered through a Milli-Q biocel system) through the tubing and valvesthat carried the nigericin to the chamber, as well as the chamber itself./ [$ u7 g3 I* [9 ]
' A3 Q& ]3 O& c) q: X  r
Data Analysis
1 B( P' M, ~% l/ h2 N" Q
Statistical analyses were performed using the Analysis Toolpack ofMicrosoft Excel. Two sets of data were considered significantly different ifthe P value of the paired or unpaired t- test was number of tubules( n ) from which it was calculated.7 n) s8 U0 G0 H! s- k

RESULTS

Control J HCO 3 and J V Studies
, R. ~/ V  L4 N' c" z3 C
In the J HCO 3 / J V experiments described in the next two subsections, we constantly perfused thelumen of the tubule with an equilibrated 5% CO 2 /22 mM at pH 7.40( Table 1, solution 2 )containing [ 3 H]methoxyinulin. We usually collected luminal samplesfor data analysis during two periods, each characterized by different bathsolutions that were delivered from different pairs of syringes, driven bydifferent syringe pumps. To verify that these two data-collection periods areequivalent, we performed control experiments in which we delivered thestandard equilibrated solution ( solution 4 ) for each of the two data-collection periods,first from one pair of syringes driven by one pump, and then from another pairof syringes driven by another pump. As summarized in Fig. 2, we observed nosignificant difference between the two data-collection periods for either J HCO 3 or J V.. I% c& N- d. ~1 i
+ Z7 k  I5 H* O8 \" l1 f2 k  g  H
Fig. 2. Control reabsorption( J HCO 3; A ) and fluid reabsorption rate( J V; B ) studies. Each experiment consisted of 2collection periods. During each, solution 4 (standard; equilibrated5% CO 2 /22 mM ) flowedthrough the bath. However, in the 2 periods this solution was delivered from 2different pairs of syringes, driven by 2 different syringe pumps, to mimic theprotocol used in Fig. 3. Valuesare means ± SE, with nos. of tubules in parentheses.The differencesbetween the 2 mean values for J HCO 3 ( P = 0.48) and between the 2 mean values for J V ( P =0.23) were not statistically significant (paired, 2-tailed t -test).8 w% S9 u( `, q9 C6 m5 b# u

Fig. 3. Effect on J HCO 3 ( A ) and J V ( B ) of the isolated removal of either bath or bath CO 2. In both A and B, the bath solutions were solution 5 (pureCO 2 OOE; hatched bars), solution 4 (standard; equilibrated5% CO 2 /22 mM; openbars), and solution 6 (pure OOE; crosshatched bars). Valuesare means ± SE, with nos. of tubules in parentheses. The differencebetween each of the hatched and crosshatched bars and the corresponding openbar is statistically significant in unpaired, 2-tailed t -tests(* P P
8 F) P; A, p$ V' w% @
Effect on J HCO 3 and J V ofIsolated Removal of Basolateral

A comparison of the open and hatched bars on Fig. 3, A and B, illustrates the effect of using the OOE approach toremove basolateral while holdingbath [CO 2 ] fixed at 5% and holding bath pH fixed at 7.40. Comparedwith those exposed to the equilibrated 5% CO 2 /22 mM solution at pH 7.40 (open bars),tubules exposed to the corresponding pure CO 2 solution (hatchedbars) had a mean J HCO 3 that was 25% higher(96.5 vs. 76.5pmol·min - 1 ·mm - 1 ). J V was 61% higher (1.03 vs. 0.64nl·min - 1 ·mm - 1 ). Viewed differently, the presence of basolateral reduces the reabsorption of both and fluid.. }7 A, ]3 L: M- b- K) o
! e. C% r$ g4 _/ `% X
Considering only the data for the equilibrated 5% CO 2 /22 mM solution at pH 7.40 (open bars in Fig. 3, A and B ), our mean J HCO 3 valueof 76.5pmol·min - 1 ·mm - 1 is similar to the value of 52.3pmol·min - 1 ·mm - 1 reported by Baum et al. ( 1 ) formidcortical and juxtamedullary proximal convoluted tubules. Similarly, ourmean J V value of 0.64nl·min - 1 ·mm - 1 is similar to the value of 0.72nl·min - 1 ·mm - 1 reported by Baum et al. and is also similar to the value of 0.5nl·min - 1 ·mm - 1 obtained by Quigley et al.( 20 ) on superficial S2segments of rabbit proximal tubules.5 {( E0 S2 [4 i( W
" m& g& Q  l3 |5 T) K/ S+ P
Effect on J HCO 3 and J V of Isolated Removal of BasolateralCO 2; O7 W" u. k0 M4 J+ A

A comparison of the open and crosshatched bars in Fig. 3, A and B, illustrates the effect of using the OOE approach to remove basolateral CO 2 while holding bath fixed at 22 mM and holding bathpH fixed at 7.40. Compared with tubules exposed to the equilibrated 5%CO 2 /22 mM solution atpH 7.40, those exposed to the corresponding pure solution had a J HCO 3 that was nearly 40% lower (48.4 vs. 76.5pmol·min - 1 ·mm - 1 ). J V was nearly 25% lower (0.49 vs. 0.64nl·min - 1 ·mm - 1 ). Viewed differently, adding basolateral CO 2 to a pure solution enhances the reabsorptionof both and fluid.

Effect on pH i of Isolated Removal of EitherBasolateral orCO 2' w" K5 E+ r8 `9 d) M# w

One would expect that tubules exposed to the different bath solutionssummarized in Fig. 3 would havedifferent values of pH i. Because such changes in pH i could conceivably affect various solute transporters and thereby alter J HCO 3 and/or J V, weexamined the effect on pH i of switching the bath solution from 5%CO 2 /22 mM at pH 7.40 tothe corresponding OOE solutions lacking either or CO 2. Figure 4 A shows anexperiment in which we used BCECF to monitor pH i in cells of anisolated perfused S2 segment. Initially, the luminal and bath solutions werebuffered with HEPES ( Table 1, solution 7 ). In a total of 38 tubules under these conditions, themean initial pH i was 7.19 ± 0.03. For two reasons, thisvalue is substantially higher than the values of 6.89-6.92 previouslyreported by this laboratory for rabbit S3 segments ( 9, 17 ). First, meanpH i values steadily increase from the S3 to S2 to S1 segments( 14 ). Second, the luminalsolution in the present study contained lactate, which, presumably like lactate in the salamander proximal tubule( 24 ) and acetate in the rabbitS3 segment ( 18 ), produces aintracellular alkalinization as it enters the cell with Na acrossthe apical membrane and exits with H across the basolateralmembrane.
! `+ p. g  N9 ]; L
Fig. 4. Effect on intracellular pH (pH i ) of the isolated removal ofeither bath or bathCO 2. A : representative experiment. See text for discussionof points a-h. B : data summary. Values are means ± SE, withnos. of tubules in parentheses. The difference between each of the hatched andcrosshatched bars and the corresponding open bar is statistically significantin unpaired, 2-tailed t -tests (** P

Switching the lumen to 5% CO 2 /22 mM ( solution 2 ) in Fig. 4 A caused anabrupt and sustained decrease in pH i ( segment ab ),reflecting the influx of CO 2 across the apical membrane, theintracellular reaction,and the continuous exit of acrossthe basolateral membrane via the electrogenic Na-HCO 3 cotransporter( 3 ). The mean pH i under these conditions was 6.99 ± 0.02 ( n = 38).
# t7 F( H1 a) A1 M$ O& |6 e2 D/ R
At point b we added equilibrated 5% CO 2 /22 mM ( solution 4 ) to the bathin the continued presence of luminal. Onone hand, we expect this maneuver to cause an abrupt pH i decreaseas the basolateral influx of CO 2 leads to the intracellulargeneration of H and and to cause a sustained pH i decrease as the elevated intracellular ( ) promotesthe efflux of through theelectrogenic Na-HCO 3 cotransporter. On the other hand, based onprevious results from this laboratory in rabbit S3 segments( 9, 10, 17 ), we expect the addition ofbasolateral tocause an increase in pH i for two reasons. First, the increase inbath  would tend to slow net efflux across the basolateralmembrane. Second, basolateral CO 2 and/or stimulates apical Na/H exchangeand H pumping. In our experiments, the alkalinizing tendenciesdominated ( bc ) as the mean pH i increased from 6.97 ± 0.02 to 7.09 ± 0.04 ( n = 18; P paired 2-tailed t -test). The latter value is also summarized by theopen bar in Fig. 4 B.The absence of a transient, CO 2 -induced acidification during bc in Fig. 4 A probably reflects the limited time resolution of data acquisition.
1 n# Q$ `5 F, V" X
Removing fromthe bath in Fig. 4 A caused a transient pH i increase due to the basolateral efflux ofCO 2, followed by a relaxation of pH i ( cd ). At point d, we switched the bath to the pure CO 2 solution( solution 5 ). The abrupt pH i decrease is the consequenceof the CO 2 influx and formation of H and. We expect the new steady-statepH i to depend on the balance between enhanced efflux (reflecting the increased ) viaelectrogenic Na-HCO 3 cotransport (which would tend to lower pH i ) and enhanced H extrusion if it is basolateralCO 2 that stimulates apical Na/H exchange and H pumping.Under the conditions of our study, the acidifying influences dominated( de ) as the mean pH i fell from 7.02 ± 0.03 to 6.90± 0.03 ( n = 17; P t -test). The latter condition, with one additional data point, isalso summarized by the hatched bar in Fig.4 B. Note that it is under this pure CO 2 condition that reabsorption wasmaximal (see Fig.3 A ).
3 k; H. \0 V' Q
Removing pure CO 2 from the bath( Fig. 4 A ) caused arebounding pH i increase ( ef ). At point f, weintroduced the pure solution( solution 6 ) to the bath. We expect the increase in bath to inhibit efflux via electrogenicNa-HCO 3 cotransport, or even to produce a net influx of. Indeed, we observed a sustainedpH i increase ( fg ) as the mean pH i rose from7.05 ± 0.06 to 7.33 ± 0.13 ( n = 6; P 0.02, paired 2-tailed t -test). The latter condition, with one additional data point, is also summarized by the crosshatched bar in Fig. 4 B. Note that itis under this pure condition that reabsorption was minimal (see Fig. 3 A ).

Finally, removing the pure solution from the bath causes pH i to drift toward its initial value( gh ).
" a5 V" v; _& G1 F' p% h/ X7 h2 f
DISCUSSION

The pioneering work of others on the impact of acid-base disturbances onrenal acid-base transport was necessarily confined by equilibrium, which made it impossible to change just one parameter in theHenderson-Hasselbalch equation. In the present experiments, we have appliedOOE technology to renal tubules and have made the first observations of theeffects of the isolated basolateral removal of either CO 2 or on J HCO 3 and pH i. We found thatbasolateral [CO 2 ] per se and basolateral per se are critical parametersfor regulating both transepithelial acid-base transport and pH i.The isolated removal of from thebasolateral solution, at a fixed [CO 2 ] and pH, raises J HCO 3 and lowers pH i. The isolatedremoval of basolateral CO 2 from the basolateral solution, at afixed  and pH, produces theopposite effects.% P. w% k% n7 @5 B0 ~) s6 q

Removing Basolateral Stimulates Reabsorption1 W1 X, b5 a3 d: m8 q

As shown in Fig. 5, which isa replot of the relationship between the J HCO 3 data in Fig. 3 A andthe pH i data in Fig.4 B, one way of interpreting the data from the presentstudy is that J HCO 3 depends mainly onpH i and that removing basolateral enhances reabsorption by loweringpH i. Although the present data are consistent with this hypothesis,the hypothesis suffers from three weaknesses.

Fig. 5. Relationship between J HCO 3 andpH i. The pH i data are from Fig. 4 B, and the J HCO 3 data are from Fig. 3 A.0 a2 R3 D. I* A' J, q3 a

First, the pH i -dependency model requires that decreasingpH i from 7.1 to 6.9 would increase apical H extrusionvia the Na/H exchanger and H pump (see Fig. 6 ) by 50%, whereas, at least in the S3 segment of the rabbit proximal tubule, actual data show thatacid extrusion is remarkably insensitive to pH i changes in theabove pH i range, particularly in the absence of ( 9, 10 ).
7 H+ X$ Q4 ?* f
Fig. 6. Model of reabsorption by theproximal tubule. Although the CO 2 sensor is shown as facing outwardfrom the basolateral membrane, the sensor could face inward from thebasolateral membrane, or it could be in the cytosol near the basolateralmembrane.

Second, the pH i -dependency model requires that decreasingpH i from 7.1 to 6.9 would increase the basolateral efflux of by 50% because changes inbasolateral efflux must, in thesteady state, parallel changes in apical H extrusion. Although thepH i dependence of the electrogenic Na-HCO 3 cotransporter(NBC) in the proximal tubule is unknown, one would expect that, if anything,lowering pH i per se would inhibit NBC, just as loweringpH i inhibits exchange ( 5, 19, 25 ). For example, loweringpH i from 7.1 to 6.9 at a fixed extracellular [CO 2 ] of 5%would lower the concentration of intracellular, the ostensible substrate for NBC,from 11 to 7 mM.
( `5 ?# S; p+ B- H
Finally, it is important to address the mechanism by which removing basolateral lowers pH i.Imagine that we start with equilibrated in thebath. Removing bath should enhancebasolateral efflux via NBC,thereby lowering pH i. Note that enhancing the basolateral efflux of is tantamount to enhancing reabsorption. According to thisanalysis, the fall in pH i is the result of enhanced reabsorption rather than the causeof it.
8 }" \) U* J6 @, I
How then might the removal of basolateral enhance reabsorption? One possibility isthat the reduction in basolateral slows or even reverses thebackflux of through the tightjunctions from the bath to the lumen. Another possibility, which is notmutually exclusive, is that the reduction in basolateral  enhances the net offloading of at the extracellular face ofNBC./ S) K" z; F: F' b/ Q

Removing Basolateral CO 2 Inhibits Reabsorption' k  K" P4 |$ d" S, ~" J+ r
; @4 I4 D0 J# m* l4 M3 Z
Given the relationship between J HCO 3 andpH i summarized in Fig.5, the most tempting explanation for why removing basolateral CO 2 inhibits reabsorption by 37% is that the removal of CO 2 causespH i to increase, which in turn inhibits apical H extrusion. The present data do not rule out this hypothesis. However, if thehypothesis is true, then we must explain how the pH i increase alsoinhibits basolateral efflux,because as the cell approaches the new steady state, apical H extrusion and basolateral effluxmust somehow fall in parallel. We expect that the rise in pH i perse should, if anything, increase the basolateral efflux of. Moreover, at a fixedintracellular [CO 2 ] ([CO 2 ] i ), a rise inpH i ought to increase, whichshould further increase the basolateral efflux of. On the other hand, the decreasein bath [CO 2 ] should lead to a fall in[CO 2 ] i, which would mitigate the rise in and therebyperhaps slow efflux. Thus, for thepH i hypothesis to be correct, removing bath CO 2 mustindeed lower, and theinhibitory effect on NBC of lowering per se mustoverwhelm the stimulatory effect of raising pH i per se.
. [% \6 R+ [# s* Z- L
As shown in Fig.4 B, removing bath CO 2 caused pH i torise from 7.09 to 7.33, and we calculate that may havesimultaneously fallen from 10.8 to 9.4mM. 5 Even if the K m for intracellular were 10 mM, a decrease in from 10.8to 9.4 mM would decrease the transport rate by only a trivial amount. Anadditional complication is that preliminary data indicate that when NBC operates with a Na : stoichiometry of 1:2, it transports rather than ( 15 ). Thus it is likely thatNBC in the proximal tubule, which appears to function with Na : stoichiometry of 1:3, transportsone Na  , one, and one. We calculate that the removal ofbath CO 2 actually causes to risefrom 6.7 to 10.1µM, 6 and predictthat, if anything, this rise in wouldstimulate NBC. In other words, for the pH i hypothesis to becorrect, the anticipated inhibitory effect of decreasing by 13%would have to outweigh the anticipated stimulatory effects of raising pH i by 0.24 and raising by 50%and produce a net 37% decrease in electrogenic Na-HCO 3 cotransport.
! X/ e/ {: m% G  S8 `1 s8 d0 n+ s
As an alternative hypothesis, we suggest that basolateral CO 2 per se may directly enhance the machinery of reabsorption, stimulating inparallel both apical H extrusion and basolateral efflux. According to thishypothesis, changes in steady-state pH i caused by removing or adding back CO 2 are the consequence, rather than the cause, ofaltered rates of H and transport. This CO 2 -triggering hypothesis is consistent withearlier observations that the application of basolateral, butnot of luminal,causes an increase in both steady-state pH i ( 17 ) and H extrusion rates ( 9, 10 ). If this hypothesis iscorrect, then it would have been the CO 2, and not the, present in the earlierequilibrated solutions that caused proximal tubule steady-state pH i andH extrusion rates to increase. Further experiments will berequired to distinguish between the pH i hypothesis and thealternative CO 2 -sensor hypothesis for producing the observedchanges in J HCO 3.
' ]4 X, f" y- ^. g" @
DISCLOSURES

This work was supported by National Institutes of Health Program ProjectGrant PO1-DK-17433. J. Zhao was supported by fellowships from the AmericanHeart Association and the American Lung Association.6 a, d% S  Z, K8 `9 w! [5 z

ACKNOWLEDGMENTS4 Z( ?) _  {) ~

We thank Dr. Raymond Quigley for advice in measuring reabsorption and fluidreabsorption rates. We are indebted to Duncan Wong for computerassistance.) w/ \! u3 L' j1 e/ w/ S' {2 O
      【参考文献】
Baum M, QuigleyR, and Quan A. Effect of luminal angiotensin II on rabbit proximalconvoluted tubule bicarbonate absorption. Am J Physiol RenalPhysiol 273:F595-F600, 1997.
. J" u% \% p8 T" x/ @/ _, `3 t0 L

Bevensee MO,Bashi E, and Boron WF. Effect of trace levels of nigericin onintracellular pH and acid-base transport in rat renal mesangial cells. J Membr Biol 169:131-139, 1999.

& a" b& {2 \7 i1 E
/ z" ^1 L! G' q1 b
Boron WF andBoulpaep EL. Intracellular pH regulation in the renal proximal tubule ofthe salamander: basolateral transport. J Gen Physiol 81:53-94, 1983.9 c, N2 o& l+ u

  m- q% D# F) [% z6 w- s" I

Boyarsky G,Ganz MB, Sterzel B, and Boron WF. pH regulation in single glomerularmesangial cells. I. Acid extrusion in absence and presence of HCO 3. Am J Physiol Cell Physiol 255:C844-C856, 1988.

Boyarsky G,Ganz MB, Sterzel B, and Boron WF. pH regulation in single glomerularmesangial cells. II. Na-dependent and -independentCl - -HCO 3 exchangers. Am J Physiol CellPhysiol 255:C857-C869, 1988.  Z& C* U# p, X; E3 U1 Z
6 I  q  e/ g8 r  \' h' W- g4 l

Brazeau P andGilman A. Effect of plasma CO 2 tension on renal tubularreabsorption of bicarbonate. Am J Physiol 175: 33-38,1953.

Burg M,Grantham J, Abramow M, and Orloff J. Preparation and study of fragments ofsingle rabbit nephrons. Am J Physiol 210: 1293-1298,1966.) D* P% v8 I* `( {6 _9 U# }

5 u- `5 k/ m8 K
: q* Z" d. l( C0 H' ^
Chaillet JR andBoron WF. Intracellular calibration of a pH-sensitive dye in isolatedperfused salamander proximal tubules. J Gen Physiol 86: 765-794,1985.
4 x" R8 a& w' ~) {- m
. d7 z$ @' d2 C% q* e- w

Chen LK andBoron WF. Acid extrusion in S3 segment of rabbit proximal tubule. I.Effect of bilateral. Am J Physiol Renal Fluid Electrolyte Physiol 268: F179-F192,1995.1 M, j9 z( b3 w# A5 W

Chen LK andBoron WF. Acid extrusion in S3 segment of rabbit proximal tubule. II.Effect of basolateral. Am J Physiol Renal Fluid Electrolyte Physiol 268: F193-F203,1995.
5 ]2 G7 J; x8 h0 _1 S: k
1 V8 R' s6 F7 x7 T  x

Cogan MG. Effects of acute alterations in P CO 2 on proximal, Cl -, andH 2 O reabsorption. Am J Physiol Renal Fluid ElectrolytePhysiol 246:F21-F26, 1984.0 \4 D% D# E' h

Dorman PJ,Sullivan WJ, and Pitts RF. The renal response to acute respiratoryacidosis. J Clin Invest 33:82-90, 1954.+ ]+ {  |4 Z  n9 C
0 N+ W' W7 h+ Z; b( y

) {1 _) l* _* l8 L! a0 x
Forrester RL,Wataji LJ, Silverman DA, and Pierre KJ. Enzymatic method for determinationof CO 2 in serum. Clin Chem 22: 243-245,1976.* ~2 A, |" w& _1 f0 _5 d
+ a4 G# d8 @$ h- y& N
, c; \  D1 L9 w; [+ |
' O6 x2 N" ]1 c; F' l- ]* M
Geibel J,Giebisch G, and Boron WF. Basolateral sodium-coupled acid-base transportmechanisms of the rabbit proximal tubule. Am J Physiol Renal FluidElectrolyte Physiol 257:F790-F797, 1989.
" D* U( {2 m% J# r4 a

Grichtchenko II and Boron WF. Surface-pH measurements in voltage-clamped Xenopus oocytes co-expressing NBCe1 and CAIV: evidence for transport (Abstract). FASEB J 16:A795, 2002.
. V% U  \) ]& P( w7 E( ?  q
0 L) Q7 R. y. i. b: n
8 s( d1 L& D6 g+ F$ r: T
McKinney TD andBurg MB. Bicarbonate and fluid absorption by renal proximal straighttubules. Kidney Int 12:1-8, 1977.
3 W3 l+ {/ V5 v4 {7 V
4 X# a) e; @' C9 ?4 |  S
% C/ g. g3 R) Y) r* N. T
Nakhoul NL,Chen LK, and Boron WF. Effect of basolateral onintracellular pH regulation in the rabbit S3 proximal tubule. J GenPhysiol 102:1171-1205, 1993.5 x# p, u; b( A4 W: _

Nakhoul NL,Lopes AG, Chaillet JR, and Boron WF. Intracellular pH regulation in the S3segment of the rabbit proximal tubule in solutions. J Gen Physiol 92:369-393, 1988.
4 d% K, C, l# h1 Z* r) Y) Q' Y
9 `' `- B/ W8 N7 n' a. w

Olsnes S,Tonnessen TI, and Sandvig K. pH-regulated anion antiport in nucleatedmammalian cells. J Cell Biol 102: 967-971,1986.0 \1 V/ \6 X4 Z1 g

/ G8 s% a: V) H. }4 b; W4 G4 p
Quigley R, BaumM, Reddy KM, Griener JC, and Falck JR. Effects of 20-HETE and19( S )-HETE on rabbit proximal straight tubule volume transport. Am J Physiol Renal Physiol 278:F949-F953, 2000./ d: Y- ?* l% y1 h) r

/ D! P$ i! }' _6 T: N% c
Ravesloot JH,Eisen T, Baron R, and Boron WF. Role of Na-H exchangers and vacuolarH pumps in intracellular pH regulation in neonatal ratosteoclasts. J Gen Physiol 105:177-208, 1995.$ Y+ U0 u. {/ L, n2 O" ^
6 e: ?/ s( h+ [' u
; C5 ]; O, `7 f& F

Richmond PH andVaughan-Jones RD. Assessment of evidence for K -H exchange in isolated type-1 cells of neonatal rat carotid body. Pflügers Arch 434:429-437, 1997./ z& t) g: Y* X/ l' S( f
, r+ l: r3 U4 Z5 F
3 V. E( h- b7 ~/ b- V% a0 Z: d9 M
9 a" X- ~+ f5 p+ c9 q
Sasaki S, BerryCA, and Rector FC Jr. Effect of luminal and peritubular concentrations andP CO 2 on reabsorption in rabbit proximal convoluted tubules perfused in vitro. J Clin Invest 70:639-649, 1982.3 q  h* [7 e4 _: A* _) F

. r0 K2 j0 b9 s* t) O
" ?8 g0 {. ^: V
Siebens AW andBoron WF. Effect of electroneutral luminal and basolateral lactatetransport on intracellular pH in salamander proximal tubules. J GenPhysiol 90:799-831, 1987.

Simchowitz L and Roos A. Regulation of intracellular pH in human neutrophils. J Gen Physiol 85:443-470, 1985.

6 V! c( n3 o; W2 \  G+ D( {
Star RA. Quantitation of total carbon dioxide in nanoliter samples by flow-throughfluorometry. Am J Physiol Renal Fluid ElectrolytePhysiol 258:F429-F432, 1990.

9 f" z# ~* P4 L7 \! V. @- J
3 c2 w  v5 Y1 W. n8 |+ X% j' _8 i: [
Thomas JA,Buchsbaum RN, Zimniak A, and Racker E. Intracellular pH measurements inEhrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210-2218, 1979.

Voipio J andKaila K. Interstitial P CO 2 and pH in rat hippocampalslices measured by means of a novel fastCO 2 /H -sensitive microelectrode based on a PVC-gelledmembrane. Pflügers Arch 423: 193-201,1993.
% o% X- \# e* {9 F; g; G
$ R; `) ?5 P! y  j
2 ]# d1 e6 q# V7 x  w( J, {: d$ Y
Zhao J, HoganEM, Bevensee MO, and Boron WF. Out-of-equilibrium solutions and their use in characterizing a new K/HCO 3 cotransporter. Nature 374:636-639, 1995.

收藏0 分享0 顶0 踩0