Datu-moduluak

F. John Gennari

University of Vermont College of Medicine, Burlington, Vermont, U.S.A.

INTRODUCTION

Maintenance of a stable intracellular pH is central to cell metabolic pro-cesses and therefore for life itself. Regulation of intracellular [H^þ] involves active transport processes, modulation of production and utilization of organic acids, and chemical buffering. Characterization of these regulatory processes is extremely difficult because of the complex nature of the intra-cellular environment and remains a work in progress. Historically, most attempts at assessment of intracellular [H^þ] have been based on the assum-ption that the interior of the cell is a uniform solution. In fact, the interior milieu is extremely heterogeneous, containing vesicles, mitochondria, and multiple other structures that are known to create microenvironments in the cell with varying pH values (Fig. 1). In addition, cells differ widely in their metabolic and transport characteristics and therefore have differences in the nature of their acid–base homeostasis. Despite these shortcomings, extensive information has been acquired about cell pH and its regulation.

This chapter reviews this information, covering both pH measurements and the nature of the transporters that regulate intracellular [H^þ] at the cell membrane and within its interior microenvironments.

25

ASSESSMENT OF INTRACELLULAR pH Historical Measurements

Our initial knowledge about cell pH came primarily from two techniques—

microelectrode impalement of cells and the use of the weak acid dimethylox-azolidinedione (DMO), whose concentration in cells is pH dependent. Both approaches have major limitations, discussed below, the most important being the assumption that the interior of the cell is a uniform solution.

Nonetheless, results obtained from these two techniques have provided the foundation for investigation into the regulation of cell pH. Both techni-ques demonstrated that cell [H^þ] is much lower than it should be if electro-chemical forces were the only determinants of its distribution.

Microelectrode Impalement

Specially designed pH-sensitive and reference microelectrodes, with tip sizes

<1 mm, are used to assess cell pH (1,2). To measure pH, a double impale-ment is required (pH and reference electrode). Obviously, such a technique has a high probability of cell injury, and problems with electrode seal also Figure 1 Schematic diagram of a generic mammalian cell, showing only the nucleus, cell membrane and the organelles discussed in this chapter. As shown in the figure, there is an inward pathway (endosomes to lysosomes) and an outward pathway (endoplasmic reticulum to secretory granules), both of which demonstrate progressive acidification. The interior of the mitochondria is highly alkaline. The pH values shown for each organelle and for the cytoplasm are approximations.

affect the accuracy of measurement (3). These limitations were partially overcome by electrode design, and by limiting measurements to relatively large cells such as the squid axon. Table 1 summarizes measurements made in muscle and nerve cells in a variety of species. With a few notable excep-tions, these measurements demonstrated intracellular pH values at or near 7.00. In four studies, pH was measured in mammalian muscle cells, and in three out of four of these studies, the pH value obtained is remarkably similar to measurements made with a less invasive technique, evaluating the pH-dependent distribution of DMO (see below and Table 2).

Table 1 Microelectrode Cell pH Measurements^a

Cell ECF Reference

Mammalian muscle

Rat 6.88 7.41 4

6.91 7.40 5

5.99 7.41 1

Mouse 7.07 7.40 6

Nonmammalian muscle

Barnacle 7.31 7.50 7

Crab 6.91 7.80 4

Axon

Snail 7.44 7.80 2

aRepresentative studies.

Table 2 Cell pH Determined by the Distribution of DMO^a

Cell ECF Reference

Human total body 6.94 7.37 8

6.77 7.40 9

6.88 7.43 10

Muscle

Human 6.87 7.40 9

6.90 7.36 11

Dog 7.04 7.37 12

6.93 7.30 13

Rat 6.92 7.49 14

6.75 7.34 15

Rabbit 6.79 7.36 16

Brain

Dog 7.05 7.36 17

Rat 7.07 7.40 18

aRepresentative studies.

Dimethyloxazolidinedione

Dimethyloxazolidinedione is a weak acid (pK⁰¼ 6.13) that diffuses across cell membranes in its undissociated form, but not when the H^þ is dissociated (3,19). Thus, when DMO is added to the extracellular compartment, the amount of DMO that enters and remains in cells at equilibrium is directly related to the transmembrane pH gradient, with concentration falling as cell pH falls. A limitation in the use of DMO to assess cell pH is the indirect nature of the measurement. For example, errors in assessing the volumes of the intra- and extracellular compartments in which the DMO is distributed will affect the estimate of cell pH. Distribution could also be affected by pro-tein binding, or to differential binding to intracellular organelles of differing pH. Finally, one cannot know with certainty whether DMO is toxic to the cell, or that it alters cell metabolism in some way that may affect pH. The limitations and utility of DMO for measuring cell pH have been reviewed in detail (19,20). Despite these limitations, estimates of cell pH using DMO have been remarkably consistent (Table 2), yielding values of 6.8–7.1.

Newer Techniques

New techniques for assessing cell pH have largely replaced the use of DMO and microelectrodes. These techniques have addressed the mechanisms maintaining the relative alkalinity of the intracellular milieu, as well as pH differences among different cell types and within the various microenvi-ronments of the cell. Most widely used is the pH-dependent fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (21). This probe is an ester that does not fluoresce until the ester linkage is cleaved.

The lipidophilic ester crosses the cell membrane and is cleaved by intracel-lular esterases, leaving the native pH-dependent fluorescent compound trapped within the cell. Other trapped pH-dependent fluorescent dyes used for cell pH measurements include acridine orange and the esterified agent, seminaphthorhodafluor-1 (SNARF-1) (22). All these probes have a rela-tively uniform cytoplasmic distribution, and thus give data similar to that obtained using DMO. Their major advantage is that they provide informa-tion about changes in pH in seconds. Techniques to characterize pH in sub-cellular compartments have used physically targeted substances to localize, for example, in lysosomes, golgi, or mitochondria (22–25). In addition, spec-tral imaging microscopy has been coupled with fluorescent dyes to localize activity to specific subcellular sites (22,25). The results obtained with these newer techniques are discussed later in the chapter.

NONEQUILIBRIUM DISTRIBUTION OF H^þ

Given the normal potential difference across most cell membranes (cell interior 90 mV negative as compared to the exterior), intracellular pH would be 5.8–6.0 if H^þ was distributed passively across the cell membrane (20).

The measurements shown in Tables 1 and 2 thus not only established values for cell pH, but also demonstrated that [H^þ] is notably lower than expected if its transcellular distribution were determined solely by electrical and che-mical forces. The only exception to this finding appears to be the red blood cell, in which cell [H^þ] is consistent with electrochemical equilibrium (19). In all other cells, regardless of the buffer properties of the intracellular com-partment, energy (i.e., O2 consumption) is continually required to either remove H^þfrom the cell or to add OH
in order to maintain the relative alkalinity of the intracellular environment. This selective ion movement is accomplished by active (or secondary active) transport of H^þand HCO3
, often linked to the transport of other ions (most notably Na^þ, K^þ, and Cl
).

REGULATION OF CELL pH

To maintain cell pH stable at a higher level than its equilibrium value, active and regulated H^þextrusion must be an ongoing process, and the rate of extrusion of H^þmust equal its rate of entry (26). Because HCO₃
can also cross the cell membrane (see below), the rate of HCO3
entry must also equal the rate of HCO3
exit. Cell buffers can minimize the change in pH induced by the addition of H^þor the loss of alkali, but these buffers will not restore pH to its baseline level. In the face of H^þentry, in fact, H^þextrusion (or alkali entry) must be sufficient to back-titrate cell buffers in order to restore pH to its resting level.

Because, as in any biological solution, [H^þ] and [OH
] in cells are determined by the strong ion difference (in addition to carbonic and organic acid concentrations) (27), one could consider regulation of cell pH from the perspective of the strong ion transport processes that establish the intracel-lular concentrations of these ions, rather than looking primarily at H^þand HCO3

movement. As an example, Na^þ- and K^þ -linked Cl
movement across cell membranes modulate intracellular [Na^þ] and [Cl
] in a fashion to promote intracellular alkalinity. Attempting to determine which strong ions are key in the regulation of cell pH is a huge undertaking, however, as they all are likely to participate. In addition, such an approach adds unnecessary complexity. In this chapter (as in most reviews on the subject), the focus is on H^þand HCO3
entry and exit from the cell or organelle.

H^þEntry into Cells

As shown in Fig. 2, H^þ has ready access to cells, through its relationship with the volatile weak acid, H2CO3, as well as through its linkage to a vari-ety of weak organic acids (e.g., lactic acid) (20,28). Carbon dioxide readily traverses the cell membrane, reacting reversibly with water to form H2CO3, a fraction of which dissociates to H^þand HCO3
. As H^þdissociates from H2CO3, the electrochemical forces at play drive it to remain in the cell and drive the associated HCO3
to leave the cell, decreasing cell pH (26). In

addition to these modes of H^þentry, H^þ channels are present in the cell membrane (Fig. 2) (29), although their role in modulating H^þ entry is unclear. These channels may in fact be an elegant way to promote H^þexit from nerve and muscle cells during depolarization (26).

The ability of H^þto enter cells has been used to study the transporters that respond to this entry. Isolated cells can be acidified acutely by a variety of techniques, including increasing bath PCO2 or using NH4Cl (20,30).

These agents induce a rapid fall in intracellular pH, followed by rapid recovery (Fig. 3). The role of various membrane transport proteins in the recovery process can then be assessed by adding specific inhibitors or removing key ions from the bath (see below).

Alkali Exit from Cells

A variety of transporters have been identified, many of which have been cloned (see Chapter 5), that carry HCO3
across the cell membrane. In mam-malian cells, most of these normally operate in mode of HCO3
exit from cells and thus will reduce intracellular pH if not counterbalanced by H^þ extrusion. Membrane transporters that facilitate alkali loss include the Na^þ-independent Cl
=HCO3
exchanger, the Na^þ–HCO3
cotransporter, Figure 2 Pathways for H^þentry and HCO3
exit from cells. Carbon dioxide (CO2) diffuses readily across the membrane and adds H^þto the cell by combining with water to form the weak acid, H2CO3. H^þalso can enter the cell on an organic anion cotransporter (circle), or via an H^þ channel (rectangle). HCO₃
exits the cell in exchange for Cl
entry on one of the AE family of exchange transporters (circle), with both ions moving down their electrochemical gradient.

the K^þ–HCO3

cotransporter, and the Cl
=organic anion exchangers. The energetics and characteristics of these transporters are reviewed in Chapter 5, and in Ref. 26. Most are only present in certain cell types (e.g., epithelial cells). Only the Cl
=HCO₃
exchanger will be discussed further here.

The AE family of electroneutral Na^þ-independent Cl
=HCO3
exchan-gers are widely extant, and in mammalian cells virtually always operate to extrude HCO3
from cells, exchanging this anion for Cl
on a one-to-one basis (Fig. 2) (26,32). The best characterized of these is the so-called band-3 protein (AE1) which is abundant in red blood cell membranes (33). The direction of exchange is dependent on the Cl
and HCO3
transcel-lular electrochemical gradients, and can be reversed by manipulating extra-cellular and=or intraextra-cellular ion concentrations (26). Because intraextra-cellular Cl
is lower and HCO3

is higher than at electrochemical equilibrium in most mammalian cells, the transporter normally brings Cl
into the cell in exchange for HCO3

exit (Fig. 2). Related Na^þ-independent Cl
=HCO3

Figure 3 Schematic representation of the change in intracellular pH induced in an isolated cell by NH₄Cl addition to and then removal from the bath. Addition of NH4Cl rapidly alkalinizes the cell due to NH3entry and recombination with intra-cellular H^þ. Following this alkalinization, NH₄^þslowly enters, causing a gradual fall in pH. With removal of NH4Cl from the bath, NH3rapidly diffuses out of the cell, releasing H^þ from NH₄^þ and markedly acidifying the cell. This drop in pH is followed by rapid recovery to baseline pH due to active H^þextrusion (line A). When the bath solution contains no CO₂or HCO₃
, inhibition of cell membrane Na^þ=H^þ, or removal of Na^þfrom the bath blocks this recovery (line B), indicating a key role for this transporter in cell pH recovery. Time across the entire abscissa is less than 10 min. Source: Abstracted from many experimental observations (see Ref. 31).

exchangers, AE2 and AE3, are present on many epithelial cells, and in smooth and cardiac muscle (26). These isoforms also normally operate to cause a loss of alkali from cells. In some experimental models, the Cl
=HCO3

exchanger appears to be activated by an increase in intracellular pH and to be inactivated by low cell pH, thus serving to regulate cell pH homeostatically in states of alkalemia (34). This property appears to occur primarily in cells of renal origin.

Regulatory Control of Cell pH

Cells contain a variety of membrane transporters that are candidates for regulatory H^þ extrusion. A few are active transporters (e.g., the H^þ -ATPase) but most are dependent on the activity of cell membrane Na^þ=K^þ ATPase to develop the necessary chemical and electrical gradients for linked H^þextrusion or alkali entry. The principal regulatory transporters are diagrammed in Fig. 4. Of these, the Na^þ=H^þexchanger is predominant.

Virtually all vertebrate cells rely primarily on this exchanger to mediate regulatory H^þ extrusion (31,32). As discussed below, notable differences

Figure 4 Examples of membrane transport proteins involved in removing acid from, or adding alkali to, mammalian cells. The Na^þ=H^þ exchanger, shown on the left, plays a central role in regulating cell pH in most cells. This exchanger depends on the electrochemical gradients favoring Na^þentry set up by the Na^þ=K^þ ATPase (not shown). Using the same gradient for Na^þentry, the Na^þ-dependent Cl
=HCO3
exchanger, shown on the right, moves HCO3
into cells as well as remov-ing H^þ. This transporter is present in muscle cells, fibroblasts, neurons and glomer-ular mesangial cells, and accounts for a varying portion of pH regulation in these cells. The H^þ-ATPase shown at the bottom is present only on a subset of cells (mainly epithelial cells), but when present appears to participate in regulation of cell pH.

exist among cell types in the presence and participation of other membrane H^þor HCO3

transporters in maintaining a low intracellular [H^þ].

Na^þ=H^þExchanger

Several isoforms of the Na^þ=H^þexchanger have been cloned and character-ized (see Chapter 5 and Ref. 35). The first of these transporters cloned, NHE1 (termed the ‘‘housekeeper’’ transporter), is ubiquitous, located on the cell membranes of both nonepithelial and epithelial cells (26,32).

NHE1 uses the Na^þ electrochemical gradient generated by the Na^þ=K^þ ATPase to extrude H^þacross the cell membrane in electroneutral exchange for Na^þentry. Recovery of cell pH after acid loading is markedly attenuated after inhibition of this exchanger in a wide variety of cells (see example in Fig. 3) (31,32,36–38). In addition to its importance in restoring cell pH in the face of an acute acid load, NHE1 is central to steady-state pH regula-tion. Inhibition of this transporter uniformly results in a sustained fall in intracellular pH (38,39). Cell membrane Na^þ=H^þexchange activity is pH dependent for several isoforms of the NHE family; the transporter is acti-vated by a fall in intracellular pH, and is largely inactiacti-vated at alkaline pH levels in the cell (35–37,40,41). In addition to intracellular pH, the trans-porter is affected by the pH in the fluid surrounding the cell; an increase in bath pH increases its activity and a decrease inhibits it (42,43).

Six isoforms of NHE family have been identified (NHE1-6) (35). Of these, NHE2 and 3 are confined to the apical membranes of intestinal and kidney epithelial cells. Both are activated by a decrease in intracellular pH and are key transporters for H^þ secretion into the gut and urine. NHE4 is confined to the basolateral membrane of stomach and renal epithelial cells and is also found in brain. This transporter appears to have no pH depen-dence. NHE5 is also found in brain tissue, and NHE6 appears to be a mito-chondrial transporter (44).

H^þ-ATPase

The vacuolar H^þ-ATPase is an electrogenic active transporter present in a subset of specialized cells in the body, including renal tubule and corneal epithelial cells, neutrophils, macrophages, and osteoclasts (Fig. 4) (26). In all these cells, H^þ-ATPase on the cell membrane can be shown to play a role in restoring intracellular pH in response to acute acidification when Na^þ=H^þ exchange is inhibited (36,45–47). Under normal conditions, cell membrane H^þ-ATPase, a relatively slow transporter, most likely works in concert with an Na^þ=H^þexchanger (most commonly NHE1) to control cell pH. A decrease in cell pH induced by increasing PCO2increases the inser-tion of vacuolar H^þ-ATPase into the apical membranes of renal tubular epithelial cells, presumably increasing their capacity for H^þ secretion (41,48,49). Exposure of renal cells to extracellular metabolic acidosis or alkalosis, however, has shown conflicting results; one study shows a

decrease in activity with metabolic acidosis (41), while another shows an increase in activity (50). In neither of these studies was intracellular pH monitored. Vacuolar H^þ-ATPases are key transporters for producing local areas of increased [H^þ] within cells, rather than maintaining the low overall cytoplasmic [H^þ] (see later) (51).

H^þ=K^þ-ATPase

Certain epithelial cells (stomach, kidney, and colon) contain an H^þ=K^þ -ATPase on their limiting membranes that secretes H^þin exchange for entry of K^þ(52). This transporter could function as a regulatory H^þ extrusion mechanism in these cells. Recovery of cell pH after acid loading in interca-lated cells of rabbit cortical collecting ducts appears to have a K^þ-dependent component that is blocked by inhibition of gastric-type H^þ=K^þ-ATPase (53). As with the H^þ-ATPase, this effect can only be demonstrated after inactivation of the Na^þ=H^þ exchanger. In other studies, expression or activity of either the gastric or colonic isoforms of this transporter was not upregulated by the induction of metabolic acidosis, but was by respira-tory acidosis (54–56). The colonic H^þ=K^þ-ATPase in the kidney is upregu-lated in states of K^þ depletion (52,55), reflecting its primary role in K^þ homeostasis. Whether the gastric isoform contributes to the maintenance of cell pH is unclear, but it is unlikely to play an important role.

Na^þ-Dependent Cl
=HCO3
Exchanger

An Na^þ-dependent Cl
=HCO3
transporter is present on the cell membrane of cultured fibroblasts, glomerular mesangial cells and neurons, carrying Na^þand HCO
3 into cells in exchange for H^þand Cl
exit (Fig. 4) (57–60).

The precise exchange is unclear, as one cannot differentiate the above exchange from one that involves 2 HCO3
or a CO3²
in rather than an H^þ out (26). Regardless of the ions carried, however, the net effect is to reduce intracellular [H^þ]. Thus, the Na^þ-dependent Cl
=HCO3

transpor-ter could play a role in maintaining cell [H^þ] below its equilibrium value.

The activity of this transporter is enhanced by a reduction in intracellular pH, and the transporter becomes inactive above a threshold pH of around 7.40 (42). Transport activity is highly dependent on the presence of Na^þin the extracellular compartment, and also appears to be related to the [HCO₃
] in the extracellular compartment (26,57,61). In glomerular mesan-gial cells, the Na^þ-dependent Cl
=HCO3
exchanger participates along with the Na^þ=H^þ exchanger in maintaining a low cell [H^þ] (59). In an HCO3
-free bathing medium, pH recovery from an acid load is mediated

The activity of this transporter is enhanced by a reduction in intracellular pH, and the transporter becomes inactive above a threshold pH of around 7.40 (42). Transport activity is highly dependent on the presence of Na^þin the extracellular compartment, and also appears to be related to the [HCO₃
] in the extracellular compartment (26,57,61). In glomerular mesan-gial cells, the Na^þ-dependent Cl
=HCO3
exchanger participates along with the Na^þ=H^þ exchanger in maintaining a low cell [H^þ] (59). In an HCO3
-free bathing medium, pH recovery from an acid load is mediated