Intracellular pH

A possible explanation for the effect of ammonia is simply the alteration of intracellular and intracompartmental pH (6,72,106,107). Ammonia in cell culture is present in both gaseous and ionic form, and these species are in equilibrium at a ratio determined by the medium pH. At normal pH values, almost all the ammonia present is in the form of ammonium ion, and the concentration of gaseous ammonia is very low (pAa = 9.2).

The regulation of intracellular (cytosolic) and intracom-partmental pH in response to added ammonia is complex (Fig. 5). Both gaseous ammonia and ammonia ion can permeate the membranes. This permeation involves both passive (diffusion) and active transport.

The permeability of gaseous ammonia is much greater than that of the ammonium ion (6,49,72,107,108). Thus, initially, gaseous ammonia permeates rapidly, raising the intracellular pH. Then, the slower penetration of ammonium ion decreases the intracellular pH. McQueen and Bailey (72,107) and Ozturk et al. (6) studied the response of hybridoma cells to added ammonia and verified this mechanism. Figure 6 shows the response of intracellular pH to ammonia addition, followed by fluorescent dye. After the addition, there is an immediate increase in intracel-lular pH. This is due to rapid gaseous ammonium diffusion into the cells. The intracellular pH decays to steady-state intracellular pH values that are lower than those observed prior to the exposure to ammonia because of the transport of ammonium ion. The steady-state intracellular pH values for this experiment are presented in Table 3.

The rate of diffusion for ammonium ion through the cell membrane is four to five orders of magnitude lower than the rate of diffusion for gaseous ammonia (109). However, ammonium ion can be actively transported across the cell membrane via [Na,K]ATPase, [Na,K,Cl]-cotransporter, and [Na,H]-exchanger, as indicated in Figure 5 (109-114). The dynamics of ammonia transport for mammalian cells were analyzed by mathematical models (107).

The transport of ammonia and ammonium ion to the cells were studied in detail by Martinelle and Haggsrom (115-117) for the murine myeloma cell (Sp2/O-Ag14). In addition to diffusional transport, these authors identified the active transport of ammonium ion via [Na,K]ATPase and [Na,K,C]-cotransporter in hybridoma cells. The presence ofK+ could inhibit the [Na,K]ATPase active transport and alter the transport of ammonium ion. When K+ (10 mM) was added, the change in intracellular pH due to ammonia addition was observed to be negligible. The authors postulate that one of the reasons for ammonia inhibition is the increased energy demand resulting from energy wasted because NH4+ is actively transported to the cells via [Na,K]ATPase, and they proposed the use of K+ for minimization of ammonia inhibition. Use of KOH instead of NaOH for pH control is proposed to increase K+ concentration in the culture.

A change in intracellular or intracompartmental pH can alter the activity of numerous enzymes and, depending on the location, different results can be obtained. The net result of ammonium ion transport to the cells is a decrease in intracellular pH. However, the ammonia transported to the cells further diffuses to compartments in the cells. Gaseous ammonia easily permeates intracellular membranes and the pH in mitochondria, Golgi, endoplasmic reticulum, and lysosymes are elevated by its presence. The pH in vesicles is normally lower than in cytosol (118,119), and the pH can be altered by ammonia. The rise in pH by the transport of ammonia gas into the lysosomes has been demonstrated (120,121).

The generation of ammonia inside the cell caused by cellular metabolism alters intracompartmental pH. Ammonia is generated mainly in mitochondria because ofglu-tamine metabolism. Ammonia gas readily diffuses out of the mitochondria, and the pH decreases. Ammonia diffuses not only to the cytoplasm of cells but also to the compartments. In all cases, ammonia that diffuses from mitochondria increases the pH in other intracellular compartments (i.e., Golgi, endoplasmic reticulum, and lysosymes).

McQueen and Bailey (72,107) have shown that the net result of ammonia addition is a decrease in intracellular pH for the hybridoma line ATCC TIB 131. When internal pH was altered by external pH, McQueen and Bailey associated ammonia effects to the variations in intracellular pH, because both ammonia addition and low external pH resulted in lower cell yields on glucose and glutamine. On the other hand, Ozturk et al. (6) individually evaluated the cell growth rate and the metabolic rates and could not relate the effects of ammonia to the effects of lowering external pH. There is a decrease in glucose consumption and an increase in glutamine uptake rates when pH was controlled below 7.2 (93). However, a decrease in intracellular pH as a result of ammonia addition increased both rates.

Figure 5. Ammonia transport and alteration of intracellular pH.

Decrease And Increase Intracellular
H4

Figure 6. Alteration of intracellular (cytosolic) pH in response to ammonia addition. Source: Data obtained from Ozturk and Pals-son (88).

Time (min)

Figure 6. Alteration of intracellular (cytosolic) pH in response to ammonia addition. Source: Data obtained from Ozturk and Pals-son (88).

Table 3. Effect of Ammonia on Intracellular pH at External pH of 7.20

[NH4+]

Peak

Steady-state

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