H+ exchange

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Regulation of cytoplasmic pH by Na+/H+ exchange Wouter H. Moolenaar Cytoplasmic p H (pH) is an important variable affecting many intracellular biochemical reactions and therefore needs to be closely regulated. Vertebrate cells maintain their p H i within the range 7.0-7. 4 by means of a transport system in the plasma membrane that mediates the electroneutral exchange of external Na + for internal H + This Na+/H ÷ exchanger responds to a fall in p H i by rapidly extruding protons out of the cell. The pH sensitivity of virtually all enzymatic reactions implies that animal cells must maintain their cytoplasmic pH (pHi) within a narrow range to provide a favourable environment for various intracellular activities. In recent years, much has been learned about the mechanisms by which pH i is controlled in a wide variety of cell types 1-5. In addition, there is growing evidence that cells may utilize pH i as a regulator of cellular functions, particularly those that are essential for the onset of cell proliferation and development 6-9. Most animal cells maintain their pH i in the range 7.0-7.4, which is considerably higher than would be expected if H + were passively distributed across the plasma membrane. For example, at an extracellular pH (pHo) of 7.4 the resting pH i of quiescent human fibroblasts is near 7.0 (Ref. 4), while their electrical transmembrane potential is more than 60 mV (interior negative) 1°. Electrochemical equilibrium for H + would then predict a pH i of less than 6.4, a cytotoxic value far below the actual pH i. Therefore, an active H + extruding system must be present to cotmteract the acidifying tendency of passive H + influx or O H efflux). Furthermore, metabolically produced acids like CO x and lactate impose an additional chronic acid load on the cell's interior, which also needs to be balanced by H ÷ ejection. In vertebrate cells, the regulation of pH i is predominantly achieved by a transport system in the plasma membrane that mediates the electroneutral, amiloride-inhibitable exchange of extracellular Na + for intracellular H ÷. This Na+/H + exchanger (or antiporter) responds to a fall in pH i by rapidly extruding H + from the cell, thereby returning pH i to its normal value. This review will briefly summarize some of the major features of pH i regulation by Na+/H + eXW.H. Moolenaar is at the Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

change in mammalian cells. For extensive reviews on the topic of pH i and its regulation the reader is referred to Refs 1-3.

Methods of investigation The most informative method of studying the mechanisms of pH i regulation in intact cells is to artificially acidify the cytoplasm and to analyse the subsequent recovery of pH i toward its initial value. This strategy requires that transient changes in pH i are monitored with adequate temporal resolution. Until recently, our knowledge of cellular pH homeostasis came mainly from studies using pH-sensitive microelectrodes in large invertebrate neurons and various muscle fibres 1-3. Although certain types of pH-sensitive microelectrodes are suited to small mammalian cells in culture as well 11, the technique is rather difficult and requires elaborate electronic instrumentation. An easier and widely used method for estimating pH i in populations of cultured cells is to measure the equilibrium distribution of a radio-labelled weak acid or base across the plasma membrane. This method, however, shows a rather poor temporal resolution and has some other limitations such as sensitivity to cellular volume changes and intracellular compartmentalization of the radioactive tracer 1. The recent introduction of novel pHsensitive dyes which can be trapped, intraceUularly provides a powerful and relatively simple way of continuously monitoring rapid changes in pH i in small cells, either in suspension or attached to a substrate. In this method, a derivative of fluorescein (with a pK a near 7.0) is introduccd into the cytoplasm, either by uptake of its membrane-permeable ester4,5,s, I2 or through endocytosis 13, and the pH-sensitive fluorescence intensity is continuously recorded. This method combines a high pH sensitivity with an excellent response time and is widely applicable. The fluorescence technique has successfully been employed to

examine the mechanisms of pH i recovery from acidification in such diverse cell types as fibroblasts4, 8, carcinoma cells 13 and lymphocytes 5. There are several methods of acidifying the cytoplasm of small cells. Lowering pH i by simply incubating the cells in acidic media is a rather slow and inefficient process. For example, most cultured cells respond to an extracellular pH shift of 0.2 unit by a change in pH i of --0.1 unit after 15-30 min. A much better method of acid loading is to suddenly expose the cells to a buffer containing a weak acid, such as bicarbonate or acetate. Undissociated acid molecules then freely permeate the plasma membrane, giving rise to excess H + upon their intracellular dissociation. A disadvantage of this acidification method is that the continuous presence of the weak acid affects the intracellular buffering capacity and thereby the kinetics of pH i recovery, and that pH i recovery is often incomplete due to an outward leak of the weak acid anion down its electrochemical gradient 1,2. Perhaps the most useful and convenient procedure of abruptly lowering pH i in any cell type is to briefly pretreat the cells with an NH4+/NH3-containing buffer, as originally described for squid axon 1,2. This elegant method lacks the disadvantages of the weak acid procedure and is based on the principle that exposing cells to NH4 + leads to the net passive influx of both NH 3 and NH4 +, whereby NH4+ acts like a proton carrier ~-3. When external NH4+ is then suddenly removed there is a dramatic fall in pHi: the accumulated NH4 + is forced to leave the cell as NH 3, thereby loading the cell's interior with an excess of protons.

Recovery of pHi from acidification Figure la shows a typical example of the time course of pH i recovery from an NHa+-induced acid load in cultured human fibroblasts, as measured by the fluorescence of intracellularly-trapped BCECF [bis(carboxyethyl)-carboxyfluorescein]. The abrupt cytoplasmic acidification is followed by a very rapid recovery of pH i to its normal value of near 7.0. Recovery of pH i is reversibly inhibited by the diuretic amiloride; it usually follows an exponential time course and is virtually complete in less than 5 min. The following lines of evidence indicate that the pH i recovery process is largely mediated by net H + extrusion through a Na+/H + exchange mechanism in the plasma membrane: (1) pH i recovery is accompanied by Na +

1986, Elsevier Science Publishers B.V., Amsterdam

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uptake and H + effiux with an apparent stoichiometry of 1:1; (2) both pH i recovery and concomitant Na + and H + fluxes are blocked by amiloride; (3) pH i recovery is completely dependent on extraeellular Na + (Li+), half-maximal recovery rates occurring at 15-50 mM Na +. At a given level of H + pumping activity, major determinants of the rate of pH i recovery are the intraceIlular buffering capacity 1 and the surface-to-volume ratio of the cell type under study. The latter parameter is very large for small and flattened ceils in monolayer culture, which explains the high rate of pH i recovery from acidification (up to - 0 . 3 pH units per minute). Figure Ib illustrates the rate of Na+/ H + exchange as a function of time during pH i recovery from acidification. Under steady-state conditions, Na+/H + exchange activity is usually very low and exactly balances the acidifying effects of passive H + influx and intracellular acid production. (Thus, it can be predicted that in rapidly metabolizing cells, like lactate-producing tumour cells, the basal Na+/H + exchange activity required to maintain a stable resting pH i will be

acid load 7O

pHi

,/ /

~

ami[oride

65 b) 10

Y o5

+= z

o

.....

-*ami(oride

Fig. 1. Regulation o f pH, by Na+/H + exchange in quiescent human fibroblasts in monolayer culture. (a) Recordings show the recovery o f pHi from an N H 4+-induced acid load toward its initial value in the absence or presence of amiloride (1 mM), as measured by the fluorescence intensity of intracellularly trapped BCECF. Full details in R e f 4. (b) Model experiment showing the transient increase in Na+/H ÷ exchange activity during p H i recovery above. Dashed line represents full inhibition by amiloride. Na+IH + exchange rate is given in arbitrary units and is usually estimated from the initial rate of amiloride-sensitive Na + uptake and/or H + exit.

higher than in metabolically inactive cells like quiescent fibroblasts.) A dramatic stimulation of the Na+/H + exchange rate occurs as soon as an acute acid load is applied, while the H + ejecting activity returns to the control level once pH i has attained its resting value again. It is clear that Na+/H + exchange represents a very efficient PHi-regulating system that accelerates H + extrusion in response to a fall in PHi, thereby protecting the cell against intracellular acidosis. A significant amount of Na + ions flows into the cell via the Na+/H + exchanger during its stimulation, but the Na+/K +ATPase removes them as fast as they enter (Fig. 2). Some properties of the Na+/H + exchanger Although nothing is known about the molecular structure of the vertebrate Na+/H + exchanger, much has been learned about its functional characteristics. Many of the basic properties of the Na+/H + exchanger have been inferred from the kinetics of pH i recovery and/or concomitant Na + and H + fluxes both in intact cells and in isolated membrane vesicles. Na+/H + exchange is driven by the steep transmembrane Na + gradient, which, in turn, is generated by the Na+/ K+-ATPase (Fig. 2). Depending on the size and direction of the transmembrane gradient for Na +, the Na+/H+ exchanger can mediate net transport of H + either into or out of the cell. Under normal ionic conditions, the exchanger is operating in its H + ejecting mode. It should be noted, however, that the value of the resting pH i in vertebrate cells is not simply determined by the magnitude of the transmembrane Na + gradient: under physiological conditions (INa+]o/ [Na+]i = 10) the transmembrane Na + gradient could theoretically raise pH i about one unit above pHo, which is much higher than the actual steady-state pH i value. Thus, the Na+/H + exchanger is normally poised far from thermodynamic equilibrium. How is the activity of the Na+/H + exchanger regulated? It appears that the major determinant of the Na+/H + exchange rate is the value of pH i. When pH i is reduced below its resting value, the activity of the Na+/H + exchanger is increasingly stimulated. In fact, the relationship between the exchange rate and pH i is approximately linear and steeper than expected for a first-order Michaelis-Menten type of reaction involving a single binding site for internal H +. Obviously, the relatively strong dependence of the Na+/H + exchange rate

H ÷

Na ÷

K*

out

Fig. 2. Schematic representation of the Na+/H + exchanger and the Na+,K+-ATPase in the plasma membrane. For each proton extruded one Na ÷ flows into the cell, which, in turn, is removed by the A TP-dependent Na +, K + pump.

on the internal H + concentration makes the exchanger ideally suited to its physiological role as a pH i regulator. Aronson et al. 14,15 have proposed that cytoplasmic H + acts as an allosteric activator of the Na+/H + exchanger. According to this concept the exchanger has at least two separate and functionally distinct H + binding sites on its cytoplasmic face. A regulatory or 'modifier' site whose occupancy triggers a conformational change that sets the exchanger in motion, and a distinct H + transport site mediating the net extrusion of H + once the exchanger is activated. The diuretic amiloride has generally been found to inhibit Na+/H + exchange activity by competing with Na + for binding to the same external site (K i - 2-5 pM). Since the reported K m for external Na + is in the range of 10-50 mu, rather high ( - 1 mM) concentrations of amiloride are required to effectively inhibit Na+/H + exchange at physiological Na + concentrations. Several amiloride analogues have been found to be up to - 1 0 0 times more potent than amiloride in blocking Na+/H + exchange and pH i recovery 16-1s. Unfortunately, however, amiloride and several of its potent analogues have non-specific side effects on various metabolic activities both in intact cells and in cell-free preparations 18A9. Hence, extreme caution is needed in attributing amiloride-sensitive changes in cell function to inhibition of N a + / H + exchange. Effects of bicarbonate In invertebrate cells like snail neuron, barnacle muscle and squid axon, the pH iregulating mechanism has an absolute requirement for extracellular H C O 3- as well as for Na ÷ (Refs 1-3). This system presumably acts by exchanging external Na + and H C O 3- for internal H ÷ and Cl-. The Na +, HCO3--CI- , H + exchanger is inhibited by stilbene derivatives but is insensitive to amiloride. It is becoming increasingly apparent that a similar HCO3--dependent system may be present in the plasma membrane of certain mammalian cells such as mouse

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skeletal muscle 20, h u m a n carcinoma cells 13 and h a m s t e r fibroblasts 21, w h e r e it acts in parallel with but i n d e p e n d e n t l y of the N a + / H + exchanger to eject H + in response to a decrease in p H i. H o w e v e r , in o t h e r m a m m a l i a n cell types, like h u m a n fibroblasts 4 and neutrophils 2z, a H C O a - d e p e n d e n t p r o t o n p u m p s e e m s to play no significant role in p H i recovery from acidification. C o n d u d i n g remarks T h e close regulation o f p H i by Na+/ H + exchange appears to be a c o m m o n property o f virtually all vertebrate cells. A high sensitivity to cytoplasmic H + is the basic instrument by which the exchanger exerts its primary function. It is interesting to note that N a + / H + is an ancient and ubiquitious transport mechanism: it is operating not only in eukaryotic cells but also in bacteria and in mitochondria (for review see Ref. 23), although it is unclear as yet w h e t h e r a similar carrier molecule is involved. Future studies will undoubtedly reveal the molecular properties and structure o f the purified and reconstituted exchanger, while gene identification should ultimately help to elucidate structure-function relationships 24. Finally, it is o f considerable signifi-

cance that Na+/H + exchange, and hence pHi, may participate in t r a n s m e m b r a n e signalling by certain surface stimulants such as mitogens and polypeptide horm o n e s 6,s,9A3, particularly those that act through the phospholipase C - m e d i a t e d hydrolysis of inositol phospholipids 11. This topic will be discussed in a forthcoming T I B S review.

Acknowledgement Research related to this review was supported by the Netherlands Cancer Foundation (Koningin Wilhelmina Fonds).

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