Jan 19, 1990 - Pancreatic acini loaded with the pa-sensitive dye. 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca'+- ...
THE JOURNAL OF BIOLOGICAL CHEMIWRV 0 1990 by The American Society for Biochemistry
Intracellular
Vol. 265, No. 22, Issue of August 5, pp. 12813-12819,199O
pH-regulatory
II. REGULATION
OF H’ AND
Printed in U.S. A.
and Molecular Biology, Inc.
HCO;
Mechanisms in Pancreatic
TRANSPORTERS
BY Ca*‘-MOBILIZING
AGONISTS* (Received
Shmuel From
Muallemz
the Department
and Peggy of Physiology,
Acinar Cells for publication,
January
19, 1990)
A. Loessberg University
of Teras,
Pancreatic acini loaded with the pa-sensitive dye 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca’+-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPEWbuffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, while in HCOS-buffered medium pHj is determined by the combined activities of a Na+/H+ exchanger, a Na*-HCO; cotransporter and a Cl-/HCO$ exchanger. In this study we found that stimulation of acini with Ca2+-mobilizing agonists in HEPES or HCOS-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPESbuffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H’ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCOb-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO;-buffered medium: (a) recovery from acid load in the presence of H2-4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (H2DIDS) (Na’/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO; influx and efflux, respectively, by Na+-HCO; cotransport) were inhibited; and (c) HCO; influx and efflux due to Cl-/HCO; exchange, which was measured by changing the Cl- or HCO; gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO; exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO; influx due to Na+HCOi cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO;-buffered medium, as much as treating the acini with HZDIDS resulted in similar agonist-mediated pHi changes in HEPESand HCOZ-buffered media. The *This work was supported by the National Institute of Health Grants DK 38938 and AR 39245. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Ctr., 5323 Harry Hines Blvd., Dallas, TX 75235.
Southwestern
Medical
Center,
Dallas,
Texas
75235
effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.
In the previous paper (1) we studied regulation of intracellular pH (pHi)’ in pancreatic acini bathed in HEPESor HCOB-buffered medium. We found that pH, regulation in HEPES-buffered medium is different than in HCOZ-buffered medium. The Na’/H’ exchanger, whose presence in pancreatic acini was demonstrated in several studies (2-4), dominates pH, regulation in HEPES-buffered medium (1). In the presence of HCO,, two additional HCO;-secreting mechanisms are activated: Na’-HCO; cotransport and Cl-/HCOy exchange. These mechanisms reduce and maintain steadystate pH, at 0.15 pH unit below that measured in HEPESbuffered medium. Acid-base regulatory mechanisms are known to be involved directly in several cellular functions in addition to regulating steady-state pHi. These include transcellular salt transport in epithelia (5), cell volume regulation (6, 7), and cell proliferation (8, 9). In pancreatic acinar cells, there is evidence that some NaCl transport, which is accompanied by fluid secretion, occurs transcellularly and is stimulated by Ca’+-mobilizing agonists (10, 11). Due to activation of K+ (12) and Cl- (13) channels by Ca2’, acinar cells shrink upon stimulation and then regain their volume (14). It is likely that pH,-regulatory mechanisms participate in the recovery of cell volume. Gastrointestinal hormones, which mobilize Ca*+ from intracellular stores and activate protein kinase C (15) are also known to promote pancreatic cell growth and proliferation (16). Of the different pHi-regulatory mechanisms in pancreatic acinar cells (l), only the regulation of the Na’/H’ exchanger by agonists was studied (3, 4). It was shown that Ca’+mobilizing agonists, like CCK-OP, caerulein, and gastrin, activate the exchanger (3, 4) by a mechanism which may involve the activation of protein kinase C (4). Therefore, it was of interest to study the effects of these agonists on pHi regulation in HEPES and HCO; media and their effect on the individual acid-base transporting mechanisms in pancreatic acini. We demonstrate here that in HEPES-buffered medium stimulation by CCK or carbachol is followed by an initial acidification and subsequent alkalinization to pH, beyond the level in resting cells. This is mainly the result of an activation of the Na+/H’ exchanger. In HCOB-buffered medium, the 1 The abbreviations used are: pH,, intracellular pH; pH,, extracellular pH; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CCK-OP, cholecystokinin octapeptide; NMG, N-methyl-D-glucamine; H*DIDS, Hz-4,4’-diisothiocyanostilbene-2,2’-disulfonic acid, BCECF, 2,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein.
12813
12814
Agonists and PHi a
initial acidification is unaffected while the subsequent alkalinization is markedly attenuated. This was not because of reduced activation of the Na’/H’ exchanger. Rather, the agonists activated the Na+/H’ exchanger in HEPESand HCOg-buffered medium to the same extent. However, in HCOT-buffered medium the agonists inhibited the Na+HCO, cotransporter and greatly activated the Cl-/HCO, exchanger, such that the rate of HCO; efflux by the Cl-/ HCO; exchanger exceeded HCO, influx due to Na+-HCO: cotransport and H+ efflux due to Na+/H+ exchange. The combined effect of agonists on the three acid-base regulatory mechanisms resulted in an attenuated increase in pHi in HCOB-buffered medium. MATERIALS
AND
NH4CI 0 PH, 745-
RESULTS
Effect of Agonists in HEPES-buffered Medium--Fig. 2 shows the effect of stimulation with CCK-OP on pHi of pancreatic acini maintained in HEPES-buffered medium. During the first 2 min of stimulation, CCK-OP induced an initial acidification from pHi of 7.28 to 7.17. Subsequently, pHi increased and reached a stable level of 7.36. Addition of amiloride to stimulated acini resulted in rapid and sustained decrease in pHi (Fig. 2~). This suggested that the Na+/H+ exchanger is involved in the increased phase of pHi. Accordingly, treatment of the acini with amiloride prior to stimulation was followed by a sustained acidification with no recovery of pHi until the removal of amiloride from the perfusion medium (Fig. 2b). In addition, removal of extracellular Na’ by perfusing the acini with solution B (NMG-Cl) prevented the alkalinization seen after the initial acidification following stimulation with CCK-OP (Fig. 2~). Finally, treatment of acini in HEPES-buffered medium with HzDIDS had no measurable effect on the pattern of CCK-OP-evoked changes in pHi (Fig. 2d). The profile of pHi changes observed after
0
719 -
695675660-
METHODS
The methods were identical to those described in the preceding article (I). Composition of solutions was the same as in the previous article, and they are similarly abbreviated. Thus, solution A is the standard, HEPES-buffered NaCl solution; solution B is HEPESbuffered Na+-free; solution C is HEPES-buffered Cl--free; solution D is the standard HCOS-buffered, NaCl solution; solution E is HCOY-buffered Na+-free; solution F is HCOa-buffered Cl--free. Cholecystokinin octapeptide (CCK-OP) was a generous gift from the Squibb Institute for Medical Research, Princeton, NJ. Comparison of H+ and HCOr flux rates required determination of intrinsic and total intracellular buffering capacity of pancreatic acinar cells. For this we adopted the method described recently for mesangial cells (17, 18). Acini attached to cover slips were first acidified by exposure to 20 mM NH&l and perfusion with solution B (NMG-Cl) until pHi stabilized. Under these conditions, no pH; regulatory mechanism is operating (1). The acini were then perfused with solution B in which 40 mM NH&l replaced 40 mM NMG-Cl. This resulted in pH, increase. Subsequently, the acini were perfused with a sequence of solutions containing decreasing concentrations of NH&l (20, 10, 5, 2.5,O) replacing equivalent amounts of NMG-Cl. A typical experiment is shown in Fig. la. The resulting changes in pH, were used to calculate the changes in NH&,, concentration. For that, it was assumed that NHsti,, = NH,(,,, and NH3c.j was calculated from the known extracellular pH (pH,) and NH&l concentration using a PK. for NH: of 8.9 at 37 “C (17). The changes in DH, due to the changes in NH&., concentration’ were used to calculate the intrinsic buffer capacity (pi) according to & = A[NH:]i,/ApHi. The resulting /3,at the different DH~ are plotted in Fig. lb (solid line). It can be seen that, as for other-cell types (17, 19, 26), 8, varies linearly with pH; between 6.3 and 7.43. The total buffering capacity, &, was calculated from & = @;+ 2.3[HCO& (5). For this purpose the clusters of pH, and pi (Fig. lb) were averaged, [HCO& was calculated for each averaged pHi and used to determine & (open squares, broken line, Fig. lb). It can be seen that in the presence of HCO;, PI was constant between pHi of 6.3 and 7.1.
(mM)
[40~20~101512.51 NMG Cl- HEPES
6.41 6.30 i
b 80 g 3 60 I CL s E 2 40 2 d G % 20 m
P Total q
-_____---.
0 ._----_______
\
,/”
B ___-__.
=-.
*f”\J\Am
A-*-o+q\A
o .-0,
Intrinsic t 6.3
I 6.5
I 6.7
, 6.9 Intracellular
I 7.1
I 7.3
I 7.5
pti
Measurement of intracellular buffer capacity. a, pancreatic acini attached to cover slips and loaded with BCECF were acidified by exposure to NH,Cl and then perfused with a HEPESbuffered, Na+-free solution B (NMG-Cl) until pHi stabilized at 6.41. Then the acini were perfused with solution B in which 40, 20, 10, 5, and 2.5 mM NH&l replaced equivalent concentration of NMG-Cl, and finally again with solution B. b, intracellular intrinsic buffer capacities calculated from the changes in pH, in a were plotted against pH,. Different symbols refer to different experiments (0, 0, A, A). Total buffer capacity (0) was calculated by averaging the clusters of intrinsic buffer capacities and pHi. The values used were: pi 37 at pHi 6.31; 32 at 6.59; 26 at 6.75; 22 at 6.94; 16 at 7.05; and j3i 12 at pHi 7.42. FIG.
1.
stimulation with CCK-OP (Fig. 2, a, c, and d) could be induced with another Ca*+-mobilizing agonist, carbachol (not shown). However, amiloride interferes with carbachol stimulation and thus the results obtained with CCK-OP are presented. The results presented in Fig. 2 suggest that an increased Na+/H’ exchange activity is responsible for the alkalinization and the stable elevation in pHi induced by CCK-OP. To provide direct evidence and evaluate the degree of stimulation of the Na+/H+ exchanger, the effect of CCK-OP stimulation on recovery from acid load was measured. Fig. 3 shows the result of such an experiment. After the NH: pulse and acidification of the cytosol to pHi of 6.8, control acini recovered pHi at a rate of 0.23 ApH/min or 6.12 mM H+/min. After stimulation with CCK-OP the acini recovered pHi from the same acid load at a rate of 11.26 mM H+/min. In three experiments with CCK-OP and two experiments with carbachol, in acini acidified to an average pHi of 6.77 f 0.08, the rate of Na’/H’ exchange activity increased by 83 f 12%. Effect of Agonists in HC&-buffered Medium-Since we showed in the previous article (1) that bathing the acini in HCO:-buffered medium uncover the activity of two additional acid-base regulatory mechanisms, the effect of CCK-OP on pHi of acini maintained in HCO;-buffered medium was tested. Fig. 4a shows that also in HCO;-buffered medium, stimula-
12815
Agonists and PHi Na Cl - HEPES
Na Cl - HCO;
5mn
H*DIDS I
I
FIG. 2. Effect of CCK-OP on pHi in HEPES-buffered medium. a. acini in solution A (NaCl-HEPES) were nerfused with solution A containing CCK-OP and then with solution A containing CCK-OP and amiloride. b, acini were perfused with solution A containing amiloride, solution A containing amiloride and CCK-OP and finally with solution A containing CCK-OP. c, acini in solution A were perfused with solution B (NMG-Cl-HEPES), solution B containing CCK-OP and solution A containing CCK-OP. d, acini in solution A were perfused with solution A containing H,DIDS and then with solution A containing H,DIDS and CCK-OP. Where present, final concentrations were 10’ M CCK-OP, 0.5 mM amiloride, and 0.2 mM HZDIDS.
CCK.OP
4. Effect of CCK-OP on pHi in HCO;-buffered medium. a, acini in solution D (NaCl-HCO;) were perfused with solution D containing CCK-OP and then solution D containing CCK-OP and amiloride. b, acini were perfused with solution D containing amiloride and then with solution D containing amiloride and CCK-OP. c, acini were perfused with solution D containing CCK-OP and then with solution D containing HZDIDS and CCK-OP. d, acini in solution D were perfused with solution D containing H,DIDS and then with solution D containing H2DIDS and CCK-OP. When present, final concentrations were lo-’ M CCK-OP, 0.5 mM amiloride, and 0.2 mM H,DIDS. FIG.
TABLE
CCK-OP-induced
I
pH,
changes in HEPESand HCOC-buffered medium Intracellular pH of acini stimulated with 10 nM CCK-OP was determined as described in Fig. 2 for HEPES-buffered medium and Fig. 4 for HCO;-buffered medium. The numbers in parentheses indicate the number of exneriments uerformed. Intracellular Conditions
2 min
0 Srni”
_
FIG. 3. Stimulation of Na+/H+ exchange by CCK-OP in HEPES-buffered medium. Acini in solution A (NaCl-HEPES) were perfused with solution A in which 20 mM NH,Cl replaced 20 mM NaCl. Then the acini were perfused with solution A. After recovery of pHi, the acini were stimulated with 10-s M CCK-OP. Where indicated, the acini were exposed again to 20 mM NH&l in the presence of CCK-OP and then NH&l was removed by perfusion with solution A containing CCK-OP. After the stabilization of pH; the acini were exposed again to NH&l and then perfused with solution A containing CCK-OP and 0.5 mM amiloride.
CCK-OP was followed by an initial acidification. However, the subsequent alkalinization was markedly reduced, as compared to that seen in HEPES-buffered medium (Fig. 2). Addition of amiloride produced a rapid and sustained acidification. Consideration of the buffer capacity in HEPESand HCO;-buffered medium (Fig. 1) indicates that addition of amiloride induced the accumulation of 2.18 and 4.36 mM of acid equivalents in the two media, respectively. This suggested to us that the attenuated increase in pH; was not due to reduced activation of the Na’/H’ exchanger, but rather due to the participation of other mechanism(s). To evaluate the contribution of the Na’/H’ exchanger to pH, change after CCK-OP we tested the effect of amiloride. Fig. 4b shows that addition of amiloride prior to CCK-OP stimulation blunted, but did not completely inhibit, pH, increase after the initial acidification. The role of the HCO; transporters was tested by addition of H,DIDS after (Fig. 4c) and before (Fig. 4d) tion
with
pH
Time after CCK-OP 10 min
HEPES +Amiloride +H,DIDS
7.28 f 0.03 (28) 7.20 + 0.05 (3) 7.29 + 0.06 (3)
7.16 f 0.06 (6)” 7.05 f 0.06 (3)” 7.14 + 0.04 (3)”
7.35 2 0.04 (6)*.’ 7.03 k 0.04 (3)” 7.33 + 0.03 (3)“,’
HCO: +Amiloride +H,DIDS
7.13 f 0.04 (62) 7.02 + 0.05 (3) 7.24 + 0.04 (3)
6.96 rt 0.06 (12)” 6.89 + 0.05 (3)” 7.13 f 0.05 (3)”
7.06 + 0.02 (12)d 6.93 f 0.03 (3)” 7.30 + 0.04 (3)’
’ * ’ ’
Significantly Significantly Significantly Significantly
different different different different
from from from from
the respective control the respective control 2 min of stimulation 2 min of stimulation
(p < 0.01). (p < 0.05). (p c 0.01). (p < 0.05).
CCK-OP stimulation. In both cases pHi increased following exposure to H*DIDS. Similar results to those shown in Fig. 4, a, c, and d, were observed when carbachol was used as the stimulant (not shown). Table I summarizes pH, changes under the various conditions illustrated in Figs. 2 and 4. It can be seen that the initial acidification due to CCK-OP was similar in HEPESand HCO,-buffered medium and could not be blocked by amiloride or H,DIDS. As expected, only in HCO;-buffered medium did HzDIDS affect the CCK-OP-induced pH, changes. Interestingly, the profile of pHi changes induced by CCK-OP after H,DIDS treatment was similar to that recorded in HEPESbuffered medium (compare Figs. 2a, 4d, and the first and last rows in Table I). This suggests that HCO;-transporting mechanisms were responsible for the reduced pH, after stimulation of acini bathed in HCOT-buffered medium with CCK-OP. Therefore, in subsequent experiments we systematically
Agonists and pHi
12816
tested the effect of agonists stimulation on each transporter. Stimulation of Na+/H+ Exchange in HC&-buffered Medium-Fig. 5 shows the effect of CCK-OP stimulation on the rate of recovery from acid load in control and HzDIDS-treated acini. Since control experiments showed that the rates of recovery from acid load after two consecutive pulses were similar, the protocol we used was to record the rate of recovery from acid load before agonist stimulation (control trail), stimulate the acini after stabilization of pHi, and measure again the rate of recovery from a second acid load in stimulated acini (test trail). Fig. 5a shows that CCK-OP stimulation increased the rate of recovery from acid load by 48%. To determine the effect of stimulation on Na+/H+ exchange, the acini were treated with H*DIDS and recovery from acid load before and after stimulation was compared. Fig. 5b shows that Na’/H’ exchange activity was increased by 73%. This increase was higher than the increase observed in the absence of H,DIDS. This provided us with the first evidence that the other mechanism participating in recovery from acid load (the Na+-HCO: cotransporter) must have been inhibited rather than stimulated by CCK-OP. Inhibition of Na+-HCO: Cotransport-To demonstrate directly the effect of CCK-OP stimulate on Na+-HCO, cotransport we first compared the rates of recovery from acid load in the presence of amiloride. Fig. 6 shows that stimulation with CCK-OP indeed inhibited this activity by 41%, which we have shown previously to be due to a Na’-HCO: cotransport (1). Another protocol used to demonstrate the effect of CCKOP on Na’-HCO; cotransport is shown in Fig. 7. In these experiments we compared HCO; efflux in the presence of PH,
FIG. 6. Inhibition by_ CCK-OP.
of HCO;
influx
due
to Na+-HCOi
cotrans-
Acini in solution D (NaCl-HCO;) _. were nerfused with solution D containing 0.5 mM amiloride. The acini were then perfused with solution D in which 20 mM NH&l replaced 20 mM NaCl and also containing amiloride. NH&l was removed by perfusion with solution D containing amiloride. After stabilization of pH; the acini were stimulated with lo-’ M CCK-OP and this concentration of CCK-OP was included in all subsequent solutions. After 2 min with CCK-OP the acini were exposed to NH&l and then perfused with solution D containing amiloride. When pH; stabilized, amiloride was removed by perfusion with solution D containing CCK-OP. _uort
a
7.27
PH.
b
7.11
CCK
I
6.59 r
I
m CCK - OP
b
HZ DIDS CCK OP
5mln of CCK-OP medium. a,
on
recovery
from
acid
load
in
acini in solution D (NaCl-HCOS) were perfused with solution D in which 20 mM NH&l replaced 20 mM NaCl. NH&l was removed by perfusion with solution D. After recovery of pHi the acini were perfused with solution D containing 10-s M CCK-OP, solution D containing NH&l, and finally solution D containing CCK-OP. b, acini in solution D were exposed to NH&I as above and then NH&l was removed by perfusion with solution D containing 0.2 mM H,DIDS. After stabilization of pHi the acini were perfused with solution D containing H,DIDS and 10-s M CCK-OP, solution D with HZDIDS, CCK-OP, and 20 mM NH&l and finally solution D containing HZDIDS and CCK-OP. HCO;-buffered
of HCO;
efflux
NMG-Cl
1
NaCI
Amilmde
due to Na+-HCOi
cotrans-
a, acini in solution D (NaCl-HCO:) were perfused with solution D containing 0.5 mM amiloride and then with Na+-free solution E (NMG-Cl-HCO,) containing amiloride and finally with solution D containing amiloride. b, acini in solution D were stimulated by perfusion with solution D containing lOma M CCK-OP. This concentration of CCK-OP was included in all subsequent solutions. After stabilization of pH; the acini were perfused with solution D containing 0.5 mM amiloride, solution E containing amiloride and finally with solution D containing amiloride. port
7.36
FIG. 5. Effect
FIG. 7. Inhibition by CCK-OP.
OP 1
Na Cl
amiloride, which was initiated by removal of extracellular Na+, in resting and stimulated cells. Fig. 7 shows that HCO; efflux due to Na’-HCO; cotransport was reduced by 74%. However, because of the stimulation with CCK-OP and treatment with amiloride, Na+-HCO; cotransport is initiated at different pHi, and thus different HCO;, in control and stimulated cells. To correct for this, the rate of HCO; efflux from control acini was estimated from the portion of the curve corresponding to pHi at which HCO, efflux was initiated in stimulated acini (pH 6.93 in Fig. 7). Such a correction indicates that Na’-HCO: efflux was inhibited by 61%. Stimulation of Cl-/HC& Exchange-Since stimulation with CCK-OP inhibited Na+-HCO; cotransport, modification of the activity of this transporter could not account for the
Agonists and PHi attenuated increase in pHi after CCK-OP stimulation in HCOh-buffered medium. Thus, it is likely that CCK-OP stimulated the Cl-/HCO: exchanger. That this prediction is correct is shown in Fig. 8 in which the effect of CCK-OP on HCO: fluxes by the Cl-/HCO: exchange was measured. HCO; influx was initiated by removal of extracellular Cland HCO; influx was measured from the decrease in pHi following the readdition of Cl- (Fig. 8). It can be seen that stimulation with CCK-OP increased the rates of HCO, fluxes and the extent of alkalinization induced by perfusing the acini with Cl-free medium (Fig. 8~). As expected, the changes in pHi observed after removal and addition of extracellular Clwere blocked by H,DIDS in the presence and absence of CCK-OP (Fig. 8b). To complement the experiments in Fig. 8 we also measured the effect of CCK-OP on HCO, efflux in the absence of extracellular HCO; (Fig. 9). For this experiment we loaded the acini with HCO; by incubation with and removal of HCO;/CO,. Stimulation of acini with CCK-OP had two effects. CCK-OP accelerated the recovery from acidification initiated by perfusion with HCO;/CO,, which was most likely due to stimulation of the Na+/H’ exchanger. CCK-OP also accelerated the rate of HCO, efflux measured after the removal of HCO,/CO,. PH, a 751
r
FIG. 8. Effect
of CCK-OP on of extracellular HCOi.
Cl-/HCO;
exchange
in
the
a, acini in solution D (NaClHCO,) were perfused with Cl--free solution F (Na-Glu-HCO:) and then with solution D. After pH, returned to the initial level, the acini were stimulated by perfusing with solution D containing lo-’ M CCKOP and then perfused with solution F containing CCK-OP and finally with solution D containing CCK-OP. b, the protocol of solution changes and cell stimulation was exactly the same as in Experiment a except that 0.2 mM H,DIDS was included in all perfusion solutions.
FIG. 9. Effect
of CCK-OP on extracellular HCO;.
Cl-/HCO;
exchange
in
the
Acini in solution A (NaClHEPES) were perfused with solution D (NaCl-HCO;), and then with solution A. After stabilization of pH, the acini were stimulated with lo-' M CCK-OP for 4 min before perfusion with solutions D and A also containing lOmaM CCK-OP. absence
of
TABLE II R&es
of HCOi
and H’ transport in resting and stimulated bathed in HCO;-buffered medium
acini
Influx rate of Cl-/HCO; exchange refers to HCO; influx measured from the increase in pH, upon removal of external Cl- (n = 5) while efflux rate was estimated from the rate of pH, decrease upon readdition of Cl- (n = 5) (Fig. 8). HCO; influx rate due to Na+-HCOY cotransport was measured in the presence of amiloride in acini acidified to pH, of 6.6-6.7 as described in Fig. 6 (n = 4). HCO, efflux rate of the Na+-HCO: cotransporter was estimated by following pH, changes after removal of external Na+ from amiloride-treated acini as described in Fig. 7 (n = 4). Na+/H+ exchange rate was determined in acini acidified to pH, of 6.6-6.7 and treated with HPDIDS as described in Fig. 5 (n = 6). All the above rates refer to experiments performed in HCO;-buffered media. The rates of change of pH, are equivalent to the rates of change of H+ or HCO; fluxes since pt for pHi values between 6.3 and 7.1 in the presence of HCO, are the same (Fig. 1). In the absence of HCO,, stimulation increased Na+/H’ exchange rate by 83 f 12%. Conditions Na+-HCOi Na+/H+ Cl-/HCO; ApH/min Influx Control 0.35 + 0.06 0.131 + 0.03 0.137 * 0.04 CCK-OP 0.87 + 0.09 0.08 f. 0.03 0.244 zk 0.06 % change +149 -t 18 -39 + 3.7 +79 318 Efflux 0.26 + 0.04 Control 0.41 + 0.065 CCK-OP 1.03 + 0.09 0.11 + 0.06 % change +151+ 17 -58 + 11 Table II summarizes the effects of CCK-OP on H’ and HCO, fluxes under all conditions tested. HCO, transport by the Cl-/HCOi exchanger is faster than the sum of H+ and HCO; transport by the Na+/H+ exchanger and Na’-HCO: cotransporter in stimulated cells. This was due to stimulation of Cll/HCO, exchange by 149-151%, inhibition of Na’HCO; cotransport and stimulation of Na’/H’ exchange by 78%. It is also apparent that stimulation with CCK-OP reduced HCO, efflux more than HCO: influx by the Na+HCO: cotransporter. The combined effects of CCK-OP on all transporters can account for the attenuated increase in pHi after CCK-OP stimulation and will result in Na+ and Clinflux into the acini.
::cb-. presence
12817
DISCUSSION In the preceding article (1) we showed the presence of three acid-base transporting mechanisms which act in concert to regulate pHi in pancreatic acinar cells. In the present studies we tested the effect of the Ca*+-mobilizing agonists (11, 15), CCK-OP and carbachol, on pHi and the activity of each transporter. Stimulation of pancreatic acini with agonists resulted in initial acidification during the first 2 min of stimulation. Subsequently, the pHi increased beyond or somewhat below the initial levels in HEPES- and HCOB-buffered media, respectively. Initial Acidification-An initial acidification prior to pHi increase due to Na+/H’ exchange activation was observed in many cell types stimulated with Ca’+-mobilizing agonists (2124) or growth factors (25-27). Here we extended these observations to show that the initial acidification occurs in HEPES- and HCOF-buffered medium (Table I). We could not obtain conclusive evidence as to the origin of this acidification. Thus, it was observed in Na’containing and Na’-free media, it was not blocked by H,DIDS or amiloride and it was observed when pH, was reduced from 7.28 to approximately 7.0 (Fig. 2~). Therefore, we can conclude that it was not mediated by any of the acid-base transporting mechanisms described in this and the preceding article (1). It was suggested previously that a Ca’+/H+ exchange activity led to a similar
12818
Agonists and pHi
acidification observed in vascular smooth muscle cells (28). However, this seems to be unlikely in pancreatic acini for the following reasons. Chemical (29) and 45Ca (30) measurements indicate that agonists mobilize approximately 1 pmol of Ca’+, while the acidification in HCO;-buffered medium required the removal of approximately 5.7 pmol of HCO; or the uptake of 5.7 prnol of H’. Furthermore, treatment of acini with low concentration of ionomycin, which depletes intracellular Ca2+ stores without an increase in free cytosolic Ca2+ (31), did not prevent the initial acidification (not shown). Hence, it seems that in pancreatic acini the initial acidification is not mediated by a Ca*+/H+ exchange mechanism. Activation of a plasma membrane-located channel which is not sensitive to H*DIDS and amiloride and does not transport Na+ but conducts H’ and HCO; can explain the acidification. Alternatively, the acidification can be the result of increased metabolism or release of acid from acidified intracellular organelles (32) to the cytosol. At present we have no evidence to support either possibility. Nu+/H+ Exchange-Evidence for activation of the Na+/H+ exchanger by CCK-OP stimulation were obtained in HEPESand HCOB-buffered medium. In HEPES-buffered medium, where only the Na+/H+ exchanger is functional, pHi increased to a steady-state level of 7.43 f 0.04 due to CCK-OP stimulation. Similar pHi increase was observed in HCO, medium when the acini were treated with HzDIDS (Table I). Since the buffer capacity in HCO, medium, pHi 7.3 is 3-fold higher than in HEPES medium (Fig. l), the exchanger must have removed three times more H’ in HCO; medium to increase pH, to similar levels. Yet, the rate of Na’/H’ exchange of stimulated acini acidified to pHi of 6.8 in both media was similar (11.26 and 10.98 mM H+/min in HEPES and HCO; media, respectively) and the degree of activation by agonist was the same (Table II). This suggests that the rate of Na’/ H+ exchange of stimulated acini was not the limiting factor in determining the final pHi attained by stimulated cells. Rather, it is probably the pHi dependence of the exchanger. It was shown in a variety of cells that NaL,‘,t/HL exchange is reduced as pHi increases (1, 6, 7, 9). In the present studies we have not addressed the mechanism by which the Na+/H+ exchanger is activated during cell stimulation. However, previous studies with guinea pig pancreatic acinar cells showed that the effect of hormonal stimulation could be mimicked by activation of protein kinase C but not elevation of Ca*+ (4). Such stimulation probably involves a concomitant but opposite change in the apparent affinity for intracellular H’ and Na’ (27, 33). Na+-HCO: Cotransport-In pancreatic acini, the Cl--independent, Na+-HCO, cotransporter (1) was inhibited by stimulation with Ca2+ mobilizing agonists. The contribution of this transporter to pHi regulation in stimulated cells is further reduced due to the initial reduction in pH, to 6.96 (Table I). At this pHi the reversal potential for the transporter is -76 mV (assuming small change in intracellular Na’ activity following 2 min of stimulating). Hence, under these conditions the transporter actually switched from an acid loader (HCO; efflux) to a base loaded (HCO: influx). Evidence for this can be seen in Fig. 4b and Table II where small recovery of pHi occurred after a lo-min stimulation of acini in the presence of amiloride. An interesting finding was that HCO; influx was inhibited by 40% while HCO: efflux was inhibited by 60% (Table II). Stimulation of rat pancreatic acini with Ca’+-mobilizing agonists depolarizes the cells by as much as 20 mV (12), due to activation of a Ca’+-activated nonselective cation channels
(14). This change in membrane potential is expected to stimulate HCO, influx and inhibit HCO: efflux since the transporter is electrogenic (34-38). Therefore, it is likely that stimulation with CCK-OP inhibited the turnover rate of the cotransporter by about 50%, but the additional change in membrane potential resulted in the apparent reduced inhibition of HCO; influx and increased inhibition of HCO; efflux. To verify that a different degree of inhibition of HCO; influx and efflux is restricted to the electrogenic Na’-HCO; cotransporter, we also compared HCO; influx and efflux by the electroneutral Cl-/HCO, exchanger. Indeed, these fluxes were similarly stimulated by CCK-OP. The Na’-HCO? cotransporter make a minor contribution to acid-base transport in unstimulated cells, since the reversal potential for cotransport is near the resting potential (1, 12, 14). Because of its electrogenic nature it is sensitive to the membrane potential (37, 38). In this way, it may serve a unique role in regulating pHi where it can be used as an acid or base loader, depending on the change in membrane potential evoked by cell stimulation. In case an agonist hyperpolarizes the cells, the Na’-HCO: cotransporter will serve as an acid loader by removing HCO, from the cytosol. When agonists depolarize the cells, as in the case of rat pancreatic acini, the Na’-HCO: cotransporter serves as a base loader by conducting HCO, influx. Cl-/HCO, Exchange-The transporter stimulated most by CCK-OP was the Cl-/HCO; exchanger. Stimulation of this exchanger can be observed when it is engaged in HCO, influx or efflux. The rate of HCO; efflux by this exchanger exceeds the overall rates of base load due to Na’/H’ exchange in resting cells and Na’/H’ exchange and Na’-HCO; cotransport in stimulated cells (Table II). This is likely responsible for the reduced pH: of resting acini and the attenuated pHi increase after 10 min stimulation with CCK-OP in HCO;buffered medium (Table I). The effect of 8-arginine vasopressin on pH,-regulatory mechanisms of renal mesangial cells was reported recently (39). Similar to our findings, it was reported that 8-arginine vasopressin stimulated the Na’/H’ and Cl-/HCO: exchangers. These studies also showed the stimulation of a Na+dependent Cl-/HCO: exchange by 8-arginine vasopressin. However, in MSE-1 fibroblasts, growth factors activated the Na’/H’ exchanger but failed to activate the Na+-dependent Cl-/HCO: exchanger (40). In renal mesagial cells (39), as in pancreatic acini (present studies), the agonist stimulated Cl-/ HCO: exchange more than it stimulated the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO; exchange. Stimulation of Cl-/HCO: exchange by Ca2+ (41), activation of protein kinase C (42) and inhibition of the exchanger by CAMP (43) were reported. Activation and inhibition of the exchanger involved a shift in the pHi dependence of the exchanger (41-43). Kinetic analysis suggests that the exchanger is regulated by OH- binding to an intracellular regulatory site different from the transport site (41). Activation of the exchanger appears to involve modification of the regulatory site (41, 43). It is possible that Ca*+-mobilizing agonists activate the Cl-/HCO, exchanger in pancreatic acini by the same mechanism. However, it is important to note that the kinetic studies were performed on Cl-/Clexchange in the absence of HCO: (42,43) or on OH- transport of an exchanger which can transport OH- and HCOy (41). Therefore, whether such a mechanism also applies to the exchanger in pancreatic acini, which can not transport OH- (l), needs to be tested directly. Physiological Significance-The acid-base transporting mechanisms and their regulation by Ca’+-mobilizing agonists described here may influence directly at least three short and
A~OT&S
long term functions in pancreatic acini, apart from regulating pHi. These are volume regulation, NaCl transport, and cell growth. The free cytosolic Ca2+ increase evoked by agonist stimulation activated Ca*+-dependent Cl- and cations or K’ channels in pancreatic acini (12, 14). This is followed by a rapid KC1 efflux and cell shrinkage (14). At subsequent stimulation periods the acini incorporate K’, Na+, and Cl-. It was shown that part of this uptake is mediated by a furosemidesensitive Na,K,2Cl cotransport (10, 14). Activation of the Na’/H’ and Cl-/HCO: exchangers described here also results in net NaCl uptake and can help in recovery of cell volume. The roles of the Na+/H+ exchanger and possibly the Cl-/ HCO, exchanger in regulatory volume increase have been documented in many cell types (6, 7). The activated Na’/H+ and Cl-/HCO; exchangers can also provide the acinar cells with the mechanisms required for the transcellular route of NaCl transport. Electrophysiological studies suggest that some of the tranepithelial Na’ transport is transcellular rather than paracellular (10, 11, 44). In addition, acinar cell fluid secretion can be partially inhibited by removal of basolateral HCO;, and amiloride (45). Our studies suggest that the Na’/H+ exchanger and the HCOB-dependent, Cl-/HCO: exchanger are the mechanisms mediating NaCl uptake in the basolateral membrane, which is required for fluid secretion. Activation of these mechanisms by CCK-OP stimulation can account for part of the stimulation of fluid secretion by CCK-OP (45). Gastrointestinal peptides, such as CCK (16, 46) bombesin (23), and secretin (46), have been shown to promote pancreatic cell proliferation. CCK-OP, carbachol, and caerulein increase proliferation of pancreatic acinar cells in primary culture (48). All of these agonists increase free cytosolic Ca*+ and activate protein kinase C (11, 15). In many cells, growth and proliferation are thought to be linked to activation of the Na’/H’ exchanger and the resulting pHi increase (8, 9). Similarly, it was suggested that stimulation of the Na+/H’ exchanger by agonists and protein kinase C may be regulating proliferation of pancreatic acinar cells (4). However, most studies recorded pHi changes due to stimulating in HCO;free medium. As we show here for pancreatic acini, in HCO:buffered medium, growth factors have either no effect (40) or reduce steady-state pHi (39). Hence, it is possible that the increased influx of Na+, rather than the change in pHi is the triggering factor for cell proliferation. In this respect, activation of Na’/H’ exchange in pancreatic acini may also mediate the proliferative effect of the gastrointestinal peptides. It is difficult to propose a role for the Na+-HCO; cotransporter and its inhibition by Ca*+-mobilizing agonists, since the membranous localization of this transporter in pancreatic acini is not known. It can play opposite roles in the above activities depending on its localization. For example, in the stimulated cells, basolateral localization will permit Na’ and HCO; influx which can help in transcellular Na‘ transport while liminal localization will result in inhibition of net transcellular Na’ transport. Hence, further studies are required to determine the possible role of this transporter in acinar cell
12819
and PHi
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for manuscript
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