Rapid acid extrusion response triggered by {alpha}1 adrenoceptor in CHO cells

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Rapid acid extrusion response triggered by $alpha$ ₁ adrenoceptor in CHO cells

Takanobu Taniguchi, Rika Inagaki, Fumiko Suzuki and Ikunobu Muramatsu

Department of Pharmacology, School of Medicine, Fukui Medical University, 23 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan

MS 12413 Resubmitted 5 March 2001; accepted 27 April 2001

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Using a microphysiometer with synchronized valve switching, we investigated real-time acid extrusion from Chinese hamster ovary (CHO) cells in which human $alpha$ ₁ adrenoceptor (AR) is stably expressed, in response to noradrenaline (NA).
In the cells expressing $alpha$ _1a AR, the time course of extracellular acidification after stimulation had two phases; in the first phase it transiently reached a rate several times greater than the base rate with a peak at around 10 s, and in the second it increased to 2 times the base rate and reached a plateau in 2 min. Both phases showed a concentration-dependent increase of acidification rate in response to NA, but had distinct pEC₅₀ values; 5.6 for the transient phase and 7.2 for the steady phase.
In the cells expressing $alpha$ _1b AR, the transient phase was not detected but the steady phase was observed. The pEC₅₀ value was 7.1, although the magnitude of the response was much smaller than that with $alpha$ _1a AR.
Both 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and HOE642 inhibited the acid extrusion response by either AR in a concentration-dependent manner. EIPA and HOE642 had high pIC₅₀ values (7.4 and 7.3, respectively) for inhibition of the transient phase response via $alpha$ _1a AR. In the inhibition of the steady phase response via $alpha$ _1a AR, both drugs revealed the presence of two components in the response; one had high pIC₅₀ values (8.1 and 8.2 for EIPA and HOE642, respectively) and the other had low pIC₅₀ values (5.6 and 6.0, respectively). In contrast, the steady phase response via $alpha$ _1b AR was inhibited by EIPA and HOE642 with low pIC₅₀ values (5.3 and 5.9, respectively).
As Ca²⁺ was depleted, the $alpha$ _1a AR-induced transient phase disappeared, while the steady phase was not affected.
These results suggest that $alpha$ _1a AR drives two acid extrusion systems in CHO cells upon stimulation; one elicits the transient response, which is largely mediated by an EIPA/HOE642-sensitive and Ca²⁺-dependent Na⁺-H⁺ exchanger (NHE), presumably NHE1, and the other induces the steady acid extrusion that is mediated by NHE1 and another NHE which has low sensitivity to both EIPA and HOE642. $alpha$ _1b AR drives only the steady phase acid extrusion response, which is mainly mediated by NHEs other than NHE1.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Until recently, extracellular pH was believed to be maintained within a narrow range of around 7.4 by homeostatic mechanisms. In fact, deviation of the pH from this value in body fluid (either acidosis or alkalosis) leads to serious malfunction of cellular processes (Adrogue & Madias, 1998). However, investigations in the past few years have revealed that extra- as well as intracellular pH are not so constant as generally thought, and it has even been hypothesized that H⁺ acts, not secondarily to metabolic changes or ionic membrane transport, but as a signalling molecule to initiate or relay intercellular communications (Chesler, 1990; Deitmer & Rose, 1996).

In general, the buffering capacity of the extracellular space and net movement of acid/base equivalents determine extracellular pH. In the former, the intrinsic buffer value and volume of extracellular fluid are major factors. In the latter, many molecules such as channels, pumps and transporters are involved (Chesler, 1990; Deitmer & Rose, 1996). Although several methods such as pH-sensitive microelectrodes and magnetic resonance are generally used to estimate extracellular pH, these methods have certain drawbacks and difficulties, in particular their poor quantitative evaluation and/or low time resolution (Chesler, 1990; Smith et al. 1998). The microphysiometer is a potentiometric system that is able to detect small changes of pH in extracellular fluid accurately (McConnell et al. 1992). It has been employed to monitor cellular responses to various stimuli quantitatively; however, it does not produce real-time recordings (Taniguchi et al. 1999). We employed a microphysiometer with synchronized valve switching to estimate the real-time change of extracellular pH in Chinese hamster ovary (CHO) cells expressing human $alpha$ ₁ adrenoceptor (AR). We report a rapid acid extrusion from CHO cells in response to noradrenaline (NA), which is mediated by a Na⁺-H⁺ exchanger (NHE). We characterized the acid extrusion response and found that it occurs in two phases, involving at least two NHEs.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Materials

Alpha minimum essential medium, fetal bovine serum and G418 were from Gibco BRL (Grand Island, NY, USA); prazosin HCl and (-)-noradrenaline HCl (NA) were from Sigma (St Louis, MO, USA); 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thapsigargin, phorbol 12-myristate 13-acetate (PMA), calphostin C and U73122 were from Research Biochemicals International (Natick, MA, USA); EGTA was from Nacalai (Kyoto, Japan); HOE642 was from Aventis Pharma Ltd (Tokyo, Japan).

Cell culture

Two clones of CHO cells, aH5 and bH16, which stably express human $alpha$ _1a (370 fmol mg^-1 membrane protein) and $alpha$ _1b (350 fmol mg^-1 membrane protein) AR (Taniguchi et al. 1999), respectively, were maintained in alpha minimum essential medium containing 10 % fetal bovine serum and 200 µg ml^-1 G418 at 37 °C in a humidified atmosphere of 5 % CO₂-95 % O₂.

Measurement of extracellular acidification rate (EAR)

Cells were seeded into the microphysiometer cups at 3 times 10⁵ cells per cup, 24 h prior to the experiment (Taniguchi et al. 1999). Low-buffered balanced salt solution (LBS) was used as running medium, the composition being 1.5 mM CaCl₂, 3 mM KCl, 0.6 mM MgCl₂, 130 mM NaCl, 5 mg l^-1 phenol red, 10 mM glucose, 0.2 mM KH₂PO₄ and 0.8 mM Na₂HPO₄, pH 7.4. Figure 1A shows the raw data of the microphysiometer recording, which reflects extracellular pH; when the pump is perfusing the medium into the chamber the pH remains constant and while the pump is off the pH decreases according to the acid excretion from the cells in the chamber. The EAR was measured in each 120 s pump cycle; flow 'on' at 100 µl min^-1 for 60 s, flow 'off' for 60 s. Because of the dead space between the chamber and the valve in the microphysiometer, there is a 6 s time lag following a switch of the valves for the fluid to reach the chamber (Fischer et al. 1999). Cells were exposed to NA 1 s before flow 'off' by switching valves 7 s prior to pump 'off', allowing real-time monitoring of extracellular acidification from 1 to 61 s after NA exposure. NA was continuously added during the next cycle to assess the response from 121 to 181 s during the NA stimulation (see Fig. 1A). After baseline rate became stable, cells were stimulated with 10 µM NA 3 times over a 30 min interval and the third response was taken as a standard. Then, an increasing concentration of NA was added to obtain the concentration-response relationship. In inhibitor studies, a standard response to 10 µM NA was recorded without the inhibitor, then the cells were perfused with LBS containing inhibitor 15 min prior to and throughout the following responses to NA. EC₅₀ and IC₅₀ values were calculated, using PRISM (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± S.E.M.

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Figure 1. Real-time assessment of extracellular acidification (A) and EAR (B)
CHO cells expressing human $alpha$ _1a AR subtype were stimulated with 10 µM NA as described in Methods. Representative data from real-time monitoring of extracellular pH (as mV) are shown in A for aH5 cells (continuous line) and mock-transfected cells (dotted line). Open bars below the graph indicate that the pump is 'on'. In B, the time course of the relative increase in EAR after NA stimulation was calculated by dividing the rate at each time point by the mean value at the corresponding time points of the three preceding cycles. There is a gap in the normalized data between 61 and 121 s during the period of LBS perfusion. NA elicited a rapid and severalfold increase in EAR that was followed by a plateau and a 2-fold increase in EAR (thick line). These increases were not seen in mock-transfected cells (dotted line) and were suppressed by the presence of 1 µM prazosin (thin line).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Two-phase response

Synchronizing valve switching in the pump cycle enabled us to monitor a real-time change in extracellular pH in the microphysiometer chamber as shown in Fig. 1A. To estimate the acceleration of acid extrusion, we normalized the stimulated EAR by the mean value of three preceding cycles at each corresponding time point (Fig. 1B). Since it is impossible to estimate the pH change during the period of LBS perfusion, there is a gap in the normalized data between 61 and 121 s. As shown in Fig. 1B, NA elicited a rapid transient increase in EAR that was followed by a plateau steady phase. Both the transient and steady phases of the EAR increase were suppressed by pretreatment of aH5 cells with 1 µM prazosin (an $alpha$ ₁ AR antagonist) and were not seen in mock-transfected cells, indicating that the acid extrusion was mediated by $alpha$ _1a AR. The transient phase reached a peak at around 10 s after NA stimulation and the steady phase maintained a plateau between at least 91 and 241 s (Fig. 1 and data not shown). We examined intracellular pH at the time of stimulation with NA but found little change in aH5 cells (data not shown). This suggests that the acid extrusion following $alpha$ _1a AR stimulation is not a secondary event to elevated acid generation in the cells.

Pharmacological characterization of the acid extrusion response

In aH5 cells, both phases displayed a concentration-dependent response but they had distinct pEC₅₀ values for NA (5.6 and 7.2 for the rapid and steady phases, respectively), as shown in Fig. 2. In bH16 cells, no apparent transient phase was detected but the steady phase was observed with a pEC₅₀ value of 7.1, which is equivalent to that seen in aH5 cells, although the magnitude of the response was less than 20 % of that in aH5 cells (Fig. 3). We then examined two inhibitors of NHEs, EIPA and HOE642, in the acid extrusion responses at a submaximal dose of NA: 30 µM for the transient phase and 1 µM for the steady phase. Both inhibitors showed concentration-dependent inhibition of the acid extrusion responses and completely blocked both phases at 10 µM (Fig. 4), indicating that the NHE predominantly functions to extrude H⁺ in these responses. EIPA and HOE642 inhibited the transient phase response via $alpha$ _1a AR with high pIC₅₀ values (7.4 and 7.3, respectively). In the inhibition of the steady phase response via $alpha$ _1a AR, both drugs revealed two components of the response; one had high pIC₅₀ values (8.1 and 8.2 for EIPA and HOE642, respectively) and the other had low pIC₅₀ values (5.6 and 6.0, respectively). In contrast, they inhibited the steady phase response via $alpha$ _1b AR with low pIC₅₀ values (5.3 and 5.9, respectively). These results suggest that at least two NHEs are involved in the $alpha$ ₁ AR-induced acid extrusion response.

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Figure 2. Concentration-dependent response of EAR for NA
The aH5 cells were stimulated with an increasing concentration of NA (10 nM to 0.3 mM) and the relative increase in EAR was assessed as in Fig. 1. A representative set of data is shown in A. Responses in the transient phase (A, left) and the steady phase (A, right) were analysed for peak values and mean values between 126 and 176 s, respectively, taking the values in each response at 10 µM NA as 100 % (B). Data represent means ± S.E.M. from 3 independent experiments.

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Figure 3. Comparison of EAR responses between $alpha$ _1a and $alpha$ _1b ARs
The aH5 (a, utri ) and bH16 (b, cir ) cells were stimulated with an increasing concentration of NA (1 nM to 0.1 mM) and the relative increase in EAR was assessed as in Fig. 1. A representative EAR response at 0.1 mM NA stimulation is shown in A. Responses in the steady phase were analysed for mean values between 126 and 176 s in B. Data represent means ± S.E.M. from 3 and 8 independent experiments for $alpha$ _1a and $alpha$ _1b ARs, respectively.

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Figure 4. Effect of NHE inhibitors on the acid extrusion response
The aH5 (A and B) and bH16 (C) cells were stimulated with 30 µM NA for the transient phase (A) or with 1 µM NA for the steady phase (B and C) in the presence of EIPA (open symbols) or HOE642 (filled symbols). Both EIPA and HOE642 showed concentration-dependent inhibition of the acid extrusion response. Data represent means ± S.E.M. from 3-6 independent experiments.

Next, we investigated signal transduction pathways in the acid extrusion response via $alpha$ _1a AR. U73122, an inhibitor of phospholipase C (PLC), at 10 µM suppressed both the rapid and steady phases of acid extrusion stimulated by NA (Fig. 5B), suggesting that both phases are downstream of PLC. Elimination of extracellular Ca²⁺ abolished the transient phase in the second stimulation without affecting the steady phase (Fig. 5C). Replenishment of Ca²⁺ restored the transient phase of the response (data not shown). Thapsigargin at 3 µM also abrogated the transient phase when the stimulation was repeated but did not affect the steady phase (Fig. 5D). These results suggest that rapid recruitment of intracellular Ca²⁺ is a key event in the mechanism of the rapid activation of the NHE following $alpha$ _1a AR stimulation. We next investigated an involvement of protein kinase C (PKC) and found that neither the depletion of PKC by overnight pretreatment with PMA nor its inhibition by the addition of calphostin C (0.3 µM) affected this two-phase acidification response induced by NA (data not shown). PKC does not seem to be involved in the NA-induced acid extrusion reaction in these cells.

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Figure 5. Characteristics of the transient phase and the steady phase of the EAR response
As shown in A, responses to 10 µM NA were first recorded without an inhibitor (dotted line in B-D), and the cells were then perfused with LBS containing an inhibitor: 10 µM U73122 in B, 0.3 mM EGTA in C, and 3 µM thapsigargin in D, 15 min prior to the next stimulation with NA (thin line for the first stimulation and thick line for the second in B-D). In the case of EGTA addition (C), CaCl₂ was eliminated from LBS. Responses in the transient and steady phases are shown on the left and right, respectively. Representative data from 3-5 experiments are shown.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

Our data demonstrate that acid extrusion in response to stimulation of $alpha$ _1a AR in CHO cells is a biphasic response (Fig. 1 and Fig. 2). The difference between the pEC₅₀ values in the two phases may reflect a higher coupling efficiency in the signalling pathway for the steady phase than for the transient one. The NHE inhibitors EIPA and HOE642 showed that at least two distinct NHEs are involved in the acid extrusion response; one is highly sensitive to these inhibitors and the other is not. The pIC₅₀ values of the inhibitors were 7-8 for the sensitive one and 5-6 for the insensitive one. In addition, the transient phase response, which is mediated mainly by a sensitive NHE, was Ca²⁺ dependent but the steady phase response was not affected by Ca²⁺ depletion (Fig. 5C). Six members of the NHE family have been reported so far in mammalian cells (Counillon & Pouyssegur, 2000). One member, NHE-1, has a high sensitivity to both EIPA (Yu et al. 1993) and HOE642 (Scholz et al. 1995); in addition it has a calmodulin-binding site that enables its rapid activation in response to the increase in intracellular Ca²⁺ (Bertrand et al. 1994; Wakabayashi et al. 1994) . We speculate that NHE-1 is likely to mediate the transient phase of acid extrusion following $alpha$ _1a AR stimulation in CHO cells. Furthermore, it has been reported that the $alpha$ _1b subtype has a lower coupling efficiency in activating PLC than the $alpha$ _1a subtype, and so exhibits a much more modest response in intracellular Ca²⁺ recruitment (Minneman et al. 1994; Theroux et al. 1996). This is in accordance with the lack of a transient phase and of participation of the EIPA/HOE642-sensitive NHE1 in the acid extrusion response via $alpha$ _1b AR (Fig. 3 and Fig. 4). However, activation of NHEs by PKC or Ca²⁺ is largely dependent on cellular background (as reviewed by Noel & Pouyssegur, 1995; Wakabayashi et al. 1997) and involvement of PKC or Ca²⁺ in the stimulation of NHE by $alpha$ ₁ AR is also variable; PKC-dependent (Iwakura et al. 1990; Otani et al. 1990; Wallert & Frohlich, 1992; Martin-Requero et al. 1997; Snabaitis et al. 1999) or -independent (Owen, 1986; Puceat et al. 1993) and Ca²⁺-sensitive (Anwer & Atkinson, 1992; Martin-Requero et al. 1997) or -insensitive (Iwakura et al. 1990; Puceat et al. 1993) activation have been reported. Thus, further investigation is required to elucidate the identity of the NHEs and their regulation in $alpha$ ₁ AR-induced acid extrusion.

In general, the buffering capacity of the extracellular space and net movement of acid/base equivalents determine extracellular pH. In terrestrial animals, the extracellular fluid has a certain buffer value at around 50 mM (Chesler, 1990; Deitmer & Rose, 1996). An additional factor that affects buffering capacity is the volume of fluid in the extracellular space. In humans, for example, at 70 kg body weight, the extracellular fluid consists of approximately 10¹⁴ cells in a volume of 10 l, resulting in 10^-7 µl extracellular fluid per single cell. Buffering capacity is, thus, 5 fmol per cell in humans. In the case of microphysiometry, the buffer is 1 mM phosphate with approximately 0.5 mM buffer value and the space for cellular response in the chamber is a few microlitres (McConnell et al. 1992) for 3 times 10⁵ cells. Therefore, the buffering capacity in microphysiometry will also be approximately 5 fmol per cell. Thus, it is possible to deduce that rapid acidification in response to NA might take place in a limited local space in vivo as seen in the microphysiometer, in which approximately 0.05 pH acidification was observed within 1 min (Fig. 1A) since 1 pH acidification is equal to a 61 mV decrease in this system (McConnell et al. 1992). In fact, a pH change in the extracellular space has been reported following electrical and neurotransmitter stimulation in the nervous system (reviewed by Deitmer & Rose, 1996), acetylcholine stimulation in the adrenal medulla (Viglione et al. 1994), illumination of the retina (Oakley & Wen, 1989), ischaemia in the heart (Yan & Kleber, 1992; Marzouk et al. 1998) and a respiratory burst in neutrophils (van Zwieten et al. 1981; Borregaard et al. 1984; Wright et al. 1986). However, these authors employed a microelectrode or titration technique, which does not allow quantitative and/or real-time analysis. With the microphysiometer, quantitative and real-time analysis of extracellular pH may allow us to investigate the dynamic regulation of the acid extrusion mechanism including NHEs, which has conventionally been carried out by an acid load technique where the situation is unlikely to be physiological.

In conclusion, we have reported that $alpha$ _1a AR elicits a rapid acid extrusion in which the transient phase and the steady phase are clearly discriminated by distinct pEC₅₀ values for NA and by a dependence on intracellular Ca²⁺. NHE-1 is likely to mediate the Ca²⁺-dependent transient phase. Based on the evidence presented here, extracellular pH may rapidly fluctuate in vivo as cells are stimulated and might play an active role in intercellular signalling.

REFERENCES

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Abstract
Introduction
Methods
Results
Discussion
References

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Acknowledgements

We are grateful to Dr K. Hashimoto, Department of Pharmacology, Yamanashi Medical University, for providing information about NHE inhibitors. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

Corresponding author

I. Muramatsu: Department of Pharmacology, School of Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan.

Email: muramatu{at}fmsrsa.fukui-med.ac.jp

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