Departments of Medicine, Duke University Medical Center, Durham 27710; and Durham Veterans Affairs Medical Center, Durham, North Carolina 27705
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ABSTRACT |
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It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.
cholecystokinin; radioimmunoassay; hormone; proton exchange; STC-1
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INTRODUCTION |
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CCK IS A gastrointestinal peptide hormone secreted into the circulation on ingestion of a meal. Circulating CCK binds to its receptors on target tissues, including the pancreas, gallbladder, stomach, and various nerves to stimulate pancreatic enzyme secretion and gallbladder contraction and to regulate satiety (18). Accordingly, CCK is an important physiological regulator of the ingestion and digestion of essential nutrients.
Studies of the cellular mechanisms regulating CCK secretion have been limited by difficulties in isolating native CCK-secreting cells, which comprise <1% of the mucosal cell population of the intestine (2, 12). More recently, an intestinal cell line that secretes CCK (STC-1) has been used in our laboratory and in others (4, 15, 17, 25) to delineate the molecular mechanisms regulating CCK secretion.
Previous studies (25) have demonstrated that CCK release is dependent on Ca2+ influx through L-type Ca2+ channels, because removal of extracellular Ca2+ or addition of specific L-type Ca2+ channel blockers inhibits agonist-stimulated CCK release. Moreover, maneuvers that depolarize the membrane, including inhibition of K+ channels, result in Ca2+ influx through voltage-dependent opening of Ca2+ channels (16). Thus there is close coupling between membrane potential, intracellular Ca2+ concentration ([Ca2+]i), and secretory activity.
Electroneutral Na+/H+ exchangers (NHEs) are expressed in the plasma membrane of mammalian cells and contribute to regulation of intracellular pH (pHi), cell volume, and perhaps cell growth (1, 27). These effects are mediated directly through H+ efflux or indirectly through effects of changing [H+]i (or pHi) on ion channels or other effector pathways. The NHE-1 subtype can be activated by stimuli such as growth factors, hyperosmolarity, hormones, and neurotransmitters (5, 10, 19). However, little is known about the relationship between NHEs and hormone secretion. The current study was designed to determine whether NHE activity in STC-1 cells is involved with CCK release. The findings of the present study, in which H+ efflux is used as a measure of NHE activity, suggest important functional interactions between NHE activity and membrane potential in CCK secretion.
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METHODS |
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Cell culture. The STC-1 cell line was derived from an intestinal endocrine tumor that developed in mice carrying the transgenes for the rat insulin promoter linked to the simian virus 40 large T antigen and the polyoma virus small t antigen (22). The cell line was kindly provided by Dr. Douglas Hanahan (University of California, San Francisco, CA).
Cells were grown in solution containing 150 ml/l horse serum, 25 ml/l FCS, high-glucose DMEM, 100 kU/l penicillin, and 100 mg/l streptomycin. Cells were incubated at 37°C and aerated with a 95% O2-5% CO2 gas mixture.CCK secretion. CCK concentrations were determined using antisera no. 9322, according to a previously described RIA technique (15). Antisera no. 9322 was kindly provided by Dr. John Walsh (University of California, Los Angeles, CA). Studies examining CCK secretion were performed on 24-well plates with cells at 80-90% confluence. Cells were pretreated with test agents or control vehicles for 15 min. Secretion was measured after the 15-min treatment period unless otherwise stated.
Microphysiometry.
Na+/H+
exchange activity was measured in real time as the rate of decrease in
extracellular pH (pHo) in intact
cell monolayers placed in an eight-chamber Cytosensor microphysiometer
(Molecular Devices, Sunnyvale, CA) (23). The microphysiometer uses a
light addressable silicon sensor to detect extracellular protons, which can be derived primarily from
Na+/H+
exchange, glycolysis, and other metabolic pathways. Data are presented
as microvolts per second, which roughly correspond to millipH units per
minute. The night before experimentation, STC-1 cells were replated
onto polycarbonate membrane inserts (3 µm pore size, 12 mm size) at a
density of 300,000 cells/insert. On the day of the study,
cells were washed with serum-free, bicarbonate-free Ham's medium,
placed into the microphysiometer chambers, and perfused at 37°C
with the same medium or with a balanced salt solution. For most
studies, the pump cycle was set to perfuse cells for 60 s, followed by
a 30-s "pump-off" phase, during which proton efflux was measured
from the 6th through the 28th s. Cells were exposed to the test agent
for two or three cycles (180-270 s). Valve switches (to add or
remove test agents) were usually performed at the beginning of the pump
cycle. In some cases in which the response was expected to be both
rapid and very transient (i.e., recovery from
Na+-free conditions), the valve
switch was performed 55 or 58 s into the perfusion phase, allowing
7-10 s for solution mixing before rate measurements. Rate data
were then calculated every 90 s. The peak effect during stimulation was
expressed either as the percentage increase from average basal efflux
of four or five rate measurements before application of the test
agent(s), whereas the mean change from basal was expressed as the
change in microvolts per second. Typical basal efflux rates in
glucose-containing solutions were in the 100-130 µV/s
range, regardless of the presence of Na+.
Ca2+ fluorescence. [Ca2+]i was measured after STC-1 cells were suspended and loaded with 10 µM fura 2-AM for 20 min in DMEM at 37°C, washed, and resuspended in NaCl or choline chloride buffer at a concentration of 3 × 106 cells/ml. Fluorescence was measured by a ratiometric method using a Perkin Elmer LS5OB spectrometer. Emission was measured at 510 nm, with an excitation ratio of 340/380 nm. Test agents were added after a stable baseline was achieved. For studies of choline chloride, fura 2-loaded cells taken from the same flask were resuspended in medium containing either NaCl or choline chloride. Calibration was based on using a binding constant of fura 2 to Ca2+ of 224 nM, and maximal and minimum fluorescence was achieved with 0.1% Triton X-100 and 20 mM EGTA, respectively (11).
Whole cell patch-clamp recording.
To measure whole cell currents, we plated cells onto uncoated Corning
35 × 10 mm polystyrene plates in 2 ml of high-glucose DMEM (14).
Cells were viewed with an Olympus inverted phase-contrast microscope.
Pipettes were made from Corning 7052 glass and had resistances of
3-6 M. After gigaseal formation, signals were fed to an
Axopatch 1-D amplifier (Axon Instruments), coupled through a Digidata
1200 interface to a personal computer.
Materials. Cell culture supplies were obtained from GIBCO-BRL (Grand Island, NY). Ethylisopropyl amiloride (EIPA) was from Research Biochemicals International (Natick, MA). Fura 2-AM was from Molecular Probes (Eugene, OR). All other chemicals were from Sigma Chemical (St. Louis, MO).
Statistical analysis. Values are reported as means ± SD, and the differences were compared using an unpaired t-test or ANOVA when appropriate. P < 0.05 was considered significant.
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RESULTS |
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To determine whether Na+/H+ exchange activity affects hormone secretion, we measured the effects of the amiloride analog EIPA, an Na+/H+ exchange blocker, on CCK release from STC-1 cells (Fig. 1). Over 15 min, inhibition of H+ efflux with EIPA (20 µM) increased CCK release by 253 ± 26% over control levels (P < 0.05). In addition, inhibition of Na+/H+ exchange by substitution of choline chloride for NaCl in the extracellular buffer caused a substantial increase of 266 ± 14% in CCK secretion.
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Na+/H+ exchange is known to participate in the control of both pHi and pHo and appears to be involved in a number of critical cellular processes, such as regulation of cell volume and growth (10, 27). If the effects of EIPA on CCK secretion are mediated by inhibition of Na+/H+ exchange, it would be anticipated that EIPA would decrease H+ efflux and the rate of extracellular acidification. Consequently, the rate of extracellular acidification in STC-1 cells was measured as the decrease in pHo using a Cytosensor microphysiometer. Data were expressed as the percentage of basal acidification rate, which was 120 ± 12 µV/s. It would be expected that Na+/H+ exchange activity would be reduced not only by treatment with an amiloride analog but would be eliminated in the absence of extracellular Na+. Cells were perfused with 1) NaCl-containing buffer, 2) NaCl-containing buffer to which EIPA had been added for 120 s, or 3) buffer in which choline chloride had been substituted for NaCl. Incubation of cells in Na+-free, choline chloride-containing medium produced a low basal extracellular rate of acidification. (There was a small degree of acidification that resulted from proton accumulation during the brief period of pump switching.) On return to NaCl-containing medium, a rapid burst of proton efflux was observed. As shown in Fig. 2, the maximal acidification rate was significantly reduced by EIPA (20 µM). Figure 2 shows that under the same conditions in which CCK secretion occurs, an EIPA-inhibitable, Na+-dependent proton efflux pathway exists and supports the involvement of Na+/H+ exchange in regulation of CCK secretion.
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We have previously determined (3, 17, 20) that [Ca2+]i is an important component for secretagogue-stimulated CCK secretion. We, therefore, sought to determine whether the effects of NHE blockade, as demonstrated by removal of extracellular Na+ (choline chloride substitution), were associated with a change in [Ca2+]i. If choline substitution increases CCK release through effects on [Ca2+]i, then choline substitution would be expected to increase [Ca2+]i levels. As shown in Fig. 3, cells in choline chloride buffer consistently demonstrated higher basal [Ca2+]i compared with cells in standard medium. EIPA interfered technically with the fluorescence measurements of [Ca2+]i, precluding its use for these studies.
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If this increase in [Ca2+]i is a trigger for CCK secretion and is due to an influx of Ca2+ from the outside of the cell, it may be sensitive to the L-type Ca2+ channel blocker diltiazem. To address this possibility, we found that the increase in basal CCK release produced by EIPA and the removal of extracellular Na+ was significantly (~60%) reduced by 10 µM diltiazem (Table 1). This finding suggests that inhibition of Na+/H+ exchange stimulates CCK secretion through the opening of Ca2+ channels.
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The relationship between NHE activity and Ca2+ influx could result from a direct effect of pHi on Ca2+ influx or an indirect effect of pHi on membrane potential, because L-type Ca2+ channels in STC-1 cells are opened by membrane depolarization (14). pH-sensitive K+ channels that are closed by intracellular acidification have been identified in several cell types (6, 7, 26). Consequently, additional studies were performed to assess the possible relationship between changes in membrane potential, [Ca2+]i, and CCK release.
It is expected that inhibition of Na+/H+ exchange would lower pHi. If STC-1 cells possess a pH-sensitive K+ conductance, accumulation of H+ would inhibit K+ efflux and result in membrane depolarization, which we have previously demonstrated is a potent stimulus for CCK secretion (13, 14, 16, 17, 24). If the effects of EIPA on CCK release are related to membrane depolarization, then it would be anticipated that elevating extracellular KCl concentrations to depolarize the membrane would inhibit EIPA-stimulated CCK release. The results of these experiments are shown in Fig. 4, in which the KCl concentrations of the extracellular buffer were increased from 5 to 50 mM. Depolarization caused a significant decrease in EIPA-stimulated CCK secretion, consistent with functional coupling between NHE activity, membrane potential, and Ca2+ influx.
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It is likely that the changes in pHi are modest, yet have substantial effects on other intracellular processes, including hormone secretion. As further evidence that intracellular acidification is critical in regulating CCK release, STC-1 cells were exposed to low pHo. Acutely decreasing pHo from 7.4 to 7.0 caused a 232% increase in CCK release from basal levels (Fig. 5).
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To also address this issue, we exposed cells to sodium butyrate (5 mM). This reagent serves as a proton donor on rapid uptake by cells and causes intracellular acidification (9). As shown in Fig. 5, exposure of STC-1 cells to sodium butyrate stimulated CCK release by 150% (P < 0.05).
Taken together, these findings suggest that intracellular acidification stimulates CCK release through effects on membrane K+ permeability and potential difference. To examine whether pH directly affects membrane K+ permeability, outward currents carried by K+ were measured using whole cell patch-clamp techniques (Fig. 6). With physiological solutions (140 mM Na+ and 5 mM K+ in the bath), net outward membrane currents were present at 0 mV (Fig. 7). With these solutions, outward currents at 0 mV are likely to reflect movement of K+ out of the cell since K+ is the only ion with a more negative reversal potential. Under basal conditions (pHo 7.4, pHi 7.2), K+ current measured at 0 mV averaged 902 ± 118 pA (mean ± SE, n = 6). K+ current was significantly inhibited by lowering pHo to 7.0 (K+ current = 325 ± 132 pA, n = 5) or 6.5 (K+ current = 180 ± 152 pA, n = 4). These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions (pHo 7.4, pHi 7.0) nearly eliminated current activity (6 ± 13 pA; P < 0.002; n = 5) in the absence of any changes in pHo.
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DISCUSSION |
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The present study was designed to determine whether Na+/H+ exchange is integral to stimulation of CCK secretion. Blocking Na+/H+ exchange was achieved by EIPA treatment and substitution of Na+ salts with choline chloride, both of which increased basal CCK release. Because Ca2+ influx through L-type Ca2+ channels is critical for CCK release stimulated by many secretagogues such as phenylalanine or glucose (14), we evaluated the effects of NHE blockade on [Ca2+]i. Although EIPA interfered technically with the fluorescence measurements of [Ca2+]i, precluding its use for these studies, removal of extracellular Na+ clearly raised [Ca2+]i. Moreover, the stimulatory effects of extracellular Na+ removal and EIPA on CCK secretion were blocked by the L-type Ca2+ channel blocker diltiazem. In aggregate, these findings suggest that the increase in basal secretion caused by inhibition of NHE is dependent on an influx of extracellular Ca2+. It is emphasized, however, that Na+ removal is likely to have other effects as well. The observation that diltiazem did not completely block CCK secretion induced by Na+-free medium supports this concept. In experiments not shown, EIPA did not modify the CCK increase caused by exposure to Na+-free buffer, suggesting that both maneuvers are through a similar mechanism (e.g., Na+/H+ exchange).
In some other cell types, membrane depolarization produced by inhibition of basally active K+ channels activates voltage-gated Ca2+ influx pathways, which leads to stimulation of secretion (21, 28). In STC-1 cells, it has been shown that membrane depolarization represents an important stimulus for CCK secretion through enhanced Ca2+ influx (24). Although these former studies invoked an important role for ATP-sensitive K+ (KATP) channel activity as a major contributor to total K+ conductance and membrane potential, the existence of pH-sensitive K+ channels in STC-1 cells and their possible contribution to membrane potential was unknown. Moreover, it was not known whether membrane potential might couple NHE activity to secretion.
One possible alternative explanation is that Na+-free medium or EIPA administration may modify cell volume, which in and of itself, may have effects on hormone secretion. Moreover, the effects of EIPA on Na+/Ca2+ exchange, which may also affect secretion, are unknown.
Proton efflux that was Na+ dependent and amiloride sensitive was taken as evidence of Na+/H+ exchange activity (19). It has been well established that acute inhibition of proton efflux leads to intracellular accumulation of H+ and a decrease in pHi (27). Our observations that maneuvers that could lower pHi, such as EIPA administration and removal of extracellular Na+, reduced pHo, or sodium butyrate treatment, are consistent with the existence of pH-sensitive K+ channels in STC-1 cells. Similar to studies of KATP channels in STC-1 cells, it is likely that these pH-sensitive K+ channels contribute to membrane potential and, in turn, modulate hormone secretion (6, 7, 26). If this mechanism was participating in EIPA-stimulated CCK release then it would be expected that CCK release would be no greater than that seen with depolarizing concentrations of KCl alone. EIPA-stimulated CCK secretion was reduced with higher concentrations of extracellular KCl, indicating that the effects of Na+/H+ exchange are closely linked to membrane potential and are not observed under conditions in which the membrane is already depolarized.
To test this hypothesis, we measured the effects of pH changes on K+ current activity by whole cell electrophysiological recordings. Lowering pHo (and presumably pHi) to 7.0 or 6.5 significantly stimulated CCK release and concomitantly reduced the amplitude of outward K+ currents. These effects appeared to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These findings indicate that K+ permeability of STC-1 cells is tightly regulated by changes in pHi.
Intestinal endocrine cells are open to the gut lumen and, being downstream from the stomach, are exposed to solutions of low pH. If sufficient acidification of mucosal cells occurs, it is conceivable that this could serve to stimulate hormone secretion. The molecular mechanisms for such regulation have not previously been considered, but the present studies offer a possible explanation.
In summary, these studies suggest that stimulation of CCK secretion is functionally linked with activation of Na+/H+ exchange through a mechanism that may involve pH-sensitive changes in membrane potential (Fig. 8). We propose that inhibition of Na+/H+ exchange lowers pHi and causes membrane depolarization presumably through effects of H+ on K+ permeability. K+ channel closure, in turn, depolarizes the plasma membrane and activates L-type Ca2+ channels, leading to an increase in [Ca2+]i. This increase in [Ca2+]i ultimately causes CCK secretion. These findings imply that CCK secretion from native cells in the intestine may be modified by secretagogues or nutrients that influence cellular proton exchange.
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A role for pHi in the regulation of hormone secretion has implications for how potential secretagogues may influence CCK release. It is possible that agents that directly lower pHi as H+ donors or indirectly influence pHi through NHE blockade could affect basal CCK secretion. At the present time, the long-term consequences of such agents are unknown, but this mechanism for regulation of hormone secretion provides a potential therapeutic target.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants DK-38626, DK-46082, DK-43278, and NS-30927 and by the Department of Veterans Affairs.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. A. Liddle, Box 3913, Duke Univ. Medical Center, Durham, NC 27710.
Received 27 January 1998; accepted in final form 21 May 1998.
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