Sodium/Proton Exchange

All cell membranes contain embedded proteins that exchange sodium ions for protons across the plasma membrane in a

fixed ratio of one Na⁺ for one H⁺. It is accepted that the main mechanism for proton extrusion is the Na⁺/H⁺ antiporter which uses energy stored in the Na⁺ gradient to pump protons out from the intracellular space (Graf et al., 1988). The activity of this antiporter is determined by the intracellular pH (pH_i) and it serves to protect cells against acidification of the cytosol (Pouyssegur et al., 1988). At pH_i

>7.4, this antiporter is virtually inactive but, as the pH_i decreases from 7.4, the activity of the antiporter increases, reach- ing a maximum at a pH_iof 6.0.

Hormones

Many of the hormones which stimulate anabolic processes in cells (mitogens or growth factors) have been shown to cause an increase in pH_i. This change is typically 0.15–0.3 pH units, representing a consider- able decrease in the intracellular proton concentration. Such a change will, by itself, influence the activity of many metabolic pathways due to the pH sensitivity of many key enzymes. Pouyssegur et al. (1988) proposed a mechanism whereby binding of these hormones to receptors on the cell surface caused activation of protein kinase C (in the case of thrombin, bombesin, Fig. 7.1. Schematic diagram illustrating how various factors represented along the bottom row can influence the rate at which Na⁺enters a cell across the plasma membrane. To maintain steady-state concentrations of Na⁺within the cell, the rate at which Na⁺is pumped from the cell by Na⁺,K⁺-ATPase must equal the rate of entry of Na⁺. The energetic cost of such pumping is one-third of a molecule of ATP per Na⁺expelled.

vasopressin and bradykinin) or activation of tyrosine kinase (in the case of epidermal growth factor and fibroblast growth factor), both resulting in modification to the Na⁺/H⁺ antiporter, changing its pH sensitivity.

Insulin and insulin-like growth factor also cause similar changes in pH_i(Moore, 1983), probably through similar mechanisms (Bryer-Ash, 1988). The increase in sodium ions entering the cell is met by an increase in the rate of Na⁺extrusion by the sodium pump. There is an inhibitor of Na⁺/H⁺ exchange, amiloride, which has enabled studies of the activity of this process, although it has been reported that its inhibitory action is not specific for this antiporter but that it may inhibit Na⁺,K⁺- ATPase activity as well (Park et al., 1992).

Such studies have been undertaken in a similar manner to those described above for the sodium pump. Instead of ouabain being added to the cell suspension, amiloride is added and the reduction in oxygen con- sumption observed is assumed to be due to a decrease in sodium pump activity brought about by a decrease in Na⁺/H⁺exchange.

Thyroid hormone levels have been shown to alter the rate of ion transport across the cell membrane. Gregg and Milligan (1987) measured Na⁺,K⁺-ATPase activity in sheep that had their thyroid gland removed surgically. Supplementation of the thyroid hormone T₃to these animals, increased the activity of this enzyme by one-third. Gregg and Milligan (1982a) showed increases in Na⁺,K⁺-ATPase activity in muscle of cold-exposed sheep when compared with animals kept in warmer conditions. Cold stress causes increased thyroid hormone levels (Park et al., 1992), and increasing the energy use for sodium pumping would be one mechanism whereby heat production could be increased. It is not clear how these hormones exert their effect, but they must increase Na⁺entry into cells substantially.

Weak acids

Intracellular pH can be affected directly by weak acids. Weak acids, such as acetic and

carbonic acids, exist in equilibrium in aqueous solution:

Acetic acid:

CH₃COOH H ⁺+ CH₃COO Carbonic acid:

CO₂+ H₂O H₂CO₃H⁺+ HCO₃ The undissociated (and uncharged) forms of these acids can cross biological membranes readily by diffusion. Ketelaars and Tolkamp (1992) proposed that weak acids such as acetic and carbonic acids can act as proton ionophores, and thus their presence in extracellular media would incur an energy cost to the cell in counteracting acidification of the cytosol in the manner depicted for acetic acid in Fig. 7.2. Increased activity of Na⁺/H⁺exchange will lead to an increase in the intracellular concentration of Na⁺, stim- ulating Na⁺,K⁺-ATPase activity (Smith and Rozengurt, 1978). What is new about the explanation put forward by Ketelaars and Tolkamp is that it requires the plasma mem- brane to be permeable to small anions, e.g.

acetate, to a significant degree. It has been argued that the plasma membrane is perme- able to HCO₃ and NH₄⁺ (Boron and De Weer, 1976) with permeabilities of 5 10⁷ and 10⁶cm s¹, respectively (compared with the much greater permeabilities of 6 10³cm s¹for both CO₂and NH₃ – mole- cules which carry no charge and therefore cross biological membranes relatively easily), and that leakage of HCO₃ occurs independently of carrier-mediated transport (Boron, 1983).

Jessop and Leng (1993) examined the effect of nutrient balance on Na⁺,K⁺- ATPase activity. Sheep were fed on poor- quality diets limiting in rumen-degradable nitrogen (the effective rumen-degradable protein to fermentable metabolizable energy ratio, eRDP:FME, was 6.0), which were either supplemented with additional rumen-undegradable protein (UDP) or not.

Thus the protein to energy ratio of absorbed nutrients was expected to vary between the two dietary treatments.

Hepatocytes were prepared and incubated over a range of acetate concentrations from 0 to 2.5 mM. Total respiration was unchanged, but the proportion of total

respiration inhibited by ouabain (and hence assumed to represent Na⁺,K⁺-ATPase activity) varied with both dietary treatment and acetate concentration. Supplying addi- tional UDP (at constant eRDP:FME) lowered (P < 0.01) the proportion of total respiration attributable to sodium pump activity by a constant amount at each acetate level whilst increasing acetate increased it linearly such that the percentage inhibition could be described by the following equation: %inhibition = 16.6 + 4.05 acetate concentration (mM) (R² = 0.65, P< 0.001). This represents a change in the pattern of use of energy by liver tissue since, as total respiration rate did not alter as acetate level increased, so a greater proportion of energy production had to be diverted towards sodium pump activity.

From these results, it can be calculated that for acetate to cause an increase in sodium pump activity via the mechanism shown in Fig. 7.2, the rate of efflux of acetatewould have to be 4.3 nmol min¹ mg¹ protein, equivalent to a permeability of 1 10⁷cm s¹. This value is very close to those calculated for HCO₃ and NH₄⁺ and is in good agreement with one report of a measured permeability of the plasma membrane to acetateof 3.4 10⁷cm s¹ (Sharp and Thomas, 1981; Hume and

Thomas, 1989). For efflux of acetate to take place, there must be a suitable ‘driving force’; this would be provided by the substantial electrochemical gradient (posi- tively charged on the extracellular surface and negatively charged on the intracellular one, thus repelling negatively charged ions from the cell) that exists across the plasma membrane (equivalent to a concentration gradient of ~100 mMfor acetate).

Carbon dioxide is produced continually within mitochondria from oxidative meta- bolism. It diffuses down its concentration gradient to the extracellular fluid. The potential exists for hydration of CO₂ to carbonic acid and then dissociation to H⁺ and HCO₃. Many tissues possess the enzyme catalase which greatly speeds up attainment of this equilibrium. The pro- duction of protons and bicarbonate from CO₂ results in what is termed facilitated diffusion of CO₂ from tissues (Gros et al., 1988). The HCO₃anion exchanges for Cl across the plasma membrane and the H⁺is pumped out of the cell by the Na⁺/H⁺ antiporter. In studies with muscle tissue, where the rate of CO₂ production can increase substantially during exercise, this process of facilitated diffusion has been estimated to account for 70% of CO₂ removal from the tissue (Gros et al., 1988).

Fig. 7.2. Representation of acetate cycling across the plasma membrane. AcH refers to acetic acid in its undissociated form, Acthe acetate anion, membrane protein 1 is the Na⁺/H⁺antiporter and membrane protein 2 is Na⁺,K⁺-ATPase.

As noted earlier, measured activities of Na⁺,K⁺-ATPase were higher when the main pH buffer of the incubation medium was HCO₃/CO₂. In such studies, the CO₂ con- centration in the extracellular fluid would be relatively high, reducing the concentra- tion gradient of CO₂ between the incuba- tion medium and intracellular fluid. Since CO₂diffuses out of cells down this concen- tration gradient, this results in lower rates of CO₂loss from cells and would increase the potential for facilitated diffusion of CO₂, increasing Na⁺,K⁺-ATPase activity.

Extracellular CO₂ levels will be lower when alternative pH buffers, such as HEPES, are used.

Sies et al. (1973), from measurements of the change in pH of media draining perfused rat liver, have shown that addi- tion of acetate (0–5 mM) to the perfusate (pH 7.4) caused a rapid change in proton uptake into hepatocytes, and using data supplied in this and an earlier publication (Sies and Noack, 1972) it can be calculated that the rates of proton uptake observed would cause changes in Na⁺,K⁺-ATPase activity similar to those reported above, assuming the previously discussed linkage between proton uptake and sodium pumping. The data from Sies’ group do not differentiate between rapid initial extru- sion of protons caused by equilibration of acetate across the plasma membrane and the proposed steady-state efflux of protons caused by acetate cycling. A similar response was seen when the perfused liver was exposed to differing levels of CO₂/HCO₃.

The ability of acetate to act as a proton ionophore will depend on the concentra- tion of acetate in the extracellular fluid relative to the cell’s ability to metabolize it.

Thus the balance of nutrients available will determine metabolic efficiency, and it is to be expected that the optimal balance of nutrients will vary from tissue to tissue.

Scollan and Jessop (1995) showed that in sheep given diets which would result in an imbalance of ingested nutrients (and would be expected to be used with a lower efficiency), blood acetate was markedly higher than in those fed on a more balanced

diet. Cronjé et al. (1991) have shown that nutrient balance has a marked effect on acetate clearance, and Leng (1990) has discussed the influence of nutrient balance on the efficiency of use of metabolizable energy, pointing out the importance of the protein:energy ratio on the overall efficiency of energy use, as well as on the level of feed consumption.