pH regulation on intracellular
in barnacle muscle fibers: dependence and extracellular pH
WALTER F. BORON, WAYNE C. MCCORMICK, AND Department of Physiology and Biophysics and Department University School of Medicine, St. Louis, Missouri 63110
BORON, WALTER
AND ALBERT dependence on extraceLLuLar PH. Am. J. Physiol. 237(3): Cl%-C193, 1979 or Am. J. Physiol.: Cell Physiol. 6(2): Cl%Cl93, 1979.~Intracellular pH (pH;) regulation was studied in acid-loaded barnacle muscle fibers by monitoring recovery of pHi with a pH-sensitive microelectrode. By multiplying the rate of pHi recovery by total intracellular buffering power, the acid extrusion rate was obtained. The acid extrusion rate was greatest at low values of pHi, and declined toward zero as pHi approached normal levels. It increased as the extracellular pH (pH,) was raised either by increasing external [HCO,] ([HC&],) at constant PCO~ or by decreasing PCO~ at constant [HCO& but more so in the former case than in the latter. These observations suggest that pH, per se is an important determinant of the acid extrusion rate, but that raising [HC&],, by itself also stimulates acid extrusion. This would be expected if acid extrusion involves the inward movement of solutions HCO.7. When f 1b ers were exposed to HCOa-containing at very low or very high pH,, pHi drifted downward or upward, respectively; these drifts were inhibited by 4-acetamido-4’ isothiocyanostilbene-2,2’ disulfonic acid (SITS). Our results are discussed in terms of possible mechanisms of acid extrusion. Roes. pH regulation intracellular and
F., WAYNE C. MCCORMICK, in barnacle
muscle
fibers:
bicarbonate transport; SITS (4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonic acid); intracellular buffer power
ALBERT ROOS of Anesthesiology,
Washington
for external Na’ has been described in the snail neuron (15) and, more recently, in barnacle muscle fiber (5) Relatively little quantitative information is available concerning pHi regulation. Thomas (13) has reported that, in the snail neuron, recovery of pHi after loading with acid seems to follow an exponential time course. That is, the rate of acid extrusion (derived from the rate of pHi recovery) is proportional to the difference between the “normal” pHi and the prevailing value. Also, the rate of acid extrusion in the squid axon increases with extracellular pH (pH,) as external [HCO,] ([HCO&) is raised at constant PCO~ (4). This result has been qualitatively confirmed in both barnacle muscle (2) and snail neurons (15). We now report a quantitative study in barnacle muscle fibers in which we have examined the rate of acid extrusion as a function both of pHi and of pH,. We found that the acid extrusion rate was greatest at low values of pHi, and gradually fell towards zero as pHi approached its normal range. In addition, at a given value of pHi, the acid extrusion rate increased with pH,, whether pH, was raised by increasing [HCO& or by lowering Pco~. Portions of this work have been previously reported (5). ment
METHODS IT IS WELL established that nerve and muscle cells possess
a mechanism for regulating their intracellular pH ‘pHi). These cells maintain pHi at a more alkaline level khan would be expected if H’ were in eqwilibrium. In +)he steady state, an active transport mechanism is required to remove acid that accumulates as the result of either cellular metabolism or passive ion movement (H+ influx, OH-, and HCOT efflux). This same mechanism is also responsible for the rapid recovery of pHi following acute acid loads. We refer to this transport process as acid extrusion, realizing, of course, that it may involve the ejection of acid and/or the accumulation of alkali. Several qualitative properties of this acid extruding mechanism responsible for the pHi regulation have already been described. In the squid giant axon (4), snail neuron (l3), and barnacle muscle (Z), acid extrusion is stimulated by external HCO; (2, 4, 13) and blocked by the anion flux inhibitor 4-acetamido-4’-isothiocyanostilbene-2,2’disulfonic acid (SITS) (2, 7, 12, 14). There is also strong evidence in each of these preparations that the mode of action of the acid-extruding mechanism is an exchange of external HCOi for internal Cl- (6, 12, 15). A require0363-6143/79/oooO-oooO$O1.25
Copyright
0 1979 the American
Physiological
GeneraZ. Single muscle fibers were isolated from the giant barnacle Balanus nub&s, obtained from D. King, Friday Harbor, Washington. Each fiber was cannulated at both ends, mounted horizontally in a chamber, and superfused with artificial barnacle sea water (BSW) at 22OC. Cut ends of the fibers were isolated from the superfused central region by Vaseline seals. Electrodes for measuring pHi and membrane potential ( Vm) were inserted through the cannulas from opposite ends. The pH-sensitive electrodes were similar to those of Hinke (10); their use has been previously described (3). Fibers were exposed to C02-containing solutions delivered from glass syringes through COZ-impermeable Saran tubing (Clarkson Controls Equipment, Detroit, MI.). SoZutions. BSW had the following composition (in mM): 440 NaCl, 10 KCl, 11 CaC12, 11 MgC12, and 21 MgS04. In nominally CO*-free solutions the BSW was buffered with 5 mM N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES). Solutions equilibrated with CO2 contained only 6 mM CaClz (remainder replaced by NaCl), and were buffered to pH 6.2-8.6 with 0.4% CO* (balance oxygen) and varying amounts of HCOT Society
Cl85
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
Cl86
BORON,
(HCO; substituting for Cl-), or were buffered to pH 6.88,0 with 2.5 mM HCOT and varying amounts of COa. The pH, 8.6 solution had to be gassed with CO* for several hours for a stable pH to be reached. This solution was slightly cloudy, probably because of some calcium and magnesium precipitation; the amount of HCO; actually added thus had to be greater than the nominal 40 mM. In the NHJXBSW used in the NH&l prepulse, 50 mM NaCl was replaced by 50 mM NH&l; the solution was buffered to pH 7.70. SITS was obtained from International Chemical and Nuclear (Cleveland, OH), and 4,4’diisothiocyanostilbene-ZJ’disulfonic acid (DIDS) from Pierce Chemical (Rockford, IL). Determination of the rate of acid extrusion. Fibers were loaded with acid by exposing them to BSW containing 50 mM NH&l for 60-90 min, and then returning them to an NH&l-free solution (pH 7.4). After removal of the NH&l, pHi fell to a value substantially less than the initial one. This NH: prepulse technique was first used to acid-load squid giant axons (4), and later in mouse soleus muscle (1). Because its theoretical basis has been previously examined in detail (3), it will be only briefly reviewed here. Exposing a fiber to a solution containing NH&l (see Fig. 1) leads to a rapid initial rise in pHi (segment ab) as some of the NH3 present in the external solution enters the cell and there combines with 50 mM
-1
I
b
8.0
AND
ROOS
protons to form NH:; the increase in pHi halts when the intracellular [NH31 ([NHa]i) equals the [NH&. However, there remains a substantial electrochemical gradient favoring the net influx of NH:, and pHi slowly falls (be) as a fraction of the entering NH: dissociates to form H’ and NH:j. When NH&l is removed from the external solution, virtually all intracellular NH: (including that which had entered as such) leaves as NH3, and, as H+ is left behind, pH; falls below its initial level (compare values at a and d). The degree of undershoot is determined by the magnitude of the previous net influx of NH,‘. After fibers had been loaded with acid in this way, they were exposed to a COz-containing solution, and the subsequent recovery of pHi was monitored on a strip chart recorder. Assuming that acid extrusion was the only factor influencing the rate of recovery of pH;, we obtained the rate of acid extrusion as the product of total intracellular buffering power (see below) and the rate of pH; recovery (dpHi/dt). Because the surface-to-volume ratio of the barnacle muscle fiber is unknown (the vast majority of the surface membrane surrounds an intricate network of invaginations), rates of acid extrusion were expressed in mmol/min per liter cell water rather than per square centimeter surface membrane. Values for dpHi/dt were derived as follows. Each recording of pHi vs. time was manually traced with a rhoIOmM
NH&I
MCCORMICK,
HCOj
/0.4X
pH,
= 8-00
CO2
7.8
7,6
7.4 PHi 72
a
d FIG. 1. Acid loading of muscle fiber and recovery of pHi from acid load. An isolated barnacle muscle fiber was exposed to BSW containing 50 mM NH&l (pH,, 7.7) for 80 min, during which time pHi rapidly rose (segment ab), and then slowly declined (bc). Returning the fiber to NH:-free BSW (pH,, 7.4) caused pHi to fall to 6.59 (cd) far below the initial pHi (7.28). This acid-loaded fiber was then exposed to BSW containing 10 mM HCOJ0.4% CO2 (pH,, 8.00), which caused pHi to
recover rapidly. With very high rates of acid extrusion, such as prevailed in this experiment, the initial acidification that normally occurs upon CO? exposure is masked. Note that the time scale was changed shortly before the CO? exposure began. All figures were traced from original pen recordings. Changes in Vnl upon recovery from acid loading were as previously described (2).
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PH REGULATION
IN
BARNACLE
MUSCLE
theta transducer, linked to a PC-12 minicomputer, which converts the curve into a set of x and y coordinates. A data point was obtained every 0.25 mm along the time axis, which in all experiments was about 20 cm long; the system was accurate to the nearest 0.25 mm. The computer’s sensitivity along the ordinate (pHi axis) was such that about six ‘/4 mm divisions corresponded to 0,Ol pH unit. The set of x,y coordinates was transferred to an IBM-360 computer where the data were curve-fitted to a polynomial of degree 4 or less, The total residual variance of the fit was always less than 10B4 (mean 1.6 x lo-“). The initial portion of the pHi-vs.-time record during the recovery period was excluded from the curvefitting procedure because this portion represented the transition from a COZ-free to a COz-containing external solution. The fitted polynomial was differentiated, and values for dpHi/dt were calculated over a range of values of pHi. Intracellular buffering power. Buffering power is defined as the ratio of the amount of strong base (B) added to a solution (mmol/l) to the resultant change in pH (p = dB/dpH). The total intracellular buffering power &) is the sum of intrinsic or non-CO2 buffering power (Pi) and buffering power due to CO2 (pco,). The former can be experimentally determined by observing the change in pHi produced by exposing the cells to various levels of external NH4C1, and will be discussed below. The latter can be calculated at any PCOZ because, by definition, pco2 equals the partial derivative of the Henderson-Hasselbalch equation (d[HCOT]i/dpHi)pco,
Cl87
FIBERS
= 2.3 [HCOs]i
The intrinsic intracellular buffering power (pi) was experimentally determined in a previous study (2) over the pHi range 6.8-7.8. Because, in the present study, we wished to determine the acid extrusion rate at values of pHi less than 6.8, we extended the previous study of p; to a lower pHi range. The method employed was similar to that previously described. Fibers were prepulsed with 50 mM NH&l to lower pHi to 6.6-6.8, and then SITS poisoned to prevent acid extrusion. They were then exposed to BSW containing either 0.5, 1, or 2 mM NH&l (pH, 7.70), and the resultant changes in pHi noted. The 10 new data points are given in Table 1 (which is an extension of Table 1 in Ref. 2). “Initial” conditions refer to the pHi and [NHT]i (calculated from the mass action law, assuming [NH:j]i = [NH&) prevailing during the exposure to 0.5, 1, or 2 mM NH4C1, and “final” conditions to the pHi and [NHZ]i (the latter assumed to be zero) prevailing after removal of the NH4Cl. “Nominal AB” describes the amount of strong base (mmol/l) added to the fiber between initial and final conditions, and is given “nominal fl” is A[NHz]i/ApHi. “Fitted AB” by A[NH,‘]i; is obtained by inserting initial and final pHi into Eq. 1 of Ref. 2, and represents the result of the least-squares analysis. The dependence of pi on pHi (in the range 6.67.8), based on the 10 new data points as well as on the 23 previous ones, was found to be adequately described by the first order polynomial: pi = A + B (pH;), where A = 155.6 -t- 11.8 (SD) and B = -17.4 t 1.6. This new description of pi differs from the old chiefly at pHi < 7.0, where it predicts substantially lower values for pi. The
1. Buffering exposed to NH&I
TABLE
power
Initial Fiber
100577A 10077iA 10107iA 011978A 01 lYi#B 0119i8H 012078A 012378A 012478A 0124’78A
Final
Nominal [NH, ’ 1, 6.72 6.73 6.99 6.84 6.91 6.9i T-0’; 6.91 6.85 7.01
data for fibers
4.70 4.57 5.07 7.20 2.95 ‘i-24 8.44 :30.4 :kiz 4.87
Nominal N%+l, 6.64 6.64 6.ii 6.W 6.#i iiai 6.78 6.83 6.74 Ki9
0
0
Nominal bH,mM
NBomin$ ,m
Fitted AB, mM
-4.70 -4.57 4.07 -7.20 -2.9s -5.24 -8.44 -3.04 -3.52 -4.8’7
58.8 50.8 23.0 48.0 59.0 52.4 29.1 38.0 32.0 22.1
-3.2 -3.5 -i.9 -5.7 -1.8 -3.5 -10.2 -2.9 -4.1 -i.a
fitted pi at selected values of pHi is as follows: at pHi 6,7, pi = 39.1; at 6.9, 35.6; at 7.1, 32.1; at 7.3, 28.7; at 7.5, 25.1; and at 7.7, 21.6. RESULTS
Recouery of pHi from intracellular acid loads. Previous work on barnacle muscle fibers (2) had shown that the recovery of pHi from acid loads was enhanced by simultaneously increasing pH, and [HCO& To determine the importance of pH, per se, we studied acid extrusion in experiments in which pH, was varied by altering either [HC03], or Pco~. Acid loading was accomplished by pretreating fibers for 60-90 min with BSW containing 50 mM NH4C1, as described in METHODS. In the experiment illustrated in Fig. 1, this procedure caused pHi to fall from 7.28 (point a) to the new value of 6.59 (d). Subsequent exposure of the fiber to BSW buffered with 0.4% COz/lO mM HCOT (pH, 8.0) caused pHi to rise rapidly and then to level off near 7.32. Assuming that the recovery of pH; is influenced only by acid extrusion, then the rate of acid extrusion is the product of the pHi recovery rate and PT (see METHODS). The rate of kid extrusion thus calculated from the course of pHi recovery in Fig. 1 was greatest at low pH;, and gradually fell toward very low values as pHi returned to the normal range (see also Fig. 7). In a series of 72 similar experiments (one fiber per experiment) we examined the recovery of pHi from intracellular acid loading at five different values of pH, (6.2, 6.8, 7.4, 8.0, and 8.6). These were established by varying [HCO& at a constant level of COz (0.4%). Figure 2 shows a typical experiment at each of the values of pH, (the fibers had previously been acidloaded by pretreating with NH&l). As can be seen, the rate of pHi recovery was not only dependent on pHi, but on pH, ([HCO&) as well. For example, at pHi 6.7 in Fig. 2, the rate of recovery (thousandths of a pH unit per min) was 89 at pH, 8.6, but only 25, 11, and 3 at pH, 8.0, 7.4, kd 6.8, respectively. At pH, 6.2, pHi actually drifted downward. Mean data for all experiments at pH, 6.8,7.4, 8.0, and 8.6 are given in Table 2. Because these studies did not allow us to separate the relative contributions of pH, and [HCO&, we also performed experiments in which pH, was altered by varying Pco2 at constant [HCO& (2.5 mM); pH,‘s of 6.8, 7.4, and 8.0 were established by equilibrating the solutions with 1.6, 0.4, and 0.1% COZ, respectively, Fibers were acidloaded as before, and the rate of pHi recovery determined
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BORON,
MCCORMICK,
AND
ROOS
FIG. 2. Dependence of pHi recovery upon pH, (Pco~ constant). Fibers were previously acid-loaded by prepulsing with 50 mM NH&l-BSW, as in Fig* 1. They were then exposed to BSW equilibrated with 0.4% CO2 at pH, 8.6, 8.0, 7,4, 6.8, or 6.2; [HCOZ],, was 40, 10, 2.5, 0.63, and 0.16 mM, respectively. Experiments are picked up at the point where pHi is 6,7, and the subsequent recovery (or, in the case of pH,, 6.2, decay) is monitored. These five experiments were chosen because their rates of recovery were representative. In the pH,, 7.4 experiment, pHi eventually reached 7,3, but only after 2 ha
TABLE pH,>
2. Rates ofpHi recovery at 0.4% CO2 following acid loading [HCOII
I,,, mM
6.8
0.6
7.4
2.5
8.0
8.6 Values
10
40 are expressed
PHI 6.7
6.8
1.9 f 0.3 (5) 13.5 k 6.5 (3) 26.0 t (7)
3.3
65.9 k 11.2 (4) in thousandths
6.9
7.0
7.1
2.0 k 0.7 (71
1.6 -+ 0.8
8.6 * 2.1 (9)
9.1 t 1.5 (13)
7.7 xk 1*2 (131
5.7 t 1.0
25.5 t 2.9
22.5 t 1.8
18.2 t 1.4
12.8 t 1.2
02) 58.3 k 6.3
(8) of a pH unit/min
(22)
43.7 t, 5.5
39.7 -+ 3.8
w
at a single point pHi = 6.8. Table 3 summarizes the data. As can be seen, both the rate of pH; recovery and the calculated acid extrusion rate increased with pH,, even though [HCO, y] 0 was held constant. The data of Table 3 thus indicate that pH, strongly influences acid extrusion independent of changes in [HCO&. The dependence of acid extrusion on [HCO& per se will be examined in a later communication. In the first set of experiments, in which pH, was altered by varying [HCO& (Pco~ 0.4%), we noticed that pH, affected not only the rate of pHi recovery, but the value at which pHi eventually leveled off as well. In 11 experiments at pH, 8.0 in which pHi recovery was allowed to continue until pHi reached a stable value, the plateau pHi was 7.33 t 0.023. At pH, 7.4 the value was not significantly different, 7.32 t 0.025 ( JZ= 7). These values closely resemble the pHi of fresh fibers in nominally CO*free BSW (pH, 7.8). However, at pH, 6.8 the plateau pHi was only 6.95 t 0.023 (n = 13). On the other hand, at pH, 8.6, pHi continued to rise beyond 7.35, though at a very low rate (approximately 0.08 per h). SITS-sensitive PHi transients. When fresh fibers are incubated in HCOT-free BSW of pH 7.4, 8.0, or 8.6, pHi remains at a normal level for hours. When fibers are incubated in HCO&containing BSW of pH 7.4 or 8.0, the recovery of pHi from an acid load (which can be stopped
7.3
3.8 t 0.8
2.2 * 0.2 (41
(2)
(21)
k SE; number
7.2
(18) of fibers
w
(81
cm
8.3 t L2 (191
3.9 k 0.7 (10)
32.0 k 4.0 (191
20.7 t, 2.7 (18)
11.9 * 2.1 (15)
in parentheses.
3. Rates ofpHi recovery with [HCO& 2.5 mM following acid loading TABLE
CO?,
6.8 7.4 8.0
=
(9 )
Kate of pH, Keoovery
Kate of Acid Extrusion
1.6 0.4 0.1
4.6 t 1.2 (4) 15.3 t 2.6 (4) 30.3 t 3.3 (4)
0.20 * 0.05 0.59 zk 0.10 1.15 t 0.13
Rates of pHi recovery values are expressed in thousandths of a pH unit/mm k SE; number of fibers in parentheses. All data were obtained at pHi 6.8. External solution contained 30 mM HEPES. Rates of acid extrusion values are expressed in mmol 1-l 9min k SE. l
by SITS) halts as pHi approaches a normal level. Thus, we did not anticipate the slow rise of pHi beyond a normal value in the pH, 8.6 experiments mentioned above. Such an intracellular alkalinization occurred in the experiment of Fig. 3A, in which a fiber, not previously acid loaded, was exposed to BSW at pH, 8.6 (0.4% CO*/ 40 mM HCO;). As can be seen, the immediate COZinduced fall in pHi was followed by a much slower rise beyond the initial value. By the end of the experiment, nearly 4 h later, pHi had reached 7.7, a level substantially higher than the initial PHi. Furthermore, this intracellular alkalinization was greatly reduced by SITS (see Fig. 3B), as was the case with the pHi recoveries at lower pH, values.
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PH REGULATION
IN
I
BARNACLE
MUSCLE
Cl89
FIBERS
4OmM HCOS /O.4%
CO2
78. 77 . P Hi7.6
75.
B
SITS
I
I
15 min.
Another SITS-sensitive pHi transient is illustrated in Fig. 4A. A fresh muscle fiber was exposed to BSW buffered with 10% COJ5 mM HCO: (pH, 6.27), a treatment that led to an abrupt fall of pHi (segment ab) as CO2 entered the cell, combined with Hz0 to form H&03, and then dissociated to form H+ and HCOT. This process would have been expected to continue until internal and external levels of CO2 were the same, at which time pHi would have leveled off. Instead, as illustrated by the first CO2 pulse of Fig. 4A, the pHi continued to fall during the CO* exposure ( bc). Furthermore, on removal of COZ, pHi returned to a level below its initial one (compare a and d), reflecting the net accumulation of acid during the CO2 exposure. The continuing acidification ( bc), as well as the shortfall of pHi, had been previously observed (2). After pHi recovered from the first CO* pulse, the fiber was treated with 0.5 mM SITS for about 25 min, and exposed to CO* once again. As before, the CO2 caused pHi to drop precipitously (ef). But this time there was no continued fall of pHi ( fg), and on removal of COZ, pHi returned to nearly the initial level (compare e and h). This acidification was not only SITS sensitive, but also depended on [HCO& Figure 4B illustrates an experiment in which a fiber was successively exposed to two HCOJCOZ solutions of pH, 6.3, the first containing only one-tenth the HC03 and CO2 of the second. Exposure of the fiber to the first solution (1% CO2/0.5 mM HCO;) caused a rapid initial fall in pHi ( ab), followed by a very
I
FIG. 3, SITS-sensitive alkalinization in pH,, 8.6 BSW (40 mM HCO;/0.4% CO?). A: rise in pHi beyond normal level. Fiber was exposed to BSW containing 40 mM HCO;, equilibrated with 0.4% COZ. The pH; initially fell due to influx of CO?, but then slowly recovered. Note the very long time scale of this experiment. Similar results were obtained in two additional experiments. 13: SITS sensitivity. The fiber was acid-loaded with an NH&l pulse as in Fig. 1. The experiment is picked up during the subsequent recovery of pHi. Note that pHi continued to rise at a slow rate even after pHi passed 7.4. The fiber was then poisoned with 0.5 mM SITS while in BSW containing 40 mM HC0.i (pH,, 8.6). The subsequent rate of alkalinization was greatly reduced (compare with broken line, which indicates control rate of alkalinization). Similar results were obtained in five additional experiments.
I
slow decline ( bc). When the CO:! was removed, FHi fell short of the initial value by only 0.03. On the other hand, when the fiber was exposed to the second solution of the same pH, but containing 10% CO& mM HCOT, pHi, after the initial COz-induced acidification ( ef), fell nearly twice as rapidly as before (the ratio of slopes fg to bc was -1.8). From the observed rates of acidification, we computed the rate of HCO; or OH- efflux, or of H’ influx, taking into account PT. Because of the higher [HCOg]i due to the higher Pco~, PT was nearly twice as great in the second pulse as in the first. We found that the flux rate must have been 3.5 times greater in fg than in bc. A similar conclusion is also reached by comparing the ratio of shortfalls following removal of CO2 in the two pulses. If the acidification during bc and fg had been due to either H+ influx or OH- efflux, one would have expected the rate of acidification to be greater in the first, in which pHi was higher and thus the inward gradient for H+ greater ( Vm and pH, were the same in both). The data thus support the hypothesis that the ionic species responsible for the acidification was HCOi. Intracellular acidification follouling bluckade of acid extrusion by S1TS. As noted in the first section of RESULTS, when an acid-loaded fiber was exposed to HCOs/ CO2 BSW at pH, 6.8, pHi would rise to only about 7.0 before leveling off. In a few such experiments, we continued to monitor pHi for some time after it had leveled off, and noted that pHi would sometimes decay, even though
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
Cl90
BORON, Sm
A
M I-KOj
SEE+
5mM I-K03 I i 10% co2
2
7.2 PHi
7.0 -
MCCORMICK,
AND
ROOS
nal solution. In Fig. 5, the fiber was acid-loaded and then exposed to HC03/COz BSW (pH, 8.0) for about 90 min. SITS was then added, after which pHi began to fall. Results of similar experiments showed that the rate of the SITS-unmasked acidification was positively related to the length of time allowed for recovery of pHi before SITS poisoning. The data from experiments at pH, 6.8, 7.4, and 8.0 are plotted in Fig. 6, where the rate of acid production (the product of PT and the rate of fall in pHi) is given on the ordinate, and the delay before SITS application on the abscissa. It can be seen that at pH, 6.8 no SITS-induced acidification was detectable unless the delay was greater than 60 min, although at pH, 8.0 such
6.8I
B
’
O.5mM
1%
HCO,
5mM
10mM
HC03
/o.f!%CO2
I SITS
HCOj
4
10%co2
co2
-
-
C
h
.64r II
20 min. FIG. 4. SITS sensitive acidification in pH,, 6.3 BSW. A: SITS sensitivity. The fiber was first exposed to BSW containing 5 mM HCO; and equilibrated with 10% CO? (pH,, 6.27). After the initial CO?-induced fall in pHi ( ab), pHi continued to fall ( bc). Return to COP-free BSW caused pHi to return to a value less than the original one. The fiber was then poisoned with 0.5 mM SITS for 25 min, and then once again exposed to 5 mM HCOJlO% CO:!-BSW. This time, after its initial fall (ef), pHi was nearly constant ( fg), and, on removal of COZ, nearly returned (gh) to its initial value, A second experiment yielded similar results. B: dependence on HCO;. The fiber was first exposed to BSW containing 0.5 mM HCO;, equilibrated with I% CO? (pH,, 6.3). This caused a rapid fall in pH, ( ab), followed by a much slower acidification ( bc). Return to CO?-f ree BSW caused pHi to fall short of the initial pHi by a small amount. The fiber was then exposed to BSW containing 5 mM HCO;, equilibrated with 10% CO,; once again pH,, was 6.3. After the initial fall in pHi (ef), pH, continued to fall ( fg) at a rate significantly greater than in the first pulse (bc). Upon return to a COz-free solution, the amount by which pHi fell short of its initial value was also significantly greater than in the first pulse (compare points a and d with points e and h). Upon exposure to these CO?-containing solutions, the fiber reversibly depolarized by 5 mV. The C&free solutions were buffered with HEPES to pH 7.4.
the fiber was still exposed to the HC0,7/COz BSW. In experiments at pH, 7.4, we routinely observed downward drifts in pHi when, after pHi had leveled off, fibers were returned to COe-free BSW of the same pH,. We considered the possibility that an intracellular acid-producing mechanism, evoked by the previous process of acid loading and/or acid extrusion, was responsible for the drifts. This mechanism might be unmasked under conditions where acid extrusion is reduced or abolished. To test this hypothesis, we acid-loaded fibers with an NH&l prepulse and then followed the course of pHi in COZ-BSW for various lengths of time before adding SITS to the exter-
I
30
min. i
t
L
FIG. 5. Effects of SITS on pH, after recovery from acid-loading. A fiber was acid-loaded by prepulsing with 50 mM NH&l, as in Fig. I, and then exposed for 90 min to BSW containing 10 mM HO;, equilibrated with 0.4% CO:! (pH,, 8.0). The fiber was then poisoned with 0.5 mM SITS, which produced an eventual decay of pHi.
30
I 90
60 DELAY
1
1
120
150
I 180
(Min)
6. Rates of acid introduction. Fibers were acid-loaded with an NH&l prepulse, as in Fig. 1, and the pHi was allowed to recover in pH,, 6.8, 7.4, or 8.0 HCOi-BSW, as in Fig. 2. After recovery had proceeded for given lengths of time (delay plotted on abscissa), fibers were poisoned with 0.5 mM SITS, causing pHi to level off and then decline (as in Fig. 5). Rates of acid introduction (ordinate) were taken as the product of the acidification rate and pi%, and are thus given in pmol 1-“I min? Number of experiments is given near each point; average SE was 8.5. The data were fitted to power functions by the least-squares technique. FIG.
l
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l
PH REGULATION
IN
BARNACLE
MUSCLE
FIBERS
an acidification was already seen at 30 min. Attempts at studying the intracellular acidification at pH,, 8.6 were unsuccessful because of the failure of SITS and/or DIDS to completely block pHi recovery at low values of pHi. At higher values of pHi, SITS blocked intracellular alkalinization, but did not unmask any acidification. Not shown are the results of three experiments in which acid-loaded fibers were SITS poisoned early enough to completely prevent pHi recovery and then alternately exposed, over a 2-h period, to HCO;1/C02-BSW at pH, 8.0 and 6.2. Even 2 h after acid-loading and SITS poisoning (during which time pHi remained at a very low level), there was no significant acidification in either pH 8.0 or 6.2 HCOT/COz-BSW. Also not shown are the results of other experiments in which fresh fibers were maintained in normal BSW (pH, 7,8) for several hours, and then exposed to SITS. Although previously stable, pHi decayed after application of SITS, and the rate of decay was comparable to that observed after acid-loading. DISCUSSION
Before discussing the pH dependence of acid extrusion, we will briefly examine two phenomena that must be considered in the interpretation of the acid extrusion data, SITS-sensitive pHi transients other than acid extrusion, and intracellular acid production. Other SITS-sensitive pHi transients. A previous study of barnacle muscle (2) provided strong evidence for the net efflux of HCO? (or the equivalent movement of H’ or OH-) under conditions of very low pH,,. The present experiments extend the earlier observations on two counts. First, we found that the continuing acidification during CO2 exposure at pH, 6.3 was much faster in 10% CO2 than in 1% CO2 (compare bc and fg in Fig. 4B), suggesting that it is the movement of HCO?, rather than that of H’ or OH-, which is responsible for the continuing fall in pHi* Second, this acidification was blocked by SITS. In addition, we found that when fibers were exposed to 0.4% COJ40 mM HCO; (pH,, 8.6), pHi continued to rise beyond the normal value of about 7.35 (Fig. 3A). This alkalinization was also blocked by SITS (Fig. 3B). On the other hand, when a fiber was exposed to HCOSfree BSW at pH, 8.6, pHi remained unchanged for hours. At the present time, it is impossible to identify the mechanism(s) of these SITS-sensitive pHi transients. We can think of three plausible explanations. First, the phenomena could be due to the passive movement of an ion through a channel. Our calculations using the constant field treatment (9, 11) show that the permeability to HCOT would have to be about 7 x lo-” cm/s (treating the fiber as a simple cylinder) in order to account for either the alkalinization of Fig. 3 or the acidification of Fig. 4. This hypothetical value is not too far from the value for Pcl calculated by DiPolo (8), l-9 x 10B6 cm/s. One way to test the possibility of passive HCO; flux would be to examine the sensitivity of the pHi course to Vm. We attempted to do this but met with failure because of technical difficulties. The second possibility is that the same mechanism responsible for acid extrusion is also instrumental in producing the transients in Figs. 3 and 4. This possibility is reinforced by the demonstrated dependence of these
Cl91 transients on [HCO& as well as by their SITS sensitivity. If this possibility were correct, it might be anticipated that these transients would have a requirement for Na’ or Cl-, as has been substantiated in the case of acid extrusion (5, 12). The transients might also be affected by furosemide and cyclic AMP (6). A third explanation for the pHi transients of Figs. 3 and 4 is that one or both were mediated by a SITSsensitive carrier that is independent of the PHi regulating system, and thus unaffected by furosemide, cyclic AMP, or Na’ removal. Our present data do not allow us to distinguish among the three choices, although the second requires the fewest ad hoc assumptions. Intracellular acidproduction. Inhibition of acid extrusion by SITS in previously acid-loaded fibers unmasked a time-dependent intracellular acidification. SITS blocks the three types of pHi transients that have been shown to be HCOZ dependent (namely, acid extrusion and the transients illustrated in Fig. 3 and 4). Therefore, movement of HCOT is probably not involved in this phenomenon. Its rate increased with the length of time during which acid extrusion was allowed to proceed, and also with pH,, (Fig. 6). When acid extrusion was suppressed from the start by early application of SITS, no acidification was observed at any pH,. We also found SITSunmasked acidification in fibers maintained in normal BSW (pH, 7.8) for several hours. Without SITS, pH, remained constant (pHi 7.32), but the application of SITS resulted in a fall of pHi at a rate comparable to that observed after acid-loading (Fig. 6). We have made no attempt to analyze the nature of the SITS-unmasked acidification; our main concern was to use these results to correct our data on the rate of acid extrusion as discussed below. The pH-dependence of acid extrusion. The data of Fig. 2 and Table 2 demonstrate that the rate of pHi recovery from an acid load is greatest at low values of pHi and high values of pH,. As illustrated in Fig. 7, the calculated rate of acid extrusion follows a similar trend. The curves represent averages of our data from 63 experiments in which pH, was altered by varying [HCO;],. Details of their construction can be found in the legend of Fig. 7. Acid extrusion rates were obtained by multiplying the rates of pHi recovery by intracellular buffering power (see METHODS) and then correcting for the time-dependent SITS-unmasked acidification. This correction was a small fraction of the acid extrusion rate except when this rate was very low, As can be seen in Fig. 7, at pH, 7.4, 8.0, and 8.6 the acid extrusion rate falls linearly as pHi increases. At pH, 7.4 and 8.0 this linearity is maintained as normal pHi is approached, whereas at. pHo 8.6 the acid extrusion rate is sustained beyond normal pHi+ It should be pointed out that at pH, 8,6 the electrochemical gradient for HCO? was inward over the entire range of pHi values (avg Vm, - 50 mV), while at pH, 7.4 and 6.8 this gradient was outward; at pH, 8.0 the gradient reversed direction at pHi -7.2. Thus, acid extrusion takes place at pH,, 7.4 and 6.8 in the face of a large outward-directed electrochemical HCO; gradient. The low rate of acid extrusion at pH, 6.8 might be ascribed to the large acidifying influence of HCOT efflux. In contrast, at pH,, 8.6 acid extrusion continued at pHi values beyond
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
BORON,
PHi
7. Acid extrusion rate as a function of pHi at 4 values of pH,, (Pco? constant). Summary of data from 63 experiments, in which pHi was lowered by pretreating with NH&l and then allowed to recover while the fiber was bathed in HCOJ0.4% CGBSW at pH, 8.6, 8.0, 7.4 or 6.8. The curves at pH,, 7.4, 8.0, and 8.6 were obtained as follows: I) at a given pH,,, the acid extrusion rate for each experiment was calculated at pHi intervals of 0.025 by multiplying the rate of pHi recovery by fir, as described in METHODS; acid extrusion rate was then plotted against pHi; 2) all curves at a given pH, were normalized to the mean pHi at which acid extrusion rate was zero; these pHi values were 7.32 k 0.025 ( n. = 7) at pH,, 7.4; 7.33 k 0.023 (n = 11) at pH,, 8.0; and rate 7.38 t 0.027 ( n = 19) at pH,, 8.6. At pH,, 8.6 the acid extrusion never became zero, therefore, the pHi to which these curves were normalized was obtained by extrapolation to zero acid extrusion rate of the linear portion of the curves; and 3) all normalized data at a given pH,, were averaged at pHi intervals of 0.025, and these averaged values plotted in this Fig. The pH,, 6.8 curve is a simple average of the data without normalization. Total number of experiments: 12 at pH,, 6.8, 12 at pH,, 7.4, 20 at pH,, 8.0, and 19 at pH,, 8.6. Each point represents the average of 2-7 experiments at pH,, 6.8,3-H at 7.4, 7-20 at 8.0, and 7-19 at 8.6. Vertical bars indicate standard error; lines represent linear regressions. FIG.
normal (see also Fig. 3). This represents HC03 movement in the direction of its electrochemical gradient. Thomas (13) observed that in snail neurons, the recovery of pH, from acid loads follows an exponential time course. This implies that, if PT is constant, the acid extrusion rate is proportional to the difference between normal pHi (the threshold pHi below which acid extrusion is initiated) and the prevailing pHi. We found a similar relationship in our experiments. In the experiments of Fig. 7, changes in pH, were produced by varying [HCO& at constant Pcoz (0.4% CO& Inasmuch as these studies, by themselves, did not distinguish between the effects of pH, per se and those of [HCOs lo, a series of experiments was undertaken in which pHO was changed by varying PCO~ at constant [HCO& (2.5 mM). The results showed that, even at constant [HCO&, raising pH, caused an increase in the acid extrusion rate. Therefore, barring a specific effect of Pcoz on acid extrusion, pH, is an important determinant of the rate of acid extrusion. On the other hand, it is well known that [HCO-] 3 0 influences acid extrusion independently of pH,. Experiments on the squid axon (4), the snail neuron (13), and barnacle muscle (2)) showed that acid extrusion is substantially augmented by switching from a HCOT-free to a HCOT-containing bathing solution
MCCORMICK,
AND
ROOS
at the same PH. Recently, Thomas (15) showed that the rate of acid extrusion in snail neurons progressively increases as [HCO& is raised from 0 to 4.5 to 21 mM, all at constant pH,. Because pH, and [HCO& can individually affect acid extrusion, one would anticipate that increasing both together would have a greater effect than increasing pH, alone. This is borne out by the data of Tables 2 and 3. Increasing pH, from 6.8 to 8.0 caused the rate of acid extrusion at pHi 6.8 to rise l7-fold when [HCO& was raised at constant PCO~ (Table Z), but only 7-fold when PCO~ was lowered at constant [HCO& (Table 3). The mechanism of the pH, effect is unknown. The data of the present study and recent work on dialyzed barnacle muscle fibers (6) provide some insight into possible mechanisms of pHi regulation in barnacle muscle. The results of the latter study indicate that acid extrusion involves an exchange of external WC03 for internal Cl-, and also suggest that the same carrier can engage in both Cl-Cl and HCO&l exchange. It was hypothesized that, at a normal pHi, the carrier operates at a low rate in the Cl-Cl exchange mode, while at lower values of pHi the rate of anion-anion exchange greatly increases and the mode partially shifts to HCO&l exchange. It would be reasonable to assume that under conditions of acid loading external HCOT and Cl- compete with one another for entry. Indeed, the results of the previous study (6) showed that when [HCO& was low, the rate of acid extrusion was low and the Cl- influx was relatively high. Increasing [HCO& at constant pH, caused an increase in the rate of acid extrusion (i.e., HCO; influx), but a decrease in Cl- influx. However, at a normal pHi, when the carrier should have been engaged only in Cl-Cl exchange, a similar increase in [HCO& had no effect on either the acid extrusion rate (which remained zero) or Cl- influx. Further evidence that HCO:jCl and Cl-Cl exchange are mediated by the same carrier is that both exchanges were inhibited by SITS and by furosemide and were stimulated by cyclic AMP. One might speculate on how the SITS-sensitive pHi transients of Figs. 3 and 4 in the present paper fit into the above scheme of a common carrier for HCOS-Cl and Cl-Cl exchange. Our analysis requires two assumptions. First, the carrier normally mediates several different exchanges (Cl-Cl, external HCOS-internal Cl, external Cl-internal HCOS, and HC03-HCOa) simultaneously, and that the relative amount of each exchange is governed by pHi, pH,,, and the prevailing substrate concentrations. Given the fact that the first two of these exchanges have already been documented, this assumption seems rather reasonable. Second, we hypothesize that the stimulatory effect of raising pH, on the acid extrusion rate is brought about by causing the carrier to pick up relatively more HCOs and less Cl- from the external solution (without affecting the total rate of anion exchange, or the ions picked up on the inside). Thus, when pH, is reduced to low levels (as was the case during the SITS-sensitive acidification of Fig. 4), external HCO:j-internal Cl and HC03-HC0.7 exchange would be reduced while external Cl-internal HCOS and Cl-Cl exchange would be increased. This would account for the observed acidification in Fig. 4. Conversely, when pH, is raised to high levels (as was the case during the SITS-sensitive alkalinization of Fig. 3), Cl-Cl and external Cl-internal HCOS exchange would be
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PH REGULATION
IN
BARNACLE
reduced in favor of external HCOS exchange.
MUSCLE
HCOs-internal
We thank J. Jones for preparing the manuscript. This work was supported by National Institutes HL-00082 to A. Roos, and NIGMS-02016 (Medical Program Fellowship) and GM-06499 (National
Cl93
FIBERS
Cl and HC@-
of Health Grants Scientist Training Research Service
Award) to W. F. Boron. A. Roos is recipient of National Institutes of Health Research Award HR-19608. W. F. Boron’s present address: Department of Physiology, University School of Medicine, New Haven, CT 06510 Received
16 November
1978; accepted
in final
form
1 May
Career Yale
1979.
REFERENCES 1. AICKIN, C. C., AND R. C. THOMAS. Micro-electrode measurements of the intracellular pH and buffering power of mouse soleus muscle fibres. J. Physiol. London 267: 791-810, 1977. 2. BORON, W. F. Intracellular pH transients in giant barnacle muscle fibers. Am. J. PhysioZ. 233: C61-C73, 1977 or Am. J. Physiol.: CeZZ PhysioZ. 2: C61-C73, 1977. 3. BORON, W, F., AND P. DE WEER. Intracellular pH transients in squid giant axons caused by COZ, NH:{, and metabolic inhibitors. J, Gen. Physiol, 67: 91-112, 1976. 4. BORON, W. F., AND P. DE WEER. Active proton transport stimulated by COz/HCOa, blocked by cyanide, Nature London 259: 240241, 1976. 5. BORON, W. F., AND A. Roos. Intracellular pH regulation in giant barnacle muscle. Biophys. J. 21: 10a, 1978. 6. BORON, W. F., J. M. RUSSELL, M. S. BRODWICK, D. W. KEIFER, AND A. ROOS. Influence of CAMP on intracellular pH regulation and chloride fluxes in barnacle muscle fibres. Nature London 276: 511-513, 1978. 7. CABANTCHIK, 2. I., AND A. ROTHSTEIN. The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives. J.
Membr. Biol, 10: 311-328, 1972. 8. DIPOLO, R. Chloride fluxes in isolated dialyzed barnacle muscle fibers. J. Gen. Physiol. 60: 471-497, 1972. 9. GOLDMAN, D. Potential, impedance and rectification in membranes. J. Gen. PhysioZ. 27: 37-60, 1943. 10. HINKE, J. A. M. Cation-selective microelectrodes for intracellular use. In: GZass EZectrodes fur Hydrogen and Other Cations, edited by G. Eisenman. New York: Dekker, 1967, p. 464-477. Il. HODGKIN, A. L., AND B. KATZ. The effect of sodium on the electrical activity of the giant axon of the squid. J. Physiol. London 108: 3777, 1949. 12. RUSSELL, J. M., AND W. F. BORON. Role of chloride transport in regulation of intracellular pH. Nature London 264: 73-74, 1976. 13. TIJOMAS, R. C. The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J. Physiol. London 255: 715-735, 1976. 14. THOMAS, R. C. Ionic mechanism of the H’ pump in a snail neurone. Nature Londorz 262: 54-55, 1976. 15. THOMAS, R. C. The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. J. Physiol. London 273: 31.7-338, 1977.
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in barnacle muscle fibers: dependence and extracellular pH
WALTER F. BORON, WAYNE C. MCCORMICK, AND Department of Physiology and Biophysics and Department University School of Medicine, St. Louis, Missouri 63110
BORON, WALTER
AND ALBERT dependence on extraceLLuLar PH. Am. J. Physiol. 237(3): Cl%-C193, 1979 or Am. J. Physiol.: Cell Physiol. 6(2): Cl%Cl93, 1979.~Intracellular pH (pH;) regulation was studied in acid-loaded barnacle muscle fibers by monitoring recovery of pHi with a pH-sensitive microelectrode. By multiplying the rate of pHi recovery by total intracellular buffering power, the acid extrusion rate was obtained. The acid extrusion rate was greatest at low values of pHi, and declined toward zero as pHi approached normal levels. It increased as the extracellular pH (pH,) was raised either by increasing external [HCO,] ([HC&],) at constant PCO~ or by decreasing PCO~ at constant [HCO& but more so in the former case than in the latter. These observations suggest that pH, per se is an important determinant of the acid extrusion rate, but that raising [HC&],, by itself also stimulates acid extrusion. This would be expected if acid extrusion involves the inward movement of solutions HCO.7. When f 1b ers were exposed to HCOa-containing at very low or very high pH,, pHi drifted downward or upward, respectively; these drifts were inhibited by 4-acetamido-4’ isothiocyanostilbene-2,2’ disulfonic acid (SITS). Our results are discussed in terms of possible mechanisms of acid extrusion. Roes. pH regulation intracellular and
F., WAYNE C. MCCORMICK, in barnacle
muscle
fibers:
bicarbonate transport; SITS (4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonic acid); intracellular buffer power
ALBERT ROOS of Anesthesiology,
Washington
for external Na’ has been described in the snail neuron (15) and, more recently, in barnacle muscle fiber (5) Relatively little quantitative information is available concerning pHi regulation. Thomas (13) has reported that, in the snail neuron, recovery of pHi after loading with acid seems to follow an exponential time course. That is, the rate of acid extrusion (derived from the rate of pHi recovery) is proportional to the difference between the “normal” pHi and the prevailing value. Also, the rate of acid extrusion in the squid axon increases with extracellular pH (pH,) as external [HCO,] ([HCO&) is raised at constant PCO~ (4). This result has been qualitatively confirmed in both barnacle muscle (2) and snail neurons (15). We now report a quantitative study in barnacle muscle fibers in which we have examined the rate of acid extrusion as a function both of pHi and of pH,. We found that the acid extrusion rate was greatest at low values of pHi, and gradually fell towards zero as pHi approached its normal range. In addition, at a given value of pHi, the acid extrusion rate increased with pH,, whether pH, was raised by increasing [HCO& or by lowering Pco~. Portions of this work have been previously reported (5). ment
METHODS IT IS WELL established that nerve and muscle cells possess
a mechanism for regulating their intracellular pH ‘pHi). These cells maintain pHi at a more alkaline level khan would be expected if H’ were in eqwilibrium. In +)he steady state, an active transport mechanism is required to remove acid that accumulates as the result of either cellular metabolism or passive ion movement (H+ influx, OH-, and HCOT efflux). This same mechanism is also responsible for the rapid recovery of pHi following acute acid loads. We refer to this transport process as acid extrusion, realizing, of course, that it may involve the ejection of acid and/or the accumulation of alkali. Several qualitative properties of this acid extruding mechanism responsible for the pHi regulation have already been described. In the squid giant axon (4), snail neuron (l3), and barnacle muscle (Z), acid extrusion is stimulated by external HCO; (2, 4, 13) and blocked by the anion flux inhibitor 4-acetamido-4’-isothiocyanostilbene-2,2’disulfonic acid (SITS) (2, 7, 12, 14). There is also strong evidence in each of these preparations that the mode of action of the acid-extruding mechanism is an exchange of external HCOi for internal Cl- (6, 12, 15). A require0363-6143/79/oooO-oooO$O1.25
Copyright
0 1979 the American
Physiological
GeneraZ. Single muscle fibers were isolated from the giant barnacle Balanus nub&s, obtained from D. King, Friday Harbor, Washington. Each fiber was cannulated at both ends, mounted horizontally in a chamber, and superfused with artificial barnacle sea water (BSW) at 22OC. Cut ends of the fibers were isolated from the superfused central region by Vaseline seals. Electrodes for measuring pHi and membrane potential ( Vm) were inserted through the cannulas from opposite ends. The pH-sensitive electrodes were similar to those of Hinke (10); their use has been previously described (3). Fibers were exposed to C02-containing solutions delivered from glass syringes through COZ-impermeable Saran tubing (Clarkson Controls Equipment, Detroit, MI.). SoZutions. BSW had the following composition (in mM): 440 NaCl, 10 KCl, 11 CaC12, 11 MgC12, and 21 MgS04. In nominally CO*-free solutions the BSW was buffered with 5 mM N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES). Solutions equilibrated with CO2 contained only 6 mM CaClz (remainder replaced by NaCl), and were buffered to pH 6.2-8.6 with 0.4% CO* (balance oxygen) and varying amounts of HCOT Society
Cl85
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Cl86
BORON,
(HCO; substituting for Cl-), or were buffered to pH 6.88,0 with 2.5 mM HCOT and varying amounts of COa. The pH, 8.6 solution had to be gassed with CO* for several hours for a stable pH to be reached. This solution was slightly cloudy, probably because of some calcium and magnesium precipitation; the amount of HCO; actually added thus had to be greater than the nominal 40 mM. In the NHJXBSW used in the NH&l prepulse, 50 mM NaCl was replaced by 50 mM NH&l; the solution was buffered to pH 7.70. SITS was obtained from International Chemical and Nuclear (Cleveland, OH), and 4,4’diisothiocyanostilbene-ZJ’disulfonic acid (DIDS) from Pierce Chemical (Rockford, IL). Determination of the rate of acid extrusion. Fibers were loaded with acid by exposing them to BSW containing 50 mM NH&l for 60-90 min, and then returning them to an NH&l-free solution (pH 7.4). After removal of the NH&l, pHi fell to a value substantially less than the initial one. This NH: prepulse technique was first used to acid-load squid giant axons (4), and later in mouse soleus muscle (1). Because its theoretical basis has been previously examined in detail (3), it will be only briefly reviewed here. Exposing a fiber to a solution containing NH&l (see Fig. 1) leads to a rapid initial rise in pHi (segment ab) as some of the NH3 present in the external solution enters the cell and there combines with 50 mM
-1
I
b
8.0
AND
ROOS
protons to form NH:; the increase in pHi halts when the intracellular [NH31 ([NHa]i) equals the [NH&. However, there remains a substantial electrochemical gradient favoring the net influx of NH:, and pHi slowly falls (be) as a fraction of the entering NH: dissociates to form H’ and NH:j. When NH&l is removed from the external solution, virtually all intracellular NH: (including that which had entered as such) leaves as NH3, and, as H+ is left behind, pH; falls below its initial level (compare values at a and d). The degree of undershoot is determined by the magnitude of the previous net influx of NH,‘. After fibers had been loaded with acid in this way, they were exposed to a COz-containing solution, and the subsequent recovery of pHi was monitored on a strip chart recorder. Assuming that acid extrusion was the only factor influencing the rate of recovery of pH;, we obtained the rate of acid extrusion as the product of total intracellular buffering power (see below) and the rate of pH; recovery (dpHi/dt). Because the surface-to-volume ratio of the barnacle muscle fiber is unknown (the vast majority of the surface membrane surrounds an intricate network of invaginations), rates of acid extrusion were expressed in mmol/min per liter cell water rather than per square centimeter surface membrane. Values for dpHi/dt were derived as follows. Each recording of pHi vs. time was manually traced with a rhoIOmM
NH&I
MCCORMICK,
HCOj
/0.4X
pH,
= 8-00
CO2
7.8
7,6
7.4 PHi 72
a
d FIG. 1. Acid loading of muscle fiber and recovery of pHi from acid load. An isolated barnacle muscle fiber was exposed to BSW containing 50 mM NH&l (pH,, 7.7) for 80 min, during which time pHi rapidly rose (segment ab), and then slowly declined (bc). Returning the fiber to NH:-free BSW (pH,, 7.4) caused pHi to fall to 6.59 (cd) far below the initial pHi (7.28). This acid-loaded fiber was then exposed to BSW containing 10 mM HCOJ0.4% CO2 (pH,, 8.00), which caused pHi to
recover rapidly. With very high rates of acid extrusion, such as prevailed in this experiment, the initial acidification that normally occurs upon CO? exposure is masked. Note that the time scale was changed shortly before the CO? exposure began. All figures were traced from original pen recordings. Changes in Vnl upon recovery from acid loading were as previously described (2).
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PH REGULATION
IN
BARNACLE
MUSCLE
theta transducer, linked to a PC-12 minicomputer, which converts the curve into a set of x and y coordinates. A data point was obtained every 0.25 mm along the time axis, which in all experiments was about 20 cm long; the system was accurate to the nearest 0.25 mm. The computer’s sensitivity along the ordinate (pHi axis) was such that about six ‘/4 mm divisions corresponded to 0,Ol pH unit. The set of x,y coordinates was transferred to an IBM-360 computer where the data were curve-fitted to a polynomial of degree 4 or less, The total residual variance of the fit was always less than 10B4 (mean 1.6 x lo-“). The initial portion of the pHi-vs.-time record during the recovery period was excluded from the curvefitting procedure because this portion represented the transition from a COZ-free to a COz-containing external solution. The fitted polynomial was differentiated, and values for dpHi/dt were calculated over a range of values of pHi. Intracellular buffering power. Buffering power is defined as the ratio of the amount of strong base (B) added to a solution (mmol/l) to the resultant change in pH (p = dB/dpH). The total intracellular buffering power &) is the sum of intrinsic or non-CO2 buffering power (Pi) and buffering power due to CO2 (pco,). The former can be experimentally determined by observing the change in pHi produced by exposing the cells to various levels of external NH4C1, and will be discussed below. The latter can be calculated at any PCOZ because, by definition, pco2 equals the partial derivative of the Henderson-Hasselbalch equation (d[HCOT]i/dpHi)pco,
Cl87
FIBERS
= 2.3 [HCOs]i
The intrinsic intracellular buffering power (pi) was experimentally determined in a previous study (2) over the pHi range 6.8-7.8. Because, in the present study, we wished to determine the acid extrusion rate at values of pHi less than 6.8, we extended the previous study of p; to a lower pHi range. The method employed was similar to that previously described. Fibers were prepulsed with 50 mM NH&l to lower pHi to 6.6-6.8, and then SITS poisoned to prevent acid extrusion. They were then exposed to BSW containing either 0.5, 1, or 2 mM NH&l (pH, 7.70), and the resultant changes in pHi noted. The 10 new data points are given in Table 1 (which is an extension of Table 1 in Ref. 2). “Initial” conditions refer to the pHi and [NHT]i (calculated from the mass action law, assuming [NH:j]i = [NH&) prevailing during the exposure to 0.5, 1, or 2 mM NH4C1, and “final” conditions to the pHi and [NHZ]i (the latter assumed to be zero) prevailing after removal of the NH4Cl. “Nominal AB” describes the amount of strong base (mmol/l) added to the fiber between initial and final conditions, and is given “nominal fl” is A[NHz]i/ApHi. “Fitted AB” by A[NH,‘]i; is obtained by inserting initial and final pHi into Eq. 1 of Ref. 2, and represents the result of the least-squares analysis. The dependence of pi on pHi (in the range 6.67.8), based on the 10 new data points as well as on the 23 previous ones, was found to be adequately described by the first order polynomial: pi = A + B (pH;), where A = 155.6 -t- 11.8 (SD) and B = -17.4 t 1.6. This new description of pi differs from the old chiefly at pHi < 7.0, where it predicts substantially lower values for pi. The
1. Buffering exposed to NH&I
TABLE
power
Initial Fiber
100577A 10077iA 10107iA 011978A 01 lYi#B 0119i8H 012078A 012378A 012478A 0124’78A
Final
Nominal [NH, ’ 1, 6.72 6.73 6.99 6.84 6.91 6.9i T-0’; 6.91 6.85 7.01
data for fibers
4.70 4.57 5.07 7.20 2.95 ‘i-24 8.44 :30.4 :kiz 4.87
Nominal N%+l, 6.64 6.64 6.ii 6.W 6.#i iiai 6.78 6.83 6.74 Ki9
0
0
Nominal bH,mM
NBomin$ ,m
Fitted AB, mM
-4.70 -4.57 4.07 -7.20 -2.9s -5.24 -8.44 -3.04 -3.52 -4.8’7
58.8 50.8 23.0 48.0 59.0 52.4 29.1 38.0 32.0 22.1
-3.2 -3.5 -i.9 -5.7 -1.8 -3.5 -10.2 -2.9 -4.1 -i.a
fitted pi at selected values of pHi is as follows: at pHi 6,7, pi = 39.1; at 6.9, 35.6; at 7.1, 32.1; at 7.3, 28.7; at 7.5, 25.1; and at 7.7, 21.6. RESULTS
Recouery of pHi from intracellular acid loads. Previous work on barnacle muscle fibers (2) had shown that the recovery of pHi from acid loads was enhanced by simultaneously increasing pH, and [HCO& To determine the importance of pH, per se, we studied acid extrusion in experiments in which pH, was varied by altering either [HC03], or Pco~. Acid loading was accomplished by pretreating fibers for 60-90 min with BSW containing 50 mM NH4C1, as described in METHODS. In the experiment illustrated in Fig. 1, this procedure caused pHi to fall from 7.28 (point a) to the new value of 6.59 (d). Subsequent exposure of the fiber to BSW buffered with 0.4% COz/lO mM HCOT (pH, 8.0) caused pHi to rise rapidly and then to level off near 7.32. Assuming that the recovery of pH; is influenced only by acid extrusion, then the rate of acid extrusion is the product of the pHi recovery rate and PT (see METHODS). The rate of kid extrusion thus calculated from the course of pHi recovery in Fig. 1 was greatest at low pH;, and gradually fell toward very low values as pHi returned to the normal range (see also Fig. 7). In a series of 72 similar experiments (one fiber per experiment) we examined the recovery of pHi from intracellular acid loading at five different values of pH, (6.2, 6.8, 7.4, 8.0, and 8.6). These were established by varying [HCO& at a constant level of COz (0.4%). Figure 2 shows a typical experiment at each of the values of pH, (the fibers had previously been acidloaded by pretreating with NH&l). As can be seen, the rate of pHi recovery was not only dependent on pHi, but on pH, ([HCO&) as well. For example, at pHi 6.7 in Fig. 2, the rate of recovery (thousandths of a pH unit per min) was 89 at pH, 8.6, but only 25, 11, and 3 at pH, 8.0, 7.4, kd 6.8, respectively. At pH, 6.2, pHi actually drifted downward. Mean data for all experiments at pH, 6.8,7.4, 8.0, and 8.6 are given in Table 2. Because these studies did not allow us to separate the relative contributions of pH, and [HCO&, we also performed experiments in which pH, was altered by varying Pco2 at constant [HCO& (2.5 mM); pH,‘s of 6.8, 7.4, and 8.0 were established by equilibrating the solutions with 1.6, 0.4, and 0.1% COZ, respectively, Fibers were acidloaded as before, and the rate of pHi recovery determined
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
BORON,
MCCORMICK,
AND
ROOS
FIG. 2. Dependence of pHi recovery upon pH, (Pco~ constant). Fibers were previously acid-loaded by prepulsing with 50 mM NH&l-BSW, as in Fig* 1. They were then exposed to BSW equilibrated with 0.4% CO2 at pH, 8.6, 8.0, 7,4, 6.8, or 6.2; [HCOZ],, was 40, 10, 2.5, 0.63, and 0.16 mM, respectively. Experiments are picked up at the point where pHi is 6,7, and the subsequent recovery (or, in the case of pH,, 6.2, decay) is monitored. These five experiments were chosen because their rates of recovery were representative. In the pH,, 7.4 experiment, pHi eventually reached 7,3, but only after 2 ha
TABLE pH,>
2. Rates ofpHi recovery at 0.4% CO2 following acid loading [HCOII
I,,, mM
6.8
0.6
7.4
2.5
8.0
8.6 Values
10
40 are expressed
PHI 6.7
6.8
1.9 f 0.3 (5) 13.5 k 6.5 (3) 26.0 t (7)
3.3
65.9 k 11.2 (4) in thousandths
6.9
7.0
7.1
2.0 k 0.7 (71
1.6 -+ 0.8
8.6 * 2.1 (9)
9.1 t 1.5 (13)
7.7 xk 1*2 (131
5.7 t 1.0
25.5 t 2.9
22.5 t 1.8
18.2 t 1.4
12.8 t 1.2
02) 58.3 k 6.3
(8) of a pH unit/min
(22)
43.7 t, 5.5
39.7 -+ 3.8
w
at a single point pHi = 6.8. Table 3 summarizes the data. As can be seen, both the rate of pH; recovery and the calculated acid extrusion rate increased with pH,, even though [HCO, y] 0 was held constant. The data of Table 3 thus indicate that pH, strongly influences acid extrusion independent of changes in [HCO&. The dependence of acid extrusion on [HCO& per se will be examined in a later communication. In the first set of experiments, in which pH, was altered by varying [HCO& (Pco~ 0.4%), we noticed that pH, affected not only the rate of pHi recovery, but the value at which pHi eventually leveled off as well. In 11 experiments at pH, 8.0 in which pHi recovery was allowed to continue until pHi reached a stable value, the plateau pHi was 7.33 t 0.023. At pH, 7.4 the value was not significantly different, 7.32 t 0.025 ( JZ= 7). These values closely resemble the pHi of fresh fibers in nominally CO*free BSW (pH, 7.8). However, at pH, 6.8 the plateau pHi was only 6.95 t 0.023 (n = 13). On the other hand, at pH, 8.6, pHi continued to rise beyond 7.35, though at a very low rate (approximately 0.08 per h). SITS-sensitive PHi transients. When fresh fibers are incubated in HCOT-free BSW of pH 7.4, 8.0, or 8.6, pHi remains at a normal level for hours. When fibers are incubated in HCO&containing BSW of pH 7.4 or 8.0, the recovery of pHi from an acid load (which can be stopped
7.3
3.8 t 0.8
2.2 * 0.2 (41
(2)
(21)
k SE; number
7.2
(18) of fibers
w
(81
cm
8.3 t L2 (191
3.9 k 0.7 (10)
32.0 k 4.0 (191
20.7 t, 2.7 (18)
11.9 * 2.1 (15)
in parentheses.
3. Rates ofpHi recovery with [HCO& 2.5 mM following acid loading TABLE
CO?,
6.8 7.4 8.0
=
(9 )
Kate of pH, Keoovery
Kate of Acid Extrusion
1.6 0.4 0.1
4.6 t 1.2 (4) 15.3 t 2.6 (4) 30.3 t 3.3 (4)
0.20 * 0.05 0.59 zk 0.10 1.15 t 0.13
Rates of pHi recovery values are expressed in thousandths of a pH unit/mm k SE; number of fibers in parentheses. All data were obtained at pHi 6.8. External solution contained 30 mM HEPES. Rates of acid extrusion values are expressed in mmol 1-l 9min k SE. l
by SITS) halts as pHi approaches a normal level. Thus, we did not anticipate the slow rise of pHi beyond a normal value in the pH, 8.6 experiments mentioned above. Such an intracellular alkalinization occurred in the experiment of Fig. 3A, in which a fiber, not previously acid loaded, was exposed to BSW at pH, 8.6 (0.4% CO*/ 40 mM HCO;). As can be seen, the immediate COZinduced fall in pHi was followed by a much slower rise beyond the initial value. By the end of the experiment, nearly 4 h later, pHi had reached 7.7, a level substantially higher than the initial PHi. Furthermore, this intracellular alkalinization was greatly reduced by SITS (see Fig. 3B), as was the case with the pHi recoveries at lower pH, values.
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PH REGULATION
IN
I
BARNACLE
MUSCLE
Cl89
FIBERS
4OmM HCOS /O.4%
CO2
78. 77 . P Hi7.6
75.
B
SITS
I
I
15 min.
Another SITS-sensitive pHi transient is illustrated in Fig. 4A. A fresh muscle fiber was exposed to BSW buffered with 10% COJ5 mM HCO: (pH, 6.27), a treatment that led to an abrupt fall of pHi (segment ab) as CO2 entered the cell, combined with Hz0 to form H&03, and then dissociated to form H+ and HCOT. This process would have been expected to continue until internal and external levels of CO2 were the same, at which time pHi would have leveled off. Instead, as illustrated by the first CO2 pulse of Fig. 4A, the pHi continued to fall during the CO* exposure ( bc). Furthermore, on removal of COZ, pHi returned to a level below its initial one (compare a and d), reflecting the net accumulation of acid during the CO2 exposure. The continuing acidification ( bc), as well as the shortfall of pHi, had been previously observed (2). After pHi recovered from the first CO* pulse, the fiber was treated with 0.5 mM SITS for about 25 min, and exposed to CO* once again. As before, the CO2 caused pHi to drop precipitously (ef). But this time there was no continued fall of pHi ( fg), and on removal of COZ, pHi returned to nearly the initial level (compare e and h). This acidification was not only SITS sensitive, but also depended on [HCO& Figure 4B illustrates an experiment in which a fiber was successively exposed to two HCOJCOZ solutions of pH, 6.3, the first containing only one-tenth the HC03 and CO2 of the second. Exposure of the fiber to the first solution (1% CO2/0.5 mM HCO;) caused a rapid initial fall in pHi ( ab), followed by a very
I
FIG. 3, SITS-sensitive alkalinization in pH,, 8.6 BSW (40 mM HCO;/0.4% CO?). A: rise in pHi beyond normal level. Fiber was exposed to BSW containing 40 mM HCO;, equilibrated with 0.4% COZ. The pH; initially fell due to influx of CO?, but then slowly recovered. Note the very long time scale of this experiment. Similar results were obtained in two additional experiments. 13: SITS sensitivity. The fiber was acid-loaded with an NH&l pulse as in Fig. 1. The experiment is picked up during the subsequent recovery of pHi. Note that pHi continued to rise at a slow rate even after pHi passed 7.4. The fiber was then poisoned with 0.5 mM SITS while in BSW containing 40 mM HC0.i (pH,, 8.6). The subsequent rate of alkalinization was greatly reduced (compare with broken line, which indicates control rate of alkalinization). Similar results were obtained in five additional experiments.
I
slow decline ( bc). When the CO:! was removed, FHi fell short of the initial value by only 0.03. On the other hand, when the fiber was exposed to the second solution of the same pH, but containing 10% CO& mM HCOT, pHi, after the initial COz-induced acidification ( ef), fell nearly twice as rapidly as before (the ratio of slopes fg to bc was -1.8). From the observed rates of acidification, we computed the rate of HCO; or OH- efflux, or of H’ influx, taking into account PT. Because of the higher [HCOg]i due to the higher Pco~, PT was nearly twice as great in the second pulse as in the first. We found that the flux rate must have been 3.5 times greater in fg than in bc. A similar conclusion is also reached by comparing the ratio of shortfalls following removal of CO2 in the two pulses. If the acidification during bc and fg had been due to either H+ influx or OH- efflux, one would have expected the rate of acidification to be greater in the first, in which pHi was higher and thus the inward gradient for H+ greater ( Vm and pH, were the same in both). The data thus support the hypothesis that the ionic species responsible for the acidification was HCOi. Intracellular acidification follouling bluckade of acid extrusion by S1TS. As noted in the first section of RESULTS, when an acid-loaded fiber was exposed to HCOs/ CO2 BSW at pH, 6.8, pHi would rise to only about 7.0 before leveling off. In a few such experiments, we continued to monitor pHi for some time after it had leveled off, and noted that pHi would sometimes decay, even though
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
Cl90
BORON, Sm
A
M I-KOj
SEE+
5mM I-K03 I i 10% co2
2
7.2 PHi
7.0 -
MCCORMICK,
AND
ROOS
nal solution. In Fig. 5, the fiber was acid-loaded and then exposed to HC03/COz BSW (pH, 8.0) for about 90 min. SITS was then added, after which pHi began to fall. Results of similar experiments showed that the rate of the SITS-unmasked acidification was positively related to the length of time allowed for recovery of pHi before SITS poisoning. The data from experiments at pH, 6.8, 7.4, and 8.0 are plotted in Fig. 6, where the rate of acid production (the product of PT and the rate of fall in pHi) is given on the ordinate, and the delay before SITS application on the abscissa. It can be seen that at pH, 6.8 no SITS-induced acidification was detectable unless the delay was greater than 60 min, although at pH, 8.0 such
6.8I
B
’
O.5mM
1%
HCO,
5mM
10mM
HC03
/o.f!%CO2
I SITS
HCOj
4
10%co2
co2
-
-
C
h
.64r II
20 min. FIG. 4. SITS sensitive acidification in pH,, 6.3 BSW. A: SITS sensitivity. The fiber was first exposed to BSW containing 5 mM HCO; and equilibrated with 10% CO? (pH,, 6.27). After the initial CO?-induced fall in pHi ( ab), pHi continued to fall ( bc). Return to COP-free BSW caused pHi to return to a value less than the original one. The fiber was then poisoned with 0.5 mM SITS for 25 min, and then once again exposed to 5 mM HCOJlO% CO:!-BSW. This time, after its initial fall (ef), pHi was nearly constant ( fg), and, on removal of COZ, nearly returned (gh) to its initial value, A second experiment yielded similar results. B: dependence on HCO;. The fiber was first exposed to BSW containing 0.5 mM HCO;, equilibrated with I% CO? (pH,, 6.3). This caused a rapid fall in pH, ( ab), followed by a much slower acidification ( bc). Return to CO?-f ree BSW caused pHi to fall short of the initial pHi by a small amount. The fiber was then exposed to BSW containing 5 mM HCO;, equilibrated with 10% CO,; once again pH,, was 6.3. After the initial fall in pHi (ef), pH, continued to fall ( fg) at a rate significantly greater than in the first pulse (bc). Upon return to a COz-free solution, the amount by which pHi fell short of its initial value was also significantly greater than in the first pulse (compare points a and d with points e and h). Upon exposure to these CO?-containing solutions, the fiber reversibly depolarized by 5 mV. The C&free solutions were buffered with HEPES to pH 7.4.
the fiber was still exposed to the HC0,7/COz BSW. In experiments at pH, 7.4, we routinely observed downward drifts in pHi when, after pHi had leveled off, fibers were returned to COe-free BSW of the same pH,. We considered the possibility that an intracellular acid-producing mechanism, evoked by the previous process of acid loading and/or acid extrusion, was responsible for the drifts. This mechanism might be unmasked under conditions where acid extrusion is reduced or abolished. To test this hypothesis, we acid-loaded fibers with an NH&l prepulse and then followed the course of pHi in COZ-BSW for various lengths of time before adding SITS to the exter-
I
30
min. i
t
L
FIG. 5. Effects of SITS on pH, after recovery from acid-loading. A fiber was acid-loaded by prepulsing with 50 mM NH&l, as in Fig. I, and then exposed for 90 min to BSW containing 10 mM HO;, equilibrated with 0.4% CO:! (pH,, 8.0). The fiber was then poisoned with 0.5 mM SITS, which produced an eventual decay of pHi.
30
I 90
60 DELAY
1
1
120
150
I 180
(Min)
6. Rates of acid introduction. Fibers were acid-loaded with an NH&l prepulse, as in Fig. 1, and the pHi was allowed to recover in pH,, 6.8, 7.4, or 8.0 HCOi-BSW, as in Fig. 2. After recovery had proceeded for given lengths of time (delay plotted on abscissa), fibers were poisoned with 0.5 mM SITS, causing pHi to level off and then decline (as in Fig. 5). Rates of acid introduction (ordinate) were taken as the product of the acidification rate and pi%, and are thus given in pmol 1-“I min? Number of experiments is given near each point; average SE was 8.5. The data were fitted to power functions by the least-squares technique. FIG.
l
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
l
PH REGULATION
IN
BARNACLE
MUSCLE
FIBERS
an acidification was already seen at 30 min. Attempts at studying the intracellular acidification at pH,, 8.6 were unsuccessful because of the failure of SITS and/or DIDS to completely block pHi recovery at low values of pHi. At higher values of pHi, SITS blocked intracellular alkalinization, but did not unmask any acidification. Not shown are the results of three experiments in which acid-loaded fibers were SITS poisoned early enough to completely prevent pHi recovery and then alternately exposed, over a 2-h period, to HCO;1/C02-BSW at pH, 8.0 and 6.2. Even 2 h after acid-loading and SITS poisoning (during which time pHi remained at a very low level), there was no significant acidification in either pH 8.0 or 6.2 HCOT/COz-BSW. Also not shown are the results of other experiments in which fresh fibers were maintained in normal BSW (pH, 7,8) for several hours, and then exposed to SITS. Although previously stable, pHi decayed after application of SITS, and the rate of decay was comparable to that observed after acid-loading. DISCUSSION
Before discussing the pH dependence of acid extrusion, we will briefly examine two phenomena that must be considered in the interpretation of the acid extrusion data, SITS-sensitive pHi transients other than acid extrusion, and intracellular acid production. Other SITS-sensitive pHi transients. A previous study of barnacle muscle (2) provided strong evidence for the net efflux of HCO? (or the equivalent movement of H’ or OH-) under conditions of very low pH,,. The present experiments extend the earlier observations on two counts. First, we found that the continuing acidification during CO2 exposure at pH, 6.3 was much faster in 10% CO2 than in 1% CO2 (compare bc and fg in Fig. 4B), suggesting that it is the movement of HCO?, rather than that of H’ or OH-, which is responsible for the continuing fall in pHi* Second, this acidification was blocked by SITS. In addition, we found that when fibers were exposed to 0.4% COJ40 mM HCO; (pH,, 8.6), pHi continued to rise beyond the normal value of about 7.35 (Fig. 3A). This alkalinization was also blocked by SITS (Fig. 3B). On the other hand, when a fiber was exposed to HCOSfree BSW at pH, 8.6, pHi remained unchanged for hours. At the present time, it is impossible to identify the mechanism(s) of these SITS-sensitive pHi transients. We can think of three plausible explanations. First, the phenomena could be due to the passive movement of an ion through a channel. Our calculations using the constant field treatment (9, 11) show that the permeability to HCOT would have to be about 7 x lo-” cm/s (treating the fiber as a simple cylinder) in order to account for either the alkalinization of Fig. 3 or the acidification of Fig. 4. This hypothetical value is not too far from the value for Pcl calculated by DiPolo (8), l-9 x 10B6 cm/s. One way to test the possibility of passive HCO; flux would be to examine the sensitivity of the pHi course to Vm. We attempted to do this but met with failure because of technical difficulties. The second possibility is that the same mechanism responsible for acid extrusion is also instrumental in producing the transients in Figs. 3 and 4. This possibility is reinforced by the demonstrated dependence of these
Cl91 transients on [HCO& as well as by their SITS sensitivity. If this possibility were correct, it might be anticipated that these transients would have a requirement for Na’ or Cl-, as has been substantiated in the case of acid extrusion (5, 12). The transients might also be affected by furosemide and cyclic AMP (6). A third explanation for the pHi transients of Figs. 3 and 4 is that one or both were mediated by a SITSsensitive carrier that is independent of the PHi regulating system, and thus unaffected by furosemide, cyclic AMP, or Na’ removal. Our present data do not allow us to distinguish among the three choices, although the second requires the fewest ad hoc assumptions. Intracellular acidproduction. Inhibition of acid extrusion by SITS in previously acid-loaded fibers unmasked a time-dependent intracellular acidification. SITS blocks the three types of pHi transients that have been shown to be HCOZ dependent (namely, acid extrusion and the transients illustrated in Fig. 3 and 4). Therefore, movement of HCOT is probably not involved in this phenomenon. Its rate increased with the length of time during which acid extrusion was allowed to proceed, and also with pH,, (Fig. 6). When acid extrusion was suppressed from the start by early application of SITS, no acidification was observed at any pH,. We also found SITSunmasked acidification in fibers maintained in normal BSW (pH, 7.8) for several hours. Without SITS, pH, remained constant (pHi 7.32), but the application of SITS resulted in a fall of pHi at a rate comparable to that observed after acid-loading (Fig. 6). We have made no attempt to analyze the nature of the SITS-unmasked acidification; our main concern was to use these results to correct our data on the rate of acid extrusion as discussed below. The pH-dependence of acid extrusion. The data of Fig. 2 and Table 2 demonstrate that the rate of pHi recovery from an acid load is greatest at low values of pHi and high values of pH,. As illustrated in Fig. 7, the calculated rate of acid extrusion follows a similar trend. The curves represent averages of our data from 63 experiments in which pH, was altered by varying [HCO;],. Details of their construction can be found in the legend of Fig. 7. Acid extrusion rates were obtained by multiplying the rates of pHi recovery by intracellular buffering power (see METHODS) and then correcting for the time-dependent SITS-unmasked acidification. This correction was a small fraction of the acid extrusion rate except when this rate was very low, As can be seen in Fig. 7, at pH, 7.4, 8.0, and 8.6 the acid extrusion rate falls linearly as pHi increases. At pH, 7.4 and 8.0 this linearity is maintained as normal pHi is approached, whereas at. pHo 8.6 the acid extrusion rate is sustained beyond normal pHi+ It should be pointed out that at pH, 8,6 the electrochemical gradient for HCO? was inward over the entire range of pHi values (avg Vm, - 50 mV), while at pH, 7.4 and 6.8 this gradient was outward; at pH, 8.0 the gradient reversed direction at pHi -7.2. Thus, acid extrusion takes place at pH,, 7.4 and 6.8 in the face of a large outward-directed electrochemical HCO; gradient. The low rate of acid extrusion at pH, 6.8 might be ascribed to the large acidifying influence of HCOT efflux. In contrast, at pH,, 8.6 acid extrusion continued at pHi values beyond
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
BORON,
PHi
7. Acid extrusion rate as a function of pHi at 4 values of pH,, (Pco? constant). Summary of data from 63 experiments, in which pHi was lowered by pretreating with NH&l and then allowed to recover while the fiber was bathed in HCOJ0.4% CGBSW at pH, 8.6, 8.0, 7.4 or 6.8. The curves at pH,, 7.4, 8.0, and 8.6 were obtained as follows: I) at a given pH,,, the acid extrusion rate for each experiment was calculated at pHi intervals of 0.025 by multiplying the rate of pHi recovery by fir, as described in METHODS; acid extrusion rate was then plotted against pHi; 2) all curves at a given pH, were normalized to the mean pHi at which acid extrusion rate was zero; these pHi values were 7.32 k 0.025 ( n. = 7) at pH,, 7.4; 7.33 k 0.023 (n = 11) at pH,, 8.0; and rate 7.38 t 0.027 ( n = 19) at pH,, 8.6. At pH,, 8.6 the acid extrusion never became zero, therefore, the pHi to which these curves were normalized was obtained by extrapolation to zero acid extrusion rate of the linear portion of the curves; and 3) all normalized data at a given pH,, were averaged at pHi intervals of 0.025, and these averaged values plotted in this Fig. The pH,, 6.8 curve is a simple average of the data without normalization. Total number of experiments: 12 at pH,, 6.8, 12 at pH,, 7.4, 20 at pH,, 8.0, and 19 at pH,, 8.6. Each point represents the average of 2-7 experiments at pH,, 6.8,3-H at 7.4, 7-20 at 8.0, and 7-19 at 8.6. Vertical bars indicate standard error; lines represent linear regressions. FIG.
normal (see also Fig. 3). This represents HC03 movement in the direction of its electrochemical gradient. Thomas (13) observed that in snail neurons, the recovery of pH, from acid loads follows an exponential time course. This implies that, if PT is constant, the acid extrusion rate is proportional to the difference between normal pHi (the threshold pHi below which acid extrusion is initiated) and the prevailing pHi. We found a similar relationship in our experiments. In the experiments of Fig. 7, changes in pH, were produced by varying [HCO& at constant Pcoz (0.4% CO& Inasmuch as these studies, by themselves, did not distinguish between the effects of pH, per se and those of [HCOs lo, a series of experiments was undertaken in which pHO was changed by varying PCO~ at constant [HCO& (2.5 mM). The results showed that, even at constant [HCO&, raising pH, caused an increase in the acid extrusion rate. Therefore, barring a specific effect of Pcoz on acid extrusion, pH, is an important determinant of the rate of acid extrusion. On the other hand, it is well known that [HCO-] 3 0 influences acid extrusion independently of pH,. Experiments on the squid axon (4), the snail neuron (13), and barnacle muscle (2)) showed that acid extrusion is substantially augmented by switching from a HCOT-free to a HCOT-containing bathing solution
MCCORMICK,
AND
ROOS
at the same PH. Recently, Thomas (15) showed that the rate of acid extrusion in snail neurons progressively increases as [HCO& is raised from 0 to 4.5 to 21 mM, all at constant pH,. Because pH, and [HCO& can individually affect acid extrusion, one would anticipate that increasing both together would have a greater effect than increasing pH, alone. This is borne out by the data of Tables 2 and 3. Increasing pH, from 6.8 to 8.0 caused the rate of acid extrusion at pHi 6.8 to rise l7-fold when [HCO& was raised at constant PCO~ (Table Z), but only 7-fold when PCO~ was lowered at constant [HCO& (Table 3). The mechanism of the pH, effect is unknown. The data of the present study and recent work on dialyzed barnacle muscle fibers (6) provide some insight into possible mechanisms of pHi regulation in barnacle muscle. The results of the latter study indicate that acid extrusion involves an exchange of external WC03 for internal Cl-, and also suggest that the same carrier can engage in both Cl-Cl and HCO&l exchange. It was hypothesized that, at a normal pHi, the carrier operates at a low rate in the Cl-Cl exchange mode, while at lower values of pHi the rate of anion-anion exchange greatly increases and the mode partially shifts to HCO&l exchange. It would be reasonable to assume that under conditions of acid loading external HCOT and Cl- compete with one another for entry. Indeed, the results of the previous study (6) showed that when [HCO& was low, the rate of acid extrusion was low and the Cl- influx was relatively high. Increasing [HCO& at constant pH, caused an increase in the rate of acid extrusion (i.e., HCO; influx), but a decrease in Cl- influx. However, at a normal pHi, when the carrier should have been engaged only in Cl-Cl exchange, a similar increase in [HCO& had no effect on either the acid extrusion rate (which remained zero) or Cl- influx. Further evidence that HCO:jCl and Cl-Cl exchange are mediated by the same carrier is that both exchanges were inhibited by SITS and by furosemide and were stimulated by cyclic AMP. One might speculate on how the SITS-sensitive pHi transients of Figs. 3 and 4 in the present paper fit into the above scheme of a common carrier for HCOS-Cl and Cl-Cl exchange. Our analysis requires two assumptions. First, the carrier normally mediates several different exchanges (Cl-Cl, external HCOS-internal Cl, external Cl-internal HCOS, and HC03-HCOa) simultaneously, and that the relative amount of each exchange is governed by pHi, pH,,, and the prevailing substrate concentrations. Given the fact that the first two of these exchanges have already been documented, this assumption seems rather reasonable. Second, we hypothesize that the stimulatory effect of raising pH, on the acid extrusion rate is brought about by causing the carrier to pick up relatively more HCOs and less Cl- from the external solution (without affecting the total rate of anion exchange, or the ions picked up on the inside). Thus, when pH, is reduced to low levels (as was the case during the SITS-sensitive acidification of Fig. 4), external HCO:j-internal Cl and HC03-HC0.7 exchange would be reduced while external Cl-internal HCOS and Cl-Cl exchange would be increased. This would account for the observed acidification in Fig. 4. Conversely, when pH, is raised to high levels (as was the case during the SITS-sensitive alkalinization of Fig. 3), Cl-Cl and external Cl-internal HCOS exchange would be
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (094.231.218.054) on January 1, 2019.
PH REGULATION
IN
BARNACLE
reduced in favor of external HCOS exchange.
MUSCLE
HCOs-internal
We thank J. Jones for preparing the manuscript. This work was supported by National Institutes HL-00082 to A. Roos, and NIGMS-02016 (Medical Program Fellowship) and GM-06499 (National
Cl93
FIBERS
Cl and HC@-
of Health Grants Scientist Training Research Service
Award) to W. F. Boron. A. Roos is recipient of National Institutes of Health Research Award HR-19608. W. F. Boron’s present address: Department of Physiology, University School of Medicine, New Haven, CT 06510 Received
16 November
1978; accepted
in final
form
1 May
Career Yale
1979.
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