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Originally published In Press as doi:10.1074/jbc.M414355200 on July 7, 2005

J. Biol. Chem., Vol. 280, Issue 35, 30864-30872, September 2, 2005
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Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH*

Debbie Willoughby, Nanako Masada, Andrew J. Crossthwaite, Antonio Ciruela, and Dermot M. F. Cooper{ddagger}

From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom

Received for publication, December 21, 2004 , and in revised form, June 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca2+ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pHi), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pHi on Ca2+-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na+/H+ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pHi is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular pH (pHi)1 is a fundamental determinant of cell function that, until recently, was considered to be tightly regulated due to rapid H+ diffusion, buffering, and ion transport mechanisms. However, it is now recognized that modest fluctuations in pHi can arise during "physiological" cellular activity (1–9). Furthermore, recent studies have provided evidence for pHi microdomains just beneath the plasma membrane, which can exhibit transient pH shifts on the order of several tenths of a pH unit, as a consequence of the local activity of plasma membrane transporters (10–12). Fluctuations of [H+] within these microdomains could potentially exert profound effects on the numerous cellular proteins residing within or near the plasma membrane, including the cAMP-producing adenylyl cyclase (AC). Early in vitro studies of plasma membrane preparations demonstrated that the catalytic activity of AC is steeply pH-dependent, over the range of pH 6.0–8.5, where acidification or alkalinization, respectively, decreases or increase cAMP synthesis (13–15).

In addition to the pH dependence of AC catalytic activity, the Ca2+ sensitivity of the Ca2+-regulated ACs is also likely to be highly pH-sensitive. Four of the nine AC isoforms cloned to date are regulated by submicromolar concentrations of Ca2+. AC1 and AC8 are stimulated by Ca2+ acting via calmodulin, whereas AC5 and AC6 are inhibited, independently of calmodulin (16). Since the binding of Ca2+ to both calmodulin and AC5/6 depends on the interaction with specific aspartate residues (17, 18), these enzymes could be expected to be quite sensitive to modest changes in pH. In the intact cell, the Ca2+-sensitive ACs are regulated exclusively by capacitative Ca2+ entry (CCE) or Ca2+ entry through voltage-gated Ca2+ channels (19–22). The selectivity of the cyclases for discrete modes of Ca2+ entry rather than other sources of cytosolic Ca2+ increase alludes to the co-localization of the ACs with preferred Ca2+ entry sites, a quality that is at least partly dependent on the selective localization of Ca2+-sensitive ACs in cholesterolrich domains of the plasma membrane, known as lipid rafts or caveolae (23, 24). It is conceivable then that, during periods of cellular activity, the overall degree of cAMP production might reflect local changes in both [Ca2+] and pHi.

In the present study, we first established that the regulation by Ca2+ of an endogenous AC6 and a heterologously expressed AC8 were markedly modulated by modest alterations of pH in in vitro measurements. We then explored whether this clear pH sensitivity would be manifest in the intact cell. To our surprise, although we could mimic the same amplitude of global pHi change as we had brought about in our in vitro experiments, the response of the ACs to CCE was largely unperturbed. Evaluation of a number of possible mechanisms revealed that the Na+/H+ exchanger (NHE1) became active during our imposed acid shifts. Furthermore, it was located in lipid rafts, along with AC8 in HEK 293 cells (and AC6 in C6-2B glioma cells). Inhibition of NHE1 activity allowed the expected effect on AC8 activity of acid shifts to be observed. The presence of NHE1 and Ca2+-sensitive AC8 or of AC6 in the same subcellular domain may generate a privileged microenvironment that insulates the cyclases from fluctuations in cellular pH.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—BCECF/AM, Fura-2/AM, Fura-2 free acid, Fura-FF free acid, and Pluronic F-127 were purchased from Molecular Probes (Leiden, Netherlands). Cariporide was a gift from Dr. Juergen Puenter at Avetis Pharma (Frankfurt, Germany). Polyclonal AC8 antibody was a gift from Dr. J. J. Cali, as previously described (24). Polyclonal caveolin antibody was obtained from BD Transduction Laboratories (Erembodegem, Belgium), and polyclonal {beta}-adaptin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal NHE1 antibody (clone 4E9) was purchased from Chemicon International (Hofheim, Germany). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Amersham Biosciences (Little Chalfont, UK), and horseradish peroxidase-conjugated goat anti-mouse IgG was from Promega (Madison, WI). All other agents were purchased from Sigma (Poole, UK) unless stated otherwise.

Cell Culture and Transfection of HEK 293 Cells—C6-2B rat glioma cells were grown in F-10 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum. This cell line expresses almost exclusively type 6 adenylyl cyclase, with trace amounts of AC3 (25). HEK 293 cells (European Collection of Cell Cultures, Porton Down, UK) were grown in minimum essential medium supplemented with 10% (v/v) fetal bovine serum. All cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. HEK 293 cells were plated on 100-mm dishes at ~70% confluence 1 day prior to transfection with 2 µg of AC8 cDNA using Effectene according to the manufacturer's procedures. Transiently transfected cells were used 2 days post-transfection. The transiently expressed AC8 was responsible for all of the Ca2+-stimulated AC activity in the HEK 293 cells, since nontransfected cells exhibited a weak inhibition of AC activity with increasing calcium concentrations (data not shown).

Measurement of Adenylyl Cyclase Activity—Determination of adenylyl cyclase activity in vitro was performed as described previously (26) with some modifications. The adenylyl cyclase activity of isolated C6-2B glioma and AC8-expressing HEK 293 cell membranes (4 µg of protein) was measured in the presence of the following components: 12 mM phosphocreatine, 2.5 units of creatine phosphokinase, 0.1 mM cAMP, 1.4 mM MgCl2, 0.1 mM ATP, 0.04 mM GTP, 0.5 mM isobutylmethylxanthine, 1 µCi of [{alpha}-32P]ATP, forskolin (20 and 2 µM respectively), and calmodulin (0 and 1 µM, respectively). Specifically, 25 µl of 50 mM Tris buffer, pH 2.2, 7.5, or 8.8, was added to establish the final pH of the reaction mixture at 7.14, 7.50, or 7.85, as indicated. Free Ca2+ concentrations were established from a series of CaCl2 solutions buffered with 200 µM EGTA and confirmed by calibration with Fura-2 and Fura-FF (Molecular Probes). The reaction mixture (final volume, 100 µl) was incubated at 30 °C for 20 min. Reactions were terminated with sodium lauryl sulfate (0.5%); [3H]cAMP (~6000 cpm) was added as a recovery marker, and the [32P]cAMP formed was quantitated as described previously (27). Data points are presented as mean activities ± S.D. of triplicate determinations.

Measurement of cAMP Accumulation—cAMP accumulation in intact cells was measured according to the method of Evans et al. (28) as described previously (29) with some modifications. C6-2B glioma cells on 24-well plates were incubated in F-10 medium, and AC8-expressing HEK 293 cells were incubated in minimal essential medium (90 min at 37 °C) with [2-3H]adenine (1.5 µCi/well) to label the ATP pool. The cells were then washed once and incubated with a nominally Ca2+-free Krebs buffer containing 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin adjusted to pH 7.4 with 2 M Tris base. The use of Ca2+-free Krebs buffer in experiments denotes the addition of 100 µM EGTA to the nominally Ca2+-free Krebs buffer. All experiments were carried out at 30 °C in the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 µM), which was preincubated with the cells for 10 min prior to a 1-min assay. Cells were preincubated for 4 min with the Ca2+-ATPase inhibitor, thapsigargin, at a final concentration of 100 nM. This has the effect of passively emptying the Ca2+ stores, establishing a low basal [Ca2+]i, and priming the cells for capacitative Ca2+ entry (30). 10 mM propionate, vehicle solution, or 20 mM trimethylamine was added 2 min prior to the assay to shift the pH of the cells to ~7.15, 7.50, and 7.85, respectively. cAMP accumulation was measured over a 1-min period beginning with the addition of 10 µM forskolin along with various concentrations of Ca2+. Assays were terminated by the addition of ice-cold 10% (w/v, final concentration) trichloroacetic acid. Unlabeled cAMP (100 µl, 10 mM), ATP (10 µl, 65 mM), and [{alpha}-32P]ATP (~7000 cpm) were added to monitor recovery of cAMP and ATP. The [3H]ATP and [3H]cAMP content of the supernatant were quantified as described previously (27). Accumulation of cAMP is expressed as the conversion of [3H]ATP into [3H]cAMP. Results are presented as the means ± S.D. of triplicate determinations.

pHi and [Ca2+]i Measurements—pHi and [Ca2+]i were measured in cell populations using a PerkinElmer Life Sciences 50B spectrofluorimeter. In brief, cells were detached with phosphate-buffered saline (12.1 mM Na2HPO4, 4 mM KH2PO4, 130 mM NaCl at pH 7.4) containing 0.01% EDTA and loaded with either 4 µM BCECF/AM (pHi measurements) or 2 µM Fura-2/AM ([Ca2+]i measurements) plus 0.02% Pluronic F-127 for 45 min at room temperature. The cells were then washed twice, aliquoted into samples containing ~1 x 106 cells, and finally resuspended in 3 ml of nominally Ca2+-free Krebs buffer and placed in a stirred cuvette just prior to the experiment. For Ca2+-free Krebs buffer, 100 µM EGTA was added. Test substances were added from 100-fold concentration stocks. BCECF 490/440 nm ratio fluorescence values were calibrated using the high K+/nigericin method (31). The resultant pH values were in good agreement with those evaluated from an in vitro calibration of BCECF free acid. Fura-2 340/380 nm fluorescence ratios were converted to [Ca2+]i values as described previously (29).

Reverse Transcription-PCR Experiments—RNA was isolated with the SV total RNA isolation system (Promega). Primers for NHE1, NHE2, and NHE3 isoforms were designed and synthesized on the basis of published human and rat cDNA sequences and are shown in Table II. Reverse transcription-PCRs were performed by using total cellular RNA. As a control for genomic DNA contamination of the RNA preparations, parallel samples were not reverse transcribed. PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide.


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  		TABLE II
Primers used for NHE isoforms based on human and rat cDNA sequences

 
Nondetergent Isolation of Raft and Nonraft Plasma Membranes— Raft and nonraft plasma membranes were separated by a procedure that exploited their different buoyancies as previously described (32). Briefly, cells were pelleted (195 x g, 5 min) and resuspended in ice-cold sodium carbonate solution (500 mM Na2CO3, 150 mM NaCl, pH 11.0) before sonication (Sonic Dismembrater, Fisher Scientific) at 4 °C, 30 s at setting 2, 30 s at setting 3, and 30 s at setting 4 with 3 min between each sonication (33). The suspension was transferred to a tight fitting Dounce homogenizer and homogenized with 20 strokes. The homogenate was adjusted to 40% sucrose by the addition of 60% sucrose in MES buffer (25 mM MES, 150 mM NaCl, 250 mM Na2CO3, pH 6.4). The extract was placed below a 5 and 30% discontinuous sucrose gradient prepared in cold MES buffer and centrifuged in a Beckman SW55 rotor at 24,000 rpm for 16 h at 4 °C. Fractions (10 x 0.5 ml) were collected from the top of the gradient, and sucrose concentration of an aliquot (40 µl) of each fraction was measured in an Abbe refractometer. The remainder of the isolated fractions were diluted in 5 volumes of MES buffer, centrifuged in a Beckman SW55 rotor at 50,000 rpm for 1 h at 4 °C, and prepared for electrophoresis. The pelleted membranes were resuspended in 1% SDS and added to a loading buffer, giving a final concentration of 62.5 mM Tris, 150 mM dithiothreitol, 20% glycerol, and 0.002% bromphenol blue, pH 6.8. Samples were heated to 100 °C for 5 min and stored at –80 °C.

Immunoblotting—Proteins were resolved using 7.5 and 12% SDS-polyacrylamide gels except for AC8, where 8 M urea was also included (34). Proteins were transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was incubated in blocking buffer (20 mM Tris, pH 7.5, 150 mM NaCl (TBS)) containing 5% skimmed milk powder, for 30 min, followed by two 10-min washes in TBS supplemented with 0.05% (v/v) Tween 20 (TTBS). Membranes were incubated overnight at room temperature with anti-AC8 antibody (1:5000), anti-caveolin polyclonal antibody (1:5000), anti-NHE1 monoclonal antibody (1:1000), or {beta}-adaptin polyclonal antibody (1:1000) in TTBS containing 1% skimmed milk powder (antibody buffer). The membranes were washed (2 x 10 min) in TTBS and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000 dilution of stock) or goat anti-mouse IgG conjugated to horseradish peroxidase (1:3000) in antibody buffer for 1 h. Finally, the membranes were washed in TTBS (2 x 10 min), rinsed in TBS, and treated with ECL reagent (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Ca2+ Sensitivity of AC8 and AC6 at Different pH— Modest changes in [H+] are known to affect the catalytic activity of adenylyl cyclases (13–15). To investigate a further pH sensitivity of ACs, with respect to their regulation by Ca2+, activities of Ca2+-stimulable AC8 and Ca2+-inhibitable AC6 were assayed in vitro over a range of [Ca2+] at different pH values (Fig. 1). Ca2+ dose-response curves were compared at pH 7.14, 7.50, and 7.85. These values were chosen, because they are within the pH range that ACs may experience under physiological conditions. Transiently expressed AC8 in HEK 293 cell membranes (Fig. 1A) and endogenously expressed AC6 in C6 glioma cell membranes (Fig. 1B) both exhibited clear pH sensitivities with respect to their peak activities and sensitivity to Ca2+. Under alkaline conditions (pH 7.85), stimulation of AC8 activity by Ca2+/calmodulin was increased to ~980% of basal activity, compared with a 610% increase above basal level in controls (pH 7.50; Fig. 1A). By contrast, acidification (pH 7.14) decreased the stimulation by Ca2+/calmodulin to just 330% of basal. The apparent Kd value for Ca2+ stimulation of AC8 was also quite dependent on pH, with EC50 values of 0.06, 0.10, and 0.16 µM Ca2+ for pH 7.85, 7.50, and 7.14 respectively.



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  		FIG. 1.
Effects of pH on calcium-regulated adenylyl cyclases in vitro. The adenylyl cyclase activity of isolated AC8-expressing HEK 293 cell membranes (A) and C6-2B glioma cell membranes (B) was measured over a range of [Ca2+] at pH 7.14, 7.50, and 7.85 buffered with 50 mM Tris. The cyclases were stimulated with 2 µM (A) or 20 µM (B) forskolin in the presence of the nonselective phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM), plus 1 µM calmodulin for AC8 experiments. Acidification (pH 7.14) lowered adenylyl cyclase activity and decreased the sensitivity of both AC8 and AC6 to Ca2+-dependent modulation compared with controls (pH 7.50). Under alkaline conditions (pH 7.85), the activity and Ca2+ sensitivity of both cyclases was enhanced. Data are plotted as mean ± S.D.

 
In C6-2B glioma cell membranes, peak AC6 activity under alkaline conditions (pH 7.85) was 170% of that seen at pH 7.50 and just 65% of control activity at pH 7.14 (Fig. 1B), consistent with previously published effects of pH on AC activity (13–15). However, in Fig. 1B we also reveal a clear pH dependence of the Ca2+ inhibition of AC6 activity, in terms of both affinity and maximal effect. As with AC8, the Ca2+ regulation of AC6 was enhanced at pH 7.85 and reduced at pH 7.14 compared with controls (pH 7.50). The corresponding IC50 values for Ca2+ were 0.19, 0.33, and 1.10 µM at pH 7.85, 7.50, and 7.14, respectively (Fig. 1B).

In Vivo Sensitivity of AC8 and AC6 to CCE at Different pHi— To explore whether Ca2+ regulation of AC8 and AC6 exhibited similar pH sensitivity in the intact cell, a series of in vivo measurements of cAMP accumulation were performed. Previous studies have shown that both cyclases are exclusively regulated by CCE in the intact cell (19–21). Similar protocols were used here to obtain dose-response curves for cAMP accumulation during varying degrees of CCE. In brief, cells were maintained in Ca2+-free Krebs Ringer, and 100 nM thapsigargin was applied to deplete intracellular endoplasmic reticulum calcium stores. After 4 min of thapsigargin treatment, CCE was evoked by the introduction of CaCl2 (0.25, 0.5, 1, 2, and 4 mM) to the extracellular Ringer solution. Production of cAMP was assayed for 1 min following the addition of CaCl2.

In a series of separate pH experiments, various concentrations of weak acid (propionate (PA)) and weak base (trimethylamine (TMA)) were tested to determine which concentrations would induce an approximate 0.3-pH unit shift in pHi (to mirror the pH values that were employed in the in vitro measurements). This {Delta}pHi shift is considered to be within the range of localized pHi changes that proteins residing within the plasma membrane might experience during periods of cellular activity. Application of 10 mM PA induced an intracellular acidification of 0.33 ± 0.03 pH units in HEK293 cells and 0.26 ± 0.03 pH units in C6-2B glioma cells. 20 mM TMA induced a pHi shift of similar amplitude in the alkaline direction (see Table I; also see Figs. 2A and 3A). Resting pHi was ~7.45 in HEK 293 cells and pH 7.40 in C6 glioma cells, according to calibrated BCECF measurements (see "Experimental Procedures" and Table I). All pH measurements and adenylyl cyclase assays were performed in nominally CO2/HCO3-free Ringer. As a consequence, the global shifts in pHi, established within 1 min of weak acid or weak base application, were relatively sustained. To determine the effects of such pHi shifts on CCE regulation of AC8 and AC6 activity, 10 mM PA or 20 mM TMA was added 2 min prior to the addition of CaCl2 and the initiation of CCE. Comparisons of the Ca2+ stimulation of AC8 at different pHi values are shown in Fig. 2B. 10 mM PA induced a clear decrease in resting pHi of HEK 293 cells (Fig. 2A, upper panel) but rather surprisingly did not affect the Ca2+ sensitivity of AC8 (pH 7.14) compared with controls (pH 7.50). The effects of alkalinization by 20 mM TMA (Fig. 2A, lower panel) were more consistent with our in vitro data and demonstrate an enhanced stimulation of AC8 (pH 7.85) during CCE evoked by 2 or 4 mM CaCl2 compared with controls (pH 7.50) (Fig. 2B, p < 0.05).


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  		TABLE I
pHi changes induced by weak acid or weak base

 
The sensitivity of AC6 to Ca2+ inhibition at different pH values was similarly muted in vivo (Fig. 3B) compared with that seen in vitro (Fig. 1B). Although a small decrease or increase in AC6 catalytic activity was seen under acid or alkaline conditions, respectively, no pH-dependent changes in Ca2+ inhibition were observed in intact C6 glioma cells. This clear discrepancy between our in vitro and in vivo studies was unexpected. Consequently, a range of hypotheses were tested in an effort to identify the mechanism(s) that could underlie the lack of pH effect in the intact cell.

In Vivo Ca2+ Sensitivity of AC8 and AC6 at Different pHo— The first hypothesis to consider was that the clear in vitro pH sensitivity of Ca2+-regulated AC activity was mediated on extracellular sites of the AC molecules, since the in vitro assays do not discriminate between intracellular and extracellular effects of pH. To address this in the intact cell, experiments described for Figs. 2B and 3B were repeated, but instead of applying weak acid or weak base, the extracellular Ringer solution (pH 7.50) was replaced with either fresh solution at pH 7.50 (controls) or solution buffered to pH 7.14 or 7.85. Acute exposure of AC8-expressing HEK 293 cells or C6 glioma cells to different extracellular pH (pHo) showed no evidence of change in the Ca2+ dose-response effects (data not shown). This observation supported our interpretation that the pH sensitivity of AC8 and AC6 in vitro was mediated by H+ interactions at cytosolic sites within the AC molecule, such as the aspartate residues within the Mg2+/Ca2+ binding domain of AC6 (18) or aspartates in the EF hands of Ca2+/calmodulin (17). More dramatic effects of pHo were revealed when C6 cells were preincubated in slightly acidic (pH 7.14) or alkaline (pH 7.85) buffered Ringer for extended periods (up to 1 h) (Fig. 4B). However, even under these conditions, HEK 293 cells did not exhibit any pH sensitivity with respect to Ca2+ stimulation (Fig. 4A). In C6 glioma cells, the general pH dependence of AC activity became more apparent with prolonged exposure to changed pHo (Fig. 4B). Furthermore, there was a marked loss of Ca2+ inhibition of AC6 at pH 7.14 and a small enhancement of Ca2+ inhibition at pH 7.85. However, it is unclear if this is due to direct pHo effects that are mediated slowly or indirect effects of pHo occurring via parallel shifts in pHi during such prolonged exposures to solutions buffered at adjusted pH values (data not shown). If the latter explanation is correct, it is unclear why similar pHi effects are not mediated by weak acid or weak base.



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  		FIG. 2.
pHi modulation of CCE-stimulated AC8 activity in intact AC8-expressing HEK 293 cells. A, weak acid (PA) and weak base (TMA) induced steady changes in intracellular pH, monitored with BCECF. B, experiments were performed to determine dose dependence of CCE-induced Ca2+ stimulation of cAMP accumulation under resting pHi conditions (pH 7.50) or in the presence of 10 mM propionate (pH 7.14) or 20 mM trimethylamine (pH 7.85). Data are normalized to cAMP accumulation at pH 7.14 during 0 Ca2+ CCE addition and plotted as mean ± S.D. *, p < 0.05.

 



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  		FIG. 3.
pHi modulation of CCE-inhibited AC6 activity in intact C6-2B glioma cells. A, PA (10 mM) and TMA (20 mM) induced changes in intracellular pH of C6-2B cell populations were measured using BCECF. B, dose dependence of CCE-induced Ca2+ inhibition of cAMP accumulation in populations of C6-2B glioma cell populations in the presence of weak acid (pH 7.14) or weak base (pH 7.85), compared with controls (pH 7.50).

 



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  		FIG. 4.
Effects of prolonged exposure to different extracellular pH on the Ca2+ sensitivities of AC8 and AC6 in vivo. Accumulation of cAMP in AC8-expressing HEK 293 cells (A) and C6-2B glioma cells (B), following varying degrees of capacitative Ca2+ entry, was determined in cells bathed in Ringer at pH 7.50, 7.14, and 7.85 for at least 30 min prior to the assays. Data are plotted as mean ± S.D. normalized to cAMP accumulation under 0 Ca2+ conditions at pH 7.14.

 
Effects of pHi Changes on CCE in HEK 293 and C6 Cells—A second hypothesis addressed the potential interrelationship between changes in pHi and CCE (35, 36). We compared the degree of CCE seen in response to 2 mM CaCl2 under control conditions and in the presence of weak acid or weak base (Fig. 5). In HEK 293 cells (Fig. 5A), acidification with 10 mM PA increased CCE by ~16%. In contrast, alkalinization by 20 mM TMA application caused CCE to decrease by ~30% compared with that seen at resting pH. Hence, it is likely that any pH sensitivity of AC8 activity that is elicited by CCE, particularly under alkaline conditions, will be dampened due to pH-dependent changes in the degree of CCE. In the presence of weak base, CCE is clearly reduced, and CCE-evoked AC8 activity is only slightly enhanced (Fig. 2A) compared with the large effect of alkaline pH seen in vitro (Fig. 1A). The effects of acidification on CCE were less significant and cannot fully explain the lack of pH sensitivity seen in vivo. The sensitivity of CCE to changes in pHi was less pronounced in C6 glioma cells (Fig. 5B). In separate experiments, the changes in cytosolic Ca2+ seen during CCE did not significantly alter global pHi (data not shown).

NHE1 Activity Protects AC8 from Local Acid Shifts in HEK 293 Cells—Although we have eliminated some possible mechanisms in explaining the discrepancies between in vitro and in vivo effects of pH, a final hypothesis considered the possibility that Ca2+-regulated ACs were protected from intracellular acid shifts due to their localization in lipid rafts or caveolae (24, 37). We hypothesized that pH is tightly regulated in these cellular microdomains, such that application of a weak acid will produce a sustained pHi decrease in the bulk cell cytosol, but localized acid extrusion within the caveolar domain could minimize local pHi changes. Since all of our experiments were performed in nominally CO2/HCO3-free Ringer, a number of potential acid-extruding mechanisms were already inhibited (e.g. Na+-dependent Cl–/HCO–3 exchange). In the absence of CO2/HCO3, the NHE is likely to be the principal acid-extruding mechanism in HEK 293 cells. In Fig. 6A, we show that inhibition of NHE with the amiloride analogue, EIPA (5 µM), clearly attenuates the recovery of HEK 293 cells from an acid load. Using cariporide, a selective NHE1 inhibitor, we further established that activity of this NHE isoform was responsible for the acid recovery phase (Fig. 6B). Application of the weak acid, PA (10 mM), induced a pHi decrease of 0.28 ± 0.01 pH units in a population of HEK 293 cells that recovered at a steady rate (0.034 ± 0.001 pH unit min–1) under control conditions. A similar fall of 0.28 pHi units was seen in response to 10 mM PA when cells were pretreated with 5 µM cariporide, and the subsequent acid recovery was clearly inhibited (0.009 ± 0.003 pH unit min–1, p < 0.01 compared with controls). Once we had established that NHE, in particular NHE1, was active in the HEK 293 cells following an acid load, we investigated whether inhibition of the acid extruder could reveal pH-dependent effects on the Ca2+-stimulable AC8 (Fig. 6C). As would have been predicted, Ca2+ stimulation of AC8 was significantly reduced (p < 0.05) when weak acid was applied in the presence of cariporide (10 µM). Under resting pHi conditions, Ca2+ stimulation of AC8 was largely unaffected by cariporide, suggesting that basal NHE activity was minimal. A similar acid-induced decrease of the Ca2+ stimulation of AC8 was also revealed when we used 5 µM EIPA, a nonselective NHE inhibitor (data not shown). Dose-dependent stimulation of AC8 by CCE in the presence of weak base was still observed.

Enrichment of NHE1 in HEK 293 and C6 Glioma Cell Caveolar (Raft) Membranes—Given the manifest importance of NHE1 in controlling the pH in the environment of AC8 (and perhaps AC6), we considered the possibility that NHE, specifically NHE1, might be localized to the same subdomain of the plasma membrane as the Ca2+-regulated cyclases. To examine the distribution of NHE1 within the cell membrane, we first confirmed the presence of this isoform in our HEK 293 and C6 glioma cells. A previous study using polyclonal antibodies to the various NHE isoforms suggested that NHE1, NHE2, and NHE3 occurred in HEK 293 cells (38), although the NHE1 isoform appears to be principally responsible for acid recovery in our cells (cf. Fig. 6). Other studies have shown that NHE1 is also the dominant isoform in C6 glioma cells (39). Here we searched for these three isoforms using reverse transcription-PCR methods (Fig. 7). The primers for NHE1, NHE2, and NHE3 isoforms (based on published human or rat cDNA sequences) are shown in Table II. Only PCR products for NHE1 were detected in both HEK 293 cells and rat C6 glioma cells, suggesting that NHE2 and NHE3 are absent in both cell types. The efficacy of the primers used for this study was validated by positive signals for all three NHE isoforms in reverse-transcribed mRNA from human and rat kidney (Fig. 7). The presence of only NHE1 in HEK 293 cells was consistent with the sensitivity of NHE activity in the cells to low micromolar concentrations of cariporide and EIPA (Fig. 6, A and B) (40).



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  		FIG. 5.
Effects of pHi on CCE in HEK 293 and C6-2B glioma cells. Cell populations were loaded with Fura-2 to measure changes in intracellular [Ca2+] during the induction of CCE by 2 mM CaCl2, in the presence and absence of weak acid or weak base. HEK 293 cells (A) and C6-2B glioma cells (B) were incubated in Ca2+-free Krebs Ringer, and at 60 s 100 nM thaspigargin (TG) was used to deplete the endoplasmic reticulum calcium stores, causing a rise in cytosolic [Ca2+]. Weak acid (10 mM PA) or weak base (20 mM TMA) were added at 180 s to induce an ~0.35-pH unit shift in intracellular pH, prior to the addition of 2 mM CaCl2 at 300 s to induce CCE. Changes in pHi induced marked effects on the degree of CCE in HEK 293 cells and relatively small effects in C6-2B glioma cells.

 



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  		FIG. 6.
NHE1 activity minimizes the effects of weak acid on Ca2+-stimulated AC8 activity. Ai and Aii, measurements of pHi changes induced by 10 mM propionate in the absence (Ai) and presence (Aii) of a nonselective NHE inhibitor, EIPA (5 µM). Bi and Bii, 10 mM propionate-induced pHi changes in the absence (Bi) and presence (Bii) of 5 µM cariporide, a selective NHE1 inhibitor, reveals a role for NHE1 activity in proton extrusion following an acid load. Data are representative of three similar experiments. C, pretreatment of cells with 10 µM cariporide results in a significant loss of Ca2+ stimulation of AC8 activity in the presence of weak acid (pH 7.14). In vivo cAMP accumulation data are plotted as mean ± S.D. *, p < 0.05.

 
Two final experiments were performed to determine whether the NHE1 protein was present in low density membrane fractions, collected using a detergent-free fractionation method. As shown in Fig. 8, Western blotting of HEK 293 cell membrane fractions using an NHE1-specific monoclonal antibody showed high expression of the exchanger in fractions 4 and 5, alongside caveolin (the marker of lipid rafts) and transiently expressed AC8. The latter is consistent with previous studies in our laboratory showing that the AC8 protein is enriched in these caveolin-containing fractions (24). A small amount of NHE1 was found in fractions 7–9, which corresponded to the bulk plasma membrane regions, as identified by the marker {beta}-adaptin (37). Again, in C6 glioma cells, Fig. 9 shows that NHE1 was also enriched in caveolin-containing fractions (4 and 5), where AC6 has been shown to reside (23). These biochemical data strongly support the hypothesis that AC8 and AC6 are localized in cellular microdomains where pHi is tightly regulated due to the high expression of acid extruders that minimize local acid shifts.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have confirmed that the regulation by Ca2+ of an endogenous AC6 and a heterologously expressed AC8 can be significantly modulated by modest alterations of pH in vitro. The exact site of interaction between protons and ACs has not yet been established, but aspartate residues within the catalytic Mg2+/Ca2+ binding domain of AC5/6 (18) have the potential to bind H+ and modulate Ca2+ inhibition of the enzyme. With respect to Ca2+ stimulation of AC8, acting via calmodulin, there are at least two potential sites for proton interaction. Calmodulin-dependent enzymes (such as AC8) may be subject to H+-induced conformational changes that are associated with the interaction of the enzymes with calmodulin (41). Furthermore, Ca2+ binding to calmodulin is pH-dependent within the physiological range, exhibiting a 10-fold decrease in the Ca2+ dissociation constant of calmodulin between pH 6.5 and 7.5 (9, 42). Such interactions might explain the reduced or enhanced Ca2+ sensitivity of AC8 to acid or base shifts, respectively. Thus, adjustment of pH by ~0.35 pH units in vitro significantly affects the regulation of ACs by Ca2+.

We investigated whether Ca2+ regulation of ACs could be significantly modulated by pH changes in the intact cell, since it is now recognized that modest fluctuations in pHi can arise due to the activation of ligand-gated ion channels (1, 2, 4), voltage-gated H+ channels (3), ion transporters (5–8), and changes in cellular metabolism (9). Furthermore, recent studies have provided evidence for pHi microdomains just beneath the plasma membrane, which can exhibit transient pH shifts on the order of several tenths of a pH unit in response to "physiological" cellular activity. These spatiotemporally localized pHi shifts are thought to arise from the local activity of the plasma membrane Ca2+-ATPase and voltage-gated H+ channels (10, 11) or as a consequence of the differential subcellular distribution of the sodium bicarbonate co-transporter or NHE in association with a type II carbonic anhydrase (12). It is expected that other channels or exchangers mediating cellular pHi changes could also give rise to relatively large pHi shifts beneath the plasma membrane. There is also potential for the feedback stimulation of pH regulatory mechanisms as a consequence of raised local cAMP levels (6, 43, 44) or the functional coupling of G protein-linked receptors to AC and acid extruders via divergent signaling mechanisms (45). Thus, there may be a clear coupling between [cAMP]i and pHi.



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  		FIG. 7.
Identification of NHE1 in HEK 293 and C6-2B glioma cells. Primers for NHE1, NHE2, and NHE3 (see Table II) were tested on cDNA reverse-transcribed from mRNA extracted from human kidney, which expresses all three NHE isoforms. Reverse transcription-PCR products from HEK 293 total cellular RNA (+) tested positive for NHE1 only. Controls were performed for DNA contamination (–), where samples were not reverse-transcribed. Primers specific for rat NHE1, NHE2, and NHE3 tested positive on cDNA from rat kidney. In contrast, cDNA from C6-2B rat glioma cells tested positive for NHE1 alone. {beta}-Actin ({beta}) was used as a positive cDNA control for all four cell types.

 
To our surprise, despite the high sensitivity of the Ca2+ regulation of the ACs to pH changes in vitro, equivalent global changes in pH in the intact cell did not alter the response of the ACs to CCE. Evaluation of a number of possible mechanisms revealed that the effects of pHi on AC Ca2+ sensitivity could be reduced in vivo due to pH-dependent alterations in the degree of CCE induced for a given extracellular [Ca2+]. The pHi sensitivity of CCE was most striking during ~0.35-pH unit alkalinization of HEK 293 cells, which resulted in a 30% decrease in CCE. These findings are in contrast to previous studies showing an enhancement of CCE under alkaline pHi conditions (36). The pH dependence of CCE in HEK 293 cells results in an underestimate of the effectiveness of pHi to modulate the Ca2+-regulated ACs and could go some way to explain the discrepancy between our in vitro and in vivo data.

Very significantly, however, we were able to prove that NHE1 was localized to the same subcellular compartments (caveolae) that expressed high levels of functional AC8. A similar distribution of NHE1 was also observed in membrane fractions from C6-2B glioma cells. This is consistent with the distribution of transfected NHE1 to caveolin-containing fractions in AP-1 cells (46). Several other studies have also shown that NHE1 is not homogeneously distributed throughout the plasma membrane. For example, NHE1 can localize to basolateral membranes of polarized epithelial cells (47) or to lamellipodia (48). The localization of NHE1 to lipid rafts or caveolae may generate microdomains that facilitate acid extrusion and protect the ACs from local fluctuations in pHi. Indeed, inhibition of the exchanger with cariporide revealed that it is the main acid-extruding mechanism in HEK 293 cells, in the absence of CO2 and . Furthermore, regulation of pHi within caveolae may be fine tuned by the selective activation of AC-coupled receptors that up- or down-regulate NHE activity (43). A recent study by Donowitz and co-workers (49) also revealed the localization of NHE3-isoform to lipid rafts. Studies in rat myocardium support the theory that specific targeting of NHE1 can create microenvironments for pH-sensitive proteins (e.g. connexin 43 and ryanodine receptor) (50). However, we propose that the targeting of NHE1 to caveolae serves to protect pH-sensitive proteins (e.g. AC8 and AC6) from acid shifts rather than to modulate their activities (50).



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  		FIG. 8.
Localization of AC8 and NHE1 in caveolin-containing raft membranes (cav) from HEK 293 cells. Nondetergent fractionation of HEK 293 cell membranes and immunoblotting was conducted as described under "Experimental Procedures." Blotting with AC8 or NHE1 antibody shows enrichment of the Ca2+-sensitive adenylyl cyclase and NHE1 in caveolin-containing membrane fractions 4 and 5, consistent with caveolar membranes. Immunoblotting with an antibody against {beta}-adaptin, a marker of nonraft membranes, produced positive bands in fractions 7–9, suggesting the presence of a small proportion of NHE1 in this domain.

 



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  		FIG. 9.
Localization of NHE1 in caveolin-containing raft membranes (cav) from C6-2B glioma cells. Nondetergent fractionation of C6 glioma cell membranes and immunoblotting was conducted as described under "Experimental Procedures." Blotting with monoclonal NHE1 antibody shows enrichment of NHE1 (~90 kDa) and caveolin in fractions 4 and 5, consistent with caveolar membranes.

 
Although the high expression of NHE1 in caveolar microdomains appears to preclude pH-dependent modulation of Ca2+-regulated ACs over the time course of our experiments, we do not discount the likelihood that dynamic shifts in caveolar pHi can be generated per se. Evidence for plasma membrane Ca2+-ATPase localization to caveolae (51–53) suggests that these subcellular domains are likely to be exposed to substantial acid loads as a consequence of calcium extrusion in exchange for protons (10, 11). Hence, it is possible that CCE, known to regulate the ACs, could also mediate a local acid shift. However, it is expected that these acidifications would rapidly collapse as a consequence of NHE1 activity. It is reasonable to conclude from our data that, although global falls in pHi are fairly sustained during weak acid application, localized recovery from the imposed acid shifts in caveolar regions may be complete prior to the start of our cAMP measurements. At present, it is not known if additional acid entry/extrusion pathways are localized to the caveolae. There is some conflicting evidence for the presence (54) or absence (55) of the proton-translocating H+-ATPase in caveolar membranes in cells originating from the kidney. Thus, it is possible that the H+-ATPase could also contribute to local pHi regulation in C6-2B glioma cells (56) and HEK 293 cells (38, 57) in the absence of CO2/HCO–3.

In conclusion, the potential for interactions between H+, Ca2+, and cAMP (9) was clearly demonstrated with respect to the pH sensitivity of Ca2+-regulated ACs in vitro. However, an abundance of NHE1 activity in the caveolae appears to generate a privileged domain that protects the ACs from local acid shifts. The physiological relevance of this situation may simply be to optimize the catalytic activity of the ACs and numerous other caveolar proteins by maintaining pHi at values that are close to pH 7.50. Only under conditions, where NHE1 activity is significantly reduced would we expect to see pH-dependent modulation of AC activity during Ca2+ entry. Thus, further evidence is gathered of the regulatory microclimate that ensures the unperturbed functioning of these critical signaling molecules.


   FOOTNOTES
 
* This work was supported by the Wellcome Trust and National Institutes of Health Grant GM32483. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1PD, UK. Tel.: 44-1223-334063; Fax: 44-1223-334040; E-mail: dmfc2{at}cam.ac.uk.

1 The abbreviations used are: pHi, intracellular pH; pHo, extracellular pH; AC, adenylyl cyclase; CCE, capacitative calcium entry; EIPA, ethyl isopropyl amiloride; NHE, sodium hydrogen exchanger; PA, propionate; TMA, trimethylamine; BCECF, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
Human and rat kidney RNA were gifts from Drs. Morris Brown and Cambridge Biotechnology, respectively. Cariporide was a generous gift from Dr. Juergen Puenter at Aventis Pharma (Frankfurt, Germany).



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