pH regulation in spread cells and round cells.

The aim of this work was to characterize the changes in pH regulation that lead to increased intracellular pH (pHi) in well-spread cells on tissue culture plastic relative to cells on a nonadhesive surface. Bicarbonate was not required for maintenance of a control steady state pHi or of the difference in pHi between round and spread cells. In the absence of bicarbonate, lowering the sodium content of the medium led to decreased pHi and elimination of the difference between round and spread cells. In the presence or absence of bicarbonate, adding ethylisopropyl amiloride lowered pHi and eliminated the difference between round and spread cells. Measurements of recovery from acute acidification in the absence of bicarbonate confirmed that Na+/H+ exchange was enhanced in spread cells. However, recovery from both acidification and alkalinization in the presence of bicarbonate showed that bicarbonate-dependent recovery in both directions, most likely due to sodium-dependent and -independent HCO3-/Cl- exchangers, was also stimulated in spread cells. We conclude that Na+/H+ exchange has a primary role in determining steady state pHi in 3T3 cells in serum and is responsible for the lower pHi in round cells. Bicarbonate-dependent pH regulatory mechanisms are also inhibited in round cells.

The aim of this work was to characterize the changes in pH regulation that lead to increased intracellular pH (pHi) in well-spread cells on tissue culture plastic relative to cells on a nonadhesive surface. Bicarbonate was not required for maintenance of a control steady state pHi or of the difference in pHi between round and spread cells. In the absence of bicarbonate, lowering the sodium content of the medium led to decreased pHi and elimination of the difference between round and spread cells. In the presence or absence of bicarbonate, adding ethylisopropyl amiloride lowered pHi and eliminated the difference between round and spread cells. Measurements of recovery from acute acidification in the absence of bicarbonate confirmed that Na+/H+ exchange was enhanced in spread cells. Howeve covery from both acidification and alkalinizaticr, ret the presence of bicarbonate showed that bicarbdn in-dependent recovery in both directions, most lik@na@e to sodium-dependent and -independent HC0;/Clyl~ dqangers, was also stimulated in spread cells. We conciude that Na+/H+ exchange has a primary role in determining steady state pHi in 3T3 cells in serum and is responsible for the lower pHi in round cells. Bicarbonatedependent pH regulatory mechanisms are also inhibited in round cells.
It has recently been reported that cytoplasmic pH (pHJ* is altered by attachment and spreading of cells on solid surfaces (1, 2). Normal, anchorage-dependent cells were 0.15-0.3 pH units more alkaline when attached to plastic coated with fibronectin or with their endogenous fibronectin-rich extracellular matrix than when in suspension or on a nonadhesive surface, It is important to note that adhesion to tissue culture plastic or other surfaces is mediated by extracellular matrix proteins such as fibronectin or vitronectin (3), which exert their effects through membrane receptors of the integrin family (4).
spreading on pHi were carried out in bicarbonate-buffered medium, so that the results are likely to be physiologically relevant. However, it remained to be determined which plasma membrane transport proteins were responsible for these effects. Most cell types have a Na'/H' exchanger that pumps protons out of the cell. Those cells that have been examined also had a Na'-dependent HCOy/Cl-exchanger, which brings HCO; into the cell and therefore alkalinizes the cytoplasm, and a Na+-independent HCO;/Cl-exchanger, which primarily excretes HCO; and acidifies the cell (5-7). The primary aim of this study was to determine which of the pH regulatory systems was responsible for the effects of spreading on pH,. Because spreading on extracellular matrix proteins is slow compared with, for example, the action of growth factors on pHi, understanding how spreading alters pHi required an analysis of which transporters determine steady state pHi. Our results show that Na'/H+ exchange is of primary importance in maintaining steady state pHi, and is responsible for the higher pHi in well-spread cells, and that the activity of the Na+/H' and both HCO,/Cl-exchangers are increased in spread cells.

The Effect of Bicarbonate
We first determined whether bicarbonate-dependent transporters were needed to maintain the difference in pH, between spread cells on tissue culture plastic and round cells on polyHEMA.
Cells were plated in dishes coated with sufficient concentrations of the nonadhesive polymer, polyHEMA, that they remained entirely round, although they attached weakly. In order that round and spread cells could be compared in the same dish, small areas of polyHEMA in each dish were scraped with a plastic pipette tip to expose the underlying plastic.
The medium was changed from the usual bicarbonatebuffered DMEM under 5% COz to HEPES-buffered medium at the same pH, without CO,. pHi was followed in single cells before and after transfer (Fig. IA). As previously observed (6, ll), pH, underwent a rapid increase, followed by a slower return to starting values. pHi returned to approximately the same value after 5-10 min and remained level. Measurements of average pHi in spread and round cells made before and at 1 h after changing the medium showed that there was no significant change in the pHi of spread or round cells (Fig.  1B). The difference between the two, ApHi, was approximately 0.15 pHi units before and after changing the medium. Similar results were obtained in five out of five such experiments. Using medium that had been degassed to remove the small amount of COz in equilibrium with the air or medium that contained 0.1 mM DIDS to block HCO:/Cl-exchange had no effect on pHi (not shown). We conclude that with 3T3 cells in serum, bicarbonate is not required to maintain steady state pH;.

The Effect of Sodium
To test the function of the Na+/H+ exchanger, cells in medium without bicarbonate were transferred to medium containing variable concentrations of sodium, with choline added to keep ionic strength constant. Fig. 2A shows the time course for a typical spread cell after changing to medium without sodium. pHi decreased after transfer, until a new steady state pHi was reached after approximately 15 min. Measurements of average pHi in spread cells made 1 h after changing medium showed that pHi depended strongly on sodium (Fig. 2B). Cells in 0 Na+ were very acidic, but regained nearly control pH; in only 14 mM Na' (half-maximal effect at 5 mM). pHi in 0 sodium was an average of 6.81 +-0.12 (10 experiments).
When pH, was measured in round cells and spread cells, the difference, ApHi, depended on sodium over the same concentration range (Fig. 2C). In medium without sodium, ApHi was an average of 0.02 f 0.02 (5 experiments) and was identical to control in 14 mM sodium. Two control experiments showed that the elimination of ApHi was due to absence of sodium and not to the low pH; per se. First, in sodium-free medium of pH 8.0, cells had a pHi of approximately 7.1, but ApHi was still 0. Second, in medium with normal sodium but with pH 6.5, spread cells had a low pHi (approximately 6.7), but ApHi was identical to control conditions (data not shown).
These experiments lead to two conclusions. First, in bicarbonate-free medium, maintaining pHi in the normal range requires sodium, but relatively low concentrations of sodium are sufficient. Second, the difference in pHi between spread and round cells changes over the same range of sodium concentrations.
Previous work (6) has shown that the extracellular binding site on the Na+/H+ exchanger has an affinity for sodium of 15-50 mM. Thus, the results would suggest that the Na'/H' exchanger is required for maintenance of pHi and that it is responsible for the effect of spreading on pHi.
The Effect of EIPA To confirm that the Na+/H+ exchanger was responsible for the effect of spreading on pHi, the drug EIPA was used. EIPA has a high affinity and, when used at low doses, is relatively specific for the Na+/H+ exchanger (12). In initial experiments, cells in normal (bicarbonate-free) medium showed changes in pHi only at rather high concentrations of EIPA (>60 PM). To avoid potential toxicity associated with high concentrations of EIPA, we adopted the approach of L'Allemain et al. (13)  because competition with extracellular Na+ is decreased. A sodium concentration of 15 mM was chosen because it is the lowest concentration that gave a normal pH; (Fig. 1B). Cells in low sodium underwent a slow decrease in pHi after addition of EIPA, which stabilized after lo-15 min. Fig. 3A shows a typical time course for a single spread cell. Measurements of average pHi after 30-60 min showed that EIPA was maximally effective at 40 pM (Fig. 3B), which is the approximate concentration at which EIPA completely inhibited Na'/ H+ exchange in previous work (12). Average pH, in 40-60 pM EIPA was 6.97 + 0.11 (11 experiments in spread and round cells using this protocol showed that ApHi was eliminated by EIPA over the same concentration range (Fig. 3C). The average ApHi was 0.00 f 0.02 (5 experiments). We conclude that, in the absence of bicarbonate, Na+/H+ exchange contributes significantly to steady state pHi and is required for the effect of spreading on pHi.
To analyze the role of the Na'/H' exchanger under conditions where HCO;/Cl-exchangers also operate, cells in medium with bicarbonate and CO, were treated with EIPA. The medium had 25 mM sodium, with choline added to keep ionic strength constant as before. Transfer of cells from normal DMEM to this medium had no effect on pHi (not shown). Addition of EIPA to spread cells resulted in decreased pH, pH in Spread and Round Cells (Fig. 4A). The average pHi of cells in 40-60 FM EIPA was 7.06 f 0.05 (10 experiments). When round and spread cells were compared, ApHi was reduced over the same concentrations of EIPA (Fig. 4B). Average ApHi in 40-60 pM EIPA was 0.02 + 0.02 (4 experiments).
These results show that Na'/H' exchange maintains pHi at a level that is higher than HCO;/ Cl-exchangers alone and that the Na+/H+ exchanger is solely responsible for the effect of spreading on steady state pHi.

PHi Recovery
Na+/H+ Exchange-To characterize further the changes in proton transport caused by cell spreading, the recovery from acute changes in pHi was examined in round and spread cells. First, the Na'/H' exchanger was studied by measuring the recovery of cells from acidification in the absence of bicarbonate. Cells were acidified by equilibrating in medium with 7 mM NH&l for lo-15 min and then quickly switching to medium without NH&l (14). Cells were rapidly acidified by approximately 0.5 pH units and then recovered slowly toward their initial pHi. Time course measurements were made on single cells before and up to 15 min after acidification.
Typical recovery curves are shown for spread and round cells in Fig. 5A. Recovery of the round cell was significantly slower, and the cell reached a lower steady state pHi. Recovery from acidification under these conditions appears to be highly dependent on Na'/H+ exchange, since it was blocked by carrying out the experiment in medium without sodium or by adding 50 pM EIPA (not shown, but see Fig. 5B). Data for a number of recovery experiments were quantitated by fitting the recovery curves to a single exponential as described under  was calculated from the fitted constants. Average values (Table I, Half-time) showed that the half-time was 59% longer for round cells. Recovery experiments of this type have also been analyzed by comparing the rate of recovery, dpHJdr, at a given pHi for cells in different states (6, 15). Therefore, dpHJdt was calculated at pHi = 6.7 from the best-fit curves shown in Fig. 5C. It is apparent that recovery of the round cell was significantly slower than the spread cell. Recovery In-was strongly inhibited by pretreatment of cells with 0.1 mM crease DIDS for lo-15 min (Fig. 5C), but was independent of external sodium (not shown). Analysis of data for a number of experiments showed that recovery was stimulated in spread cells to about the same extent as was Na+-dependent HCO;/ Cl-exchange (Table I). min % Na+/H+ 2.9 f 1.0 (n = 10) 4.6 t 2.0 (n = 10) 59* Na'-dependent 3.3 f 1.8 (n = 11) 5.2 + 1.6 (n = 9) 57* HCO,/Cl-Na+-independent 1.6 t 0.9 (n = 10) 2.7 + 1.  (6). When the data for individual recovery experiments were analyzed in this manner, it was found that & was 19.0 + 5.0 mM/pH for spread cells and 18.3 + 3.5 mM/pH for round cells. Thus, the buffering capacity for spread and round cells under these conditions does not differ significantly.
We conclude that the differences in recovery rates reflect differences in proton flux, so that the Na'/H+ exchanger appears to be activated in spread cells relative to round cells.
Our results lead to three main conclusions. First, the difference in steady state pH, between spread cells and round cells is due to changes in the activity of the Na'/H+ exchanger. This conclusion holds for cells in medium with or without bicarbonate.
It is based on studies with low sodium and with EIPA and is supported by measurements of recovery from low pHi. It should be emphasized that round cells still have substantial capacity to recover from acid loads. They differ in that the rates are somewhat diminished relative to spread cells, and the pHi levels off at a lower value. These differences are similar to the type of changes observed after serum stimulation of quiescent attached cells (17). Note that Margolis et al. (1) reported that the change in pHi upon spreading was due to the Na'/H+ exchanger and that its activity was also necessary for cell spreading. However, both of these conclusions were apparently based on experiments that used sodiumfree medium that lowered pHi to well below the physiological range and that also would be expected to have effects on cells other than blocking Na+/H+ exchange. Our data (Ref. 2, and Footnote 2) have consistently shown that changes in pHi in the physiological range, or specific blockade of the Na+/H' exchanger, do not affect cell shape or cell spreading to an appreciable extent.

Sodium-dependent
HCO:/Cl-Exchange-To analyze bicarbonate-dependent pH regulation, the recovery of cells from acidification in the presence of bicarbonate was measured. EIPA (50 PM) was added to block Na+/H+ exchange. Recovery under these conditions has been attributed to a Na+-dependent HCO,/Cl-exchanger (7, 16). Typical recovery curves are shown in Fig. 5B, and data are summarized in Table I. It appears that recovery was significantly slower for the round cell than for the spread one, although the cells reached the same final pHi. Recovery was completely inhibited when bicarbonate was omitted, and in the absence of sodium (Fig.  5B), and was strongly inhibited by preincubating cells for lo-15 min with 0.1 mM DIDS (not shown). The half-time for recovery was 57% longer in round cells (Table I, Half-time). Alternatively, the rate of recovery at pHi = 6.7 was 59% faster in spread cells (Table I, Recovery rate). Comparison of &-, calculated as before, also showed no difference between spread cells and round cells under these conditions (14.3 + 5.0 and 14.6 f 4.3 mM/pH, respectively).
Sodium-independent HCO,/Cl-Exchange-The ability of cells to recover from acute alkalinization was also examined. Rapid recovery of pH, after transfer of cells from bicarbonate/ COZ medium to HEPES-buffered medium without CO, has been attributed to a sodium-independent HCO:/H+ exchanger (7,16). Round and spread cells were observed after changing to bicarbonate-free medium. Typical recovery curves are Second, Na'/H+ exchange does make a significant contribution to steady state pHi in the presence or absence of bicarbonate.
The role of Na+/H' exchange in setting steady state pHi, however, has been the subject of some controversy in the literature.
Evidence in favor of the idea that Na+/H+ exchange determines pHi came in part from experiments showing that when cells in medium without bicarbonate were stimulated with serum or growth factors, pHi rapidly increased due to activation of the Na+/H+ exchanger (for reviews, see Refs. 17,18). More recently, it has been shown that the effect of serum on pH, is abolished by adding bicarbonate to the medium (11). Bicarbonate raises the pHi of serum-starved cells so that the change in set point of the Na+/H' exchanger is masked, implying that Na'/H' exchange did not determine pHi when bicarbonate was present. On the other hand, experiments in which pHi was measured in unsynchronized fibroblasts in the presence of bicarbonate gave the opposite result. Bright et al. (19) found that serum-treated fibroblasts were about 0.3 pH units more alkaline than starved cultures. We have also observed that mitogen-stimulated cells in medium with bicarbonate had a higher pHi than cells in the absence of mitogen.3z 4 Also, the data presented in this paper demonstrate that when cell spreading alters the activity of the Na+/ H+ exchanger, steady state pHi is altered in the presence of bicarbonate.
While the exact source of the discrepancy is not known, it would seem that measurements of pHi a few minutes ' M. A. Schwartz, E. J. Cragoe, Jr., and C. P. Lechene, unpublished results.
after addition of serum cannot be extrapolated to the entire cell cycle.
Experiments with amiloride or its derivatives have also generated disagreement concerning the importance of Na'/ H' exchange in setting pHi. One study found that these drugs only altered steady state pH, at very high concentrations, where they appeared to act by nonspecific means (20). They concluded that Na'/H' exchange was only activated by low pH, and did not function under normal conditions. Although we are in agreement with the data from these latter experiments, we do not support their conclusions. It appears that Na'/H' exchange can be partially inhibited without substantially altering steady state pHi. We found, however, that complete inhibition, either by removal of sodium from the medium or by adding EIPA to low sodium medium, resulted in a significant decrease in pHi. Other workers (6) have also shown an effect of amiloride derivatives on steady state pHi. Thus, at steady state, Na+/H+ exchange does appear to operate at a level that makes a significant contribution to pHi. Our third conclusion is that pHi recovery due to HCO?/Clexchange is increased in spread cells. Our cells alkalinized via a mechanism that has the characteristics of the previously described sodium-dependent HCO;/Cl-exchanger and acidified via what appears to be the sodium-independent HCO;/ Cl-exchanger. Both of these were inhibited in round cells to about the same extent (Table I). Ganz et al. (16) recently showed that stimulation of mesangial cells with vasopressin triggered an increase in Na'/H' exchange, in sodium-dependent HCO,/Cl-exchange, and in sodium-independent HCO;/ Cl-exchange. The net effect in their system was acidification, because the largest increase was in the rate of HCO, efflux due to sodium-independent exchange. In our system, there was no difference in steady state pH, between round and spread cells when only HCOT-dependent mechanisms were active. This may be because the changes in uptake and efflux of HCOF balance, an idea that is consistent with the result that alkalinization due to Na+-dependent HCO,/Cl-exchange, and acidification due to Na+-independent HCO;/Clexchange decreased to about the same extent in round cells. Alternatively, it may be that the HCO:/Cl-exchangers are inactive at resting pHi or that other factors such as acid production are involved. Our results are consistent with the general view that normal cells deprived of an extracellular matrix on which to spread are in a state similar to that of cells deprived of growth factors. In both cases, the absence of as yet unknown cytoplasmic signals leads to a general decrease in the activity of the transport proteins responsible for pH regulation. Other factors such as altered geometry could conceivably also play a role in the decreased rate at which round cells recover from acute changes in pH,, but this need not be the case. Round cells develop a highly convoluted surface, so that no net loss of membrane surface area need occur (21). Diffusion of protons and other ions from the interior of the cell to the plasma membrane might also be slower in round cells. The diffusion times, however, are orders of magnitude faster than the observed recovery times and should therefore not be rate-limiting.
The next stage in this work will be to identify the cytoplasmic signaling pathways that are activated by ECM and that lead to altered pHi regulation.