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W F Boron

Publications and source records attributed to W F Boron.

At least 91 records · Page 5Linked to original sources
PubMed1989

Depolarization-induced alkalinization in proximal tubules. II. Effects of lactate and SITS.

Intracellular pH and voltage microelectrodes were used to further characterize the depolarization-induced alkalinization (DIA) observed in isolated perfused proximal tubules of the tiger salamander Ambystoma tigrinum. Tubules were depolarized by raising basolateral [K+] from 2.5 to 50 mM. The solutions were air equilibrated and nominally HCO3- free (estimated [HCO3-] = 0.2 mM). In the preceding study we showed that the DIA is partially blocked by removal of Na+ from only the lumen or only the bath, but completely blocked by bilateral Na+ removal. In the present study we found that bilateral amiloride (1 mM) had no effect on the DIA, suggesting that Na-H exchange is not involved. In contrast, basolateral 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (0.5 mM) partially blocked the DIA, presumably due to inhibition of one or more of three SITS-sensitive basolateral transporters present in amphibian proximal tubules: electrogenic Na-HCO3 cotransport, Na-dependent Cl-HCO3 exchange, and H-lactate cotransport. Bilateral removal of all organic substrates, or of only lactate (Lac-) also blocked the DIA partially. As shown elsewhere (A. W. Siebens, and W. F. Boron, J. Gen. Physiol. 90: 799-831, 1987), in the absence of depolarization, luminal Lac- causes a pHi increase due to luminal Lac- entry via a Na-Lac cotransporter, followed by basolateral Lac- exit via an H-Lac cotransporter sensitive to alpha-cyano-4-hydroxycinnamate (CHC). Three lines of evidence indicate that this Na-Lac/H-Lac mechanism is involved in the DIA. 1) As noted previously, the DIA is partially blocked by luminal Na+ removal. 2) With the DIA partially blocked by basolateral SITS, removal of Lac- from only the lumen blocks the remainder of the DIA. 3) Basolateral CHC partially blocks the DIA. Our data suggest that the DIA is mediated by at least two additive mechanisms, a basolateral transporter that is SITS sensitive and Na+ dependent, and the Na-Lac/H-Lac transport system.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Effects of acetate on luminal acidification processes in the S3 segment of the rabbit proximal tubule.

We determined that, in the nominal absence of HCO-3, acetate (Ac-) doubles luminal acidification in the S3 segment of the rabbit proximal tubule. This stimulation had two components, one that was dependent on Na+ and luminal Ac- and a second that was independent of Na+ but dependent on basolateral Ac-. In the presence of 25 mM HCO-3, Ac- did not stimulate acid secretion (i.e., HCO-3 reabsorption), but actually inhibited it. The inhibition was 35% with bilateral Ac- and 15% with basolateral Ac-. The effects of Ac- were reversible both in the absence and presence of HCO-3, and are present at concentrations as low as 1 mM. We conclude that acetate (i.e., monocarboxylates) has a significant effect on luminal acidification processes both in the presence and absence of HCO-3.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Basolateral sodium-coupled acid-base transport mechanisms of the rabbit proximal tubule.

We studied Na+-coupled acid-base transport at the basolateral membrane of single, isolated, perfused rabbit proximal tubules by monitoring the time course of intracellular pH (pHi). The latter was determined using a microspectrofluorometric apparatus to alternatively excite the pH-sensitive fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF) at 440 and 490 nm, while the fluorescence emission, was measured at 530 nm. All experiments were conducted in the nominal absence of HCO-3 S1, S2, and S3 segments from both superficial and juxtamedullary nephrons were examined individually. We found that removing Na+ from both the lumen and bath (i.e., basolateral solution) caused pHi to fall from 7.24 to 6.75 in the superficial S1 segment (SS1), from 7.14 to 6.67 in the SS2, and from 7.09 to 6.69 in the SS3. Similarly, in juxtamedullary nephrons (J), bilateral Na+ removal caused pHi to fall from 7.25 to 6.76 in the JS1, from 7.16 to 6.71 in the JS2, and from 7.10 to 6.75 in the JS3. In all six proximal tubule subtypes, returning Na+ to the bath caused pHi to recover (i.e., increase). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 50 microM), an inhibitor of HCO-3 transport systems, blocked this Na+-dependent pHi recovery in all three superficial subtypes and the JS3 but had no effect in either the JS1 or JS2. On the other hand, 50 microM ethylisopropyl amiloride (EIPA), an inhibitor of Na-H exchange, blocked the Na+-dependent pHi recovery in the JS1 and JS2 but had no effect in the JS3 or any of the superficial subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-gamma-S.

The regulation of intracellular pH (pHi) is essential for normal cell function, and controlled changes in pHi may play a central role in cell activation. Sodium-dependent Cl-HCO3 exchange is the dominant mechanism of pHi regulation in the invertebrate cells examined, and also occurs in mammalian cells. The transporter extrudes acid from the cell by exchanging extracellular Na+ and HCO3- (ref. 9) (or a related species) for intracellular Cl- (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate, ref. 10) and SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonate, ref. 3), and has a stoichiometry of two intracellular H+ neutralized for each Na+ taken up and each Cl- extruded by the axon. Because the inwardly-directed Na+ concentration gradient is sufficiently large to energize both the HCO3- influx and Cl- efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl-HCO3 exchanger stoichiometrically hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pHi regulation by dialysing axons with the ATP analogue ATP-gamma-S. In many systems, ATP-gamma-S is an acceptable substrate for protein kinases, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.

Adenosine Triphosphate↗
PubMed1988

Role of monocarboxylate transport in the regulation of intracellular pH of renal proximal tubule cells.

Traditional models of acid-base transport and intracellular pH (pHi) regulation in the renal proximal tubule have been based on the existence of a Na+/H+ exchanger at the luminal membrane and a simple HCO3- conductance at the basolateral membrane. Our recent work, in which we used pH-sensitive microelectrodes or dyes to monitor pHi in isolated renal tubules perfused in the nominal absence of HCO3-, has demonstrated the existence of a novel mechanism of acid extrusion in amphibian and mammalian proximal tubule cells. The salamander proximal tubule, for example, possesses an electroneutral Na+ monocarboxylate (Na+-X-) co-transporter, but only at the luminal membrane. It also possesses an electroneutral H+-X- co-transporter, but only at the blood side or basolateral membrane. In the presence of lactate, the luminal Na+-lactate co-transporter mediates a net influx of lactate, driven by the Na+ gradient. The cell-to-blood lactate gradient, in turn, drives the coupled efflux of H+ and lactate across the basolateral membrane. The net effect is the reabsorption of lactate, the luminal uptake of Na+ and the basolateral extrusion of H+. Acid extrusion mediated by this monocarboxylate system in the salamander is comparable in magnitude to that mediated by the Na+/H+ exchanger. In the S3 segment of the rabbit proximal tubule, a similar monocarboxylate system (studied with acetate instead of lactate) extrudes acetate at twice the rate of the Na+/H+ exchanger. Thus, monocarboxylate transport, at least in the nominal absence of HCO3-, can have a major impact on pHi regulation.

Acetates↗
PubMed1988

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

Ammonia↗
PubMed1988

Acetate transport in the S3 segment of the rabbit proximal tubule and its effect on intracellular pH.

We monitored intracellular pH (pHi) in isolated perfused S3 segments of the rabbit proximal tubule, and studied the effect of acetate (Ac-) transport on pHi. pHi was calculated from the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein trapped intracellularly. All solutions were nominally HCO3(-)-free. Removal of 10 mM Ac- from bath and lumen caused pHi to rapidly rise by approximately 0.2, and then to decline more slowly to a value approximately 0.35 below the initial one. Removal of only luminal Ac- caused pHi changes very similar to those resulting from bilateral removal of Ac-. When Ac- was removed from bath only, pHi rose rapidly at first, and then continued to rise more slowly. Readdition of Ac- to bath caused pHi to rapidly fall to a value slightly higher than the one prevailing before the removal of Ac- from the bath. In experiments in which Ac- was first removed from both bath and lumen, readdition of 10 mM Ac- to only lumen caused a rapid but small acidification, followed by a slower alkalinization that brought the pHi near the value that prevailed before the bilateral removal of Ac-. The alkalinizing effects caused by the readdition of 10 or 0.5 mM Ac- were indistinguishable. When Ac- was returned to only lumen in the absence of luminal Na+, there was a small and rapid pHi decrease, but no pHi recovery. Removal of Na+ from bath did not affect the pHi transients caused by the addition of Ac- to lumen. In experiments in which Ac- was first removed bilaterally, readdition of Ac- to only bath caused a large and sustained drop in pHi, whereas the subsequent removal of Ac- from the bath caused a slight alkalinization. These pHi changes caused by readdition or removal of Ac- from baths were unaffected by the absence of external Na+. We conclude that there is a Na+/Ac- cotransporter at the luminal membrane, and pathways for acetic acid transport at both luminal and basolateral membranes. The net effect of Ac- transport on pHi is to alkalinize the cell as a result of the luminal entry of Na+/Ac-, which is followed by the luminal and basolateral exit of acetic acid.

Acetates↗
PubMed1988

pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-.

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl(-)-HCO3- exchangers.

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells.

We investigated changes in intracellular pH (pHi) of cultured rat glomerular mesangial cells (MCs) exposed to angiotensin II (ANG II) and arginine vasopressin (AVP). pHi of quiescent MCs, passage 2-5, and grown on glass cover slips, was assessed by spectrofluorometry using the pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The steady-state pHi of MCs in a pH 7.4, HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.10 +/- 0.02 (n = 68) and in a pH 7.4, HCO3-containing solution, was 7.23 +/- 0.03 (n = 47) (P less than 0.01). The pHi recovery following an NH+4-induced acid load was inhibited by removal of Na+ from the bath or by addition of the amiloride analogue, ethyl isopropyl amiloride (EIPA). These effects were observed in MCs bathed in HEPES- or in HCO3-buffered solutions, consistent with the action of a Na+-H+ exchanger. When cells were bathed in HEPES, a 10-min exposure to ANG II or AVP (10(-10) to 10(-6) M) caused early and transient acidification of MCs (maximal pH change was -0.10), followed by gradual alkalinization (maximal pHi change +0.15 above the initial value). The increase of pHi was dependent on the presence of Na+ in the bath and was inhibited by EIPA. In the presence of HCO3, ANG II or AVP induced merely a small gradual acidification of MCs (pHi change -0.05). These findings demonstrate that MCs utilize a Na+-H+ exchanger for acid extrusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1987

Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules.

We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization.

Ambystoma↗
PubMed1987

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule.

This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane potential (Vbl) and intracellular pH (pHi) of 1) lowering basolateral [HCO3-] at constant PCO2, and 2) replacing Na+ with N-methyl-D-glucamine. Vbl and pHi were measured with Ling-Gerard and liquid-membrane pH microelectrodes, respectively, in isolated tubules perfused in vitro. We found that decreasing basolateral [HCO3-] from 10 mM (pH 7.5) to 2 mM (pH 6.8) resulted in an immediate depolarization of 14.9 mV, and a pHi decrease of 0.35. SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulfonic acid, 0.5 mM) inhibited the HCO3-induced depolarization by 87% and inhibited the initial rate of the pHi decrease by 79%. Replacement of basolateral Na+ with N-methyl-D-glucamine resulted in an immediate depolarization of 11.3 mV, and a pHi decrease of 0.36. SITS inhibited the zero Na-induced depolarization by 86% and the initial rate of the pHi decrease by 81%. Nominal removal of basolateral HCO3- (replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) inhibited the zero Na-induced depolarization by 64%, whereas nominal removal of Na+ inhibited the 2 mM HCO3-induced depolarization by 67%. Replacement of all basolateral Cl- with glucuronate did not inhibit the changes in Vbl induced by changing [HCO3-] or [Na+]. Observations similar to those described above have been made previously on Ambystoma proximal tubules, and attributed to an electrogenic Na/HCO3 cotransport mechanism that carries HCO3-, Na+, and net negative charge in the same direction. We conclude that Necturus proximal tubules possess a similar, if not identical, electrogenic Na/HCO3 cotransport mechanism.

Animals↗
PubMed1986

Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl-HCO3 exchange.

An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4',5'-dimethyl-5(and -6)-carboxyfluorescein by exposing them to the dye's permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3(-)-free Ringer's solution, pHi recovered exponentially from acid loads applied by NH+4 prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer's solution, external Cl- removal caused pHi to reversibly increase by approximately equal to 0.3. This pHi increase was substantially reduced by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by approximately equal to 0.2. This pHi change was greatly diminished by DIDS, by removal of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences.

The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.

Acid-Base Equilibrium↗
PubMed1985

Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules.

We evaluated the dye 4',5'-dimethyl-5-(and -6-) carboxyfluorescein (Me2CF) for determining the intracellular pH(pHi) of isolated, perfused proximal tubules of the salamander. The intracellular absorbance spectrum, corrected for the intrinsic absorbance of the tubule, was obtained once per second. The dye was incorporated into tubule cells by exposing them to the membrane-permeable precursor 4',5'-dimethyl-5- (and -6-) carboxyfluorescein diacetate. The introduction of the dye had no significant effect on either pHi or cell voltage transients. Compared with dye contained in a cuvette, intracellular dye had a peak absorbance that was red-shifted by approximately 5 nm, and an apparent pK that was increased by approximately 0.3. These differences precluded an accurate calculation of pHi by the comparison of intracellular spectra with in vitro calibration spectra. However, when Me2CF was calibrated intracellularly, using the K-H exchanger nigericin to equalize external pH and pHi, the dye-derived, steady state pHi was within approximately 0.1 of the value obtained with pH-sensitive microelectrodes. Furthermore, when pHi was simultaneously measured with dye and microelectrodes during rapid pHi transients, the pHi time courses measured by the two methods were very similar. We conclude that the intracellular absorbance spectrum of Me2CF can be used to measure steady state pHi and rapid pHi transients reliably, provided the dye is calibrated intracellularly.

Ambystoma↗
PubMed1985

Basolateral Na-H exchange in the rabbit cortical collecting tubule.

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.

Amiloride↗