Department of Physiological Sciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom
Submitted 9 December 2002 ; accepted in final form 13 March 2003
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ABSTRACT |
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pancreas; Cl-/HCO3- exchanger; tachykinin
CFTR Cl- channels, Ca2+-activated Cl-
channels, and anion exchangers are the only transport elements that have been
identified on the apical membrane of duct cells in small interlobular and
intralobular ducts, which are probably the major sites of
HCO3- secretion
(2). Originally, it was
considered that HCO3- secretion occurred on the
exchanger with the CFTR and Ca2+-activated Cl- channels
acting to recycle Cl- across the apical membrane
(2). Computer modeling studies
suggest that such a mechanism could increase the luminal
HCO3- concentration to 70 mM
(25). To raise the luminal
HCO3- concentration to 150 mM, the concentration
secreted by the pancreas of guinea pigs, humans, and some other species
(2), additional
HCO3- is secreted directly via the channels
(10). The regulatory pathways
that stimulate pancreatic ductal HCO3- secretion are
well described (2); however,
much less is known about inhibitory pathways. Substance P (SP)
(3) and 5-OH-tryptamine
(26) strongly inhibit fluid
and HCO3- secretion from isolated pancreatic ducts,
suggesting that agents have a direct inhibitory effect on the ductal
epithelium. Such inhibitory pathways may be physiologically important in terms
of limiting the hydrostatic pressure within the lumen of the duct (thus
preventing leakage of enzymes into the parenchyma of the gland) and in terms
of switching off pancreatic secretion after a meal
(3,
26).
The undecapeptide SP has been shown to inhibit secretin-stimulated secretion from the pancreas of the dog (11, 15) and rat (12). SP-like immunoreactivity has been identified in the pancreas of the dog, rat, and mouse (21), although it has not been localized around the ducts (24). In isolated rat pancreatic ducts, 10-10 and 10-8 M SP inhibited basal fluid secretion by 54 and 92%, respectively, and the same doses of SP completely blocked the fluid secretory responses to secretin and bombesin (3). These inhibitory effects of SP were blocked by spantide, which is an SP receptor antagonist (3). SP also inhibited fluid secretion stimulated by dibutyryl cAMP and forskolin (3), which places the inhibitory effect of SP at a point in the secretory mechanism downstream from the generation of cAMP. The purpose of this study was to identify the duct cell transporter(s) that are modulated by SP.
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METHODS |
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Small intra/interlobular ducts were isolated from the pancreas of guinea pigs weighing 150250 g. The guinea pig was killed by cervical dislocation, the pancreas was removed, and the intra/interlobular ducts were isolated by enzymatic digestion and microdissection and then cultured overnight as previously described (1). During overnight culture, the ducts seal to form a closed sac that swells due to accumulation of secretions in the duct lumen (1).
Measurement of Intracellular pH
Cultured ducts were attached, using Cell-Tak, to a coverslip (24 mm2) forming the base of a perfusion chamber mounted on a Nikon Diaphot microscope (Nikon UK, Kingston upon Thames, UK). The ducts were bathed in the standard HEPES solution at 37°C and loaded with the pH-sensitive fluorescent dye BCECF by exposure to 2 µM BCECF-AM for 2030 min. After loading, the ducts were continuously perfused with solutions at a rate of 45 ml/min. Intracellular pH (pHi) was measured using a Life Sciences microspectrofluorimeter system (Life Sciences Resources, Cambridge, UK). A small area of 510 cells was excited with light at wavelengths of 490 and 440 nm, and the 490/440 fluorescence emission was measured at 535 nm. Four pHi measurements were obtained per second. In situ calibration of the fluorescence signal was performed using the high K+-nigericin technique (28). During the calibration, ducts were bathed in a high-K+ HEPES solution and extracellular pH was stepped between 5.95 and 8.46.
Determination of Buffering Capacity
The intrinsic buffering capacity (i) of duct cells was
estimated according to the NH4+ prepulse technique
(29).
i
refers to the ability of intrinsic cellular components (excluding
HCO3-/CO2) to buffer changes of
pHi. Briefly, pancreatic duct cells were exposed to various
concentrations of NH4Cl while Na+ and
HCO3- were omitted from the solution to block the
Na+-dependent pH regulatory mechanisms.
i was
estimated by the Henderson-Hasselbach equation. The total buffering capacity
(
total) was calculated as
total =
i +
HCO3- =
i + 2.3 x [HCO3-]i,
where
HCO3- is the buffering capacity
of the HCO3-/CO2 system and [HCO3
cellular concentration of HCO3-
(Fig. 1).
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Measurement of HCO3 Transport
HCO3- efflux. INHIBITOR STOP METHOD. In this series of experiments the HCO3- accumulation mechanisms on the basolateral membrane of the duct cell, the Na+/H+ exchanger, and the Na+-HCO3- cotransporter (2) were blocked by brief exposure (5 min) of the ducts to amiloride (0.2 mM) and DIDS (0.1 mM) (27). This procedure causes pHi to acidify (27). The rate of pHi acidification has been shown to reflect the rate of HCO3- efflux across the apical membrane and the buffering capacity of the cell (27).
The initial rate of intracellular acidification (dpH/dt), over the first 60 s of exposure to amiloride and DIDS, was calculated by linear regression analysis using 240 data points (4 pHi measurements per second) (see Fig. 2). Each duct was exposed to amiloride and DIDS twice, the first exposure being the control and the second the test. Secretin (10 nM) secretin and/or SP (20 nM) and/or spantide (20 nM) were given for 10 min between the two measurements. In the control group, 1% albumin was administered before the second measurement because it was used as the vehicle for secretin and SP.
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RECOVERY FROM AN ALKALINE LOAD. Ducts were alkali loaded by exposure to 3-min pulses of 20 mM NH4Cl. The initial rate of recovery from alkalosis (dpH/dt) over the first 30 s (120 pHi measurements) in the continued presence of NH4Cl was calculated as described above.
HCO3- influx. RECOVERY FROM AN ACID LOAD. Ducts were acid loaded as described above, and then the NH4Cl was removed, which caused a marked acidification of pHi. The initial rate of pHi recovery (dpH/dt) was measured over the first 30 s (120 data points) after removal of NH4Cl.
The rates of pHi change measured in these experiments were
converted to transmembrane base flux [J(B-)] using the
equation J(B-) = dpH/dt
xtotal. The
total value used in the
calculation of base fluxes was obtained from
Fig. 1 by using the
pHi value at the start of the 30- or 60-s period over which
dpH/dt was measured. We denote base influx as
J(B-) and base efflux as -J(B-).
Solutions and Chemicals
The compositions of the solutions used are shown in Table 1. HEPES-buffered solutions were gassed with 100% O2 at 37°C and pH was set to 7.4 with HCl. HCO3- buffered solutions were gassed with 95% O2 and 5% CO2 at 37°C.
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All laboratory chemicals, peptides, transport blockers, and ionophores were obtained from Sigma (Poole, Dorset, UK). Chromatographically pure collagenase was obtained from Worthington (Lakewood, NJ), and culture medium was from ICN (Aurora, OH). Nigericin was dissolved in absolute ethanol, DIDS and amiloride in DMSO, and secretin, spantide, and SP in 1% albumin. Cell-Tak was obtained from Becton Dickinson Labware (Bedford, MA). BCECF-AM was obtained from Molecular Probes (Eugene, OR) and made up asa2mM stock solution using DMSO.
Statistical Analysis
Results are expressed as means ± SE (n = no. of observations). Statistical analyses were performed using either Student's t-test (when the data consisted of 2 groups) or ANOVA (when 3 or more data groups were compared). P values <0.05 were accepted as significant.
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RESULTS |
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The resting pHi of duct cells bathed in the standard HCO3--containing buffer was 7.36 ± 0.01 (n = 8). Exposing ducts to 0.1 mM DIDS and 0.2 mM amiloride caused an acidification of pHi (Fig. 2A) due to inhibition of the basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers, which normally act to transport HCO3- into the duct cell from the blood (27). The effects of DIDS and amiloride are likely to be confined to the basolateral transporters because 1) DIDS is unlikely to gain rapid access to the lumen of the sealed ducts because of its charged sulfonic acid groups, and 2) there are no amiloride-sensitive transport steps at the apical membrane of the small interlobular ducts used in this study. In addition, basolateral DIDS will block the basolateral anion exchanger (8, 31), which will normally act to transport HCO3- out of the cell. Thus the rate at which pHi acidifies after exposure to DIDS and amiloride will reflect the intracellular buffering capacity and the rate at which HCO3- effluxes (i.e., is secreted) across the apical membrane on Cl-/HCO3- exchangers and CFTR Cl- channels (2, 27). Ca2+-activated Cl- channels should not be active under the conditions used in this study.
Removal of DIDS and amiloride caused pHi to return to the control value (Fig. 2A), after which a second exposure to the inhibitors caused pHi to acidify again at the same rate (Fig. 2A). Figure 2B shows that secretin (10 nM) markedly enhanced the rate at which pHi acidifies after addition of DIDS and amiloride. SP (20 nM) had no effect on the rate of pHi acidification in unstimulated ducts (Fig. 2C) but abolished the effect of secretin (compare Fig. 2, B and D).
In Fig. 3, the changes in pHi observed after addition of DIDS and amiloride have been converted to a rate of base efflux [-J(B-)] using the rate of pHi change and the total buffering capacity of the cell (see METHODS). Secretin (10 nM) increased -J(B-) about fivefold. SP (20 nM) had no effect on the basal -J(B-), but the same dose completely blocked the secretin-stimulated increase in -J(B-) (Fig. 3). This inhibitory effect of SP on secretin-stimulated -J(B-) could be reversed by 20 nM spantide, an SP receptor antagonist (Fig. 3). These data are consistent with secretin enhancing HCO3- secretion and the inhibition of that process by SP. Because basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers would be blocked by DIDS and amiloride, it is unlikely that SP could inhibit secretion by modulating their activity. We next sought to confirm this conclusion by investigating the effect of SP on pHi recovery after an acid load.
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Effect of SP on pHi Recovery From Acid Load
In this series of experiments we tested whether SP affected the ability of duct cells to recover from an acid load. The transporters most likely to be involved in this process are the Na+-HCO3- cotransporter, the Na+/H+ exchanger, and the H+ pump located on the basolateral membrane of the duct cell (2).
Exposure of duct cells to 20 mM NH4Cl induced an immediate rise in pHi due to the rapid entry of the lipophilic base NH3 into the duct cell (Fig. 4). After the initial alkalinization, pHi started to recover as a result of influx of the less membrane-permeant NH4+ and activation of cellular pHi regulatory mechanisms. After the removal of NH4Cl, pHi decreased rapidly. This acidification is caused by the dissociation of intracellular NH4+ into H+ and NH3, followed by the rapid diffusion of NH3 out of the cell. After the acidification, pHi starts to recover as a result of activation of pHi regulatory mechanisms (Fig. 4). Figure 4 also shows that the removal of HCO3- markedly reduced the initial rate of pHi recovery after acid loading. In the absence of both HCO3- and sodium ions, pHi recovery from an acid load was virtually abolished (Fig. 4).
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Figure 5 shows data from a series of experiments in which we measured the recovery of pHi, expressed as J(B-), after an acid load. The initial recovery J(B-) after an acid load was 17.8 ± 4.0 mM/min (n = 6) in the standard HCO3--buffered solution (Fig. 5A). The recovery J(B-) decreased markedly to 4.1 ± 0.4 mM/min (n = 6) in a HCO3--free solution and to 1.6 ± 0.4 mM/min (n = 6) in a Na+- and HCO3--free solution (Fig. 5A). These data indicate that pHi recovery after an acid load is dependent on the presence of extracellular HCO3- and Na+, implicating basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers in this process. Therefore, the basolateral H+ pump probably has only a marginal effect on pHi recovery from acid load.
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Consistent with these ideas, DIDS and amiloride alone markedly reduced
initial recovery J(B-) values after an acid load by
60 and 70%, respectively (Fig.
5B). When the inhibitors were applied together,
J(B-) was further reduced to 1.3 ± 0.02 mM/min
(Fig. 5B), which is
not significantly different from the rate of recovery observed in the absence
of both Na+ and HCO3- (1.6 ± 0.04
mM/min, n = 6, P > 0.05)
(Fig. 5A). These data
confirm that the basolateral Na+-HCO3-
cotransporter and the Na+/H+ exchanger are the major
base loaders in the duct cell. Figure
5C shows that neither SP (20 nM) nor secretin (10 nM),
nor a combination of the two agents, had any effect on the recovery
J(B-). As expected from these results, SP had no effect on
recovery from an acid load in the presence of DIDS
(Na+/H+ exchangers active) and no effect on
pHi recovery in the presence of amiloride
(Na+-HCO3- cotransporters active) (data not
shown). These data confirm that SP has no effect in the major base loading
transporter on the basolateral membrane of the duct cell.
Effect of SP on pHi Recovery From Alkaline Load
This series of experiments was performed to test whether SP has any effect on HCO3- efflux from the duct cells. Figure 6 shows that after the alkalization caused by exposure of ducts to 20 mM NH4Cl, pHi started to recover in the continued presence of NH4Cl. Figure 6 also shows that the rate of pHi recovery was markedly increased by secretin (10 nM) and that the effect of secretin was inhibited by SP (20 nM).
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Figure 7 is a summary of the data obtained in this experimental series. The initial -J(B-) of the recovery from alkaline load was 36.3 ± 6.2 mM/min (n = 6) in the standard HCO3--buffered solution (Fig. 7A). -J(B-) decreased to 0.6 ± 0.2 mM/min (n = 6) in a HCO3--free solution, and under these conditions, SP had no detectable effect (Fig. 7A). -J(B-) also decreased to 17.2 ± 3.6 mM/min (n = 6) in a Cl--free solution and to 1.6 ± 0.2 mM/min (n = 6) in a Cl-- and HCO3--free solution (Fig. 7B). These data indicate that the recovery from an alkaline load is a HCO3--dependent process and, therefore, that the influx of NH4+ (derived from extracellular NH4Cl) has only a marginal effect on pHi recovery. In addition, pHi recovery after an alkali load consists of both Cl--dependent and Cl--independent HCO3- transport processes of about equal magnitude (Fig. 7B). Neither secretin nor SP had any effect on the recovery from alkaline load in a Cl--free solution, indicating that these agonists do not modulate Cl--independent HCO3- transport (Fig. 7B).
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In the standard HCO3--buffered solution, 20 nM SP decreased -J(B-) to 18.7 ± 3.1 mM/min (n = 6), whereas 10 nM secretin stimulated -J(B-) to 83.6 ± 11.6 mM/min (n = 6) (Fig. 7C). Note that the control -J(B-) and the secretin-stimulated -J(B-) values in these experiments are about 10-fold and 4-fold higher, respectively, than those measured in the inhibitor stop studies (Fig. 2). This difference might be explained by a higher [HCO3-]i after alkali loading of the duct cell. This secretin-stimulated -J(B-) was inhibited by 20 nM SP (28.4 ± 5.2 mM/min) (n = 6), and 20 nM spantide could block the inhibitory effect of SP (59.8 ± 7.6 mM/min) (n = 6) (Fig. 7C). These data indicate that SP can inhibit a Cl--dependent HCO3- efflux mechanism in the pancreatic duct cell. The cellular model suggests that there are three candidates for this mechanism: the Cl-/HCO3- exchangers, located on the apical and basolateral membranes of the duct cell, and apical CFTR, which is known to have a finite permeability to HCO3- (23).
To check whether the basolateral anion exchanger was involved, we examined the effects of DIDS on the pHi recovery after an alkali load. We reasoned that basolateral DIDS would inhibit -J(B-) after an alkali load if the basolateral anion exchanger was active. The effect of DIDS was rather variable, but in nine experiments the disulfonic stilbene had no significant effect on -J(B-) after an alkali load (Fig. 7D). Importantly, SP significantly inhibited -J(B-) in the presence of DIDS, again suggesting that basolateral anion exchangers and Na+-HCO3- cotransporters are not targets for the peptide (Fig. 7D).
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DISCUSSION |
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In the first part of the study, we derived HCO3- secretion from the rate of intracellular acidification that occurs after the addition of DIDS and amiloride to the perfusion buffer (27). Because these two inhibitors will block the base-loading transporters (Na+-HCO3- cotransporters and Na+/H+ exchangers) and the acid-loading transporter (Cl-/HCO3- exchanger) on the basolateral side of the duct cell, the rate of acidification should reflect the rate of HCO3- efflux across the apical membrane, i.e., the rate of HCO3- secretion. This conclusion is supported by the fact that 10 nM secretin, the physiological stimulant of pancreatic HCO3- secretion (2), increased -J(B-) about fivefold. Studies by others have shown that secretin stimulates the basal rate of fluid and HCO3- secretion from isolated guinea pig ducts to a similar extent (27). We found that secretin-stimulated HCO3- efflux was completely blocked by 20 nM SP, an effect that was largely reversed by 20 nM spantide.
Next, we designed experiments to test where in the
HCO3- secretory mechanism SP exerts its effect. The fact
that SP inhibits HCO3- efflux in the presence of
basolateral DIDS and amiloride (see above) strongly suggests that the peptide
does not exert its effect by either inhibition of the basolateral base loaders
(Na+-HCO3- cotransporter and
Na+/H+ exchanger) or activation of the basolateral acid
loaders (Cl-/HCO3- exchanger). We confirmed
this by investigating the effects of SP on the recovery from an acid load.
After acid loading, [HCO3-]i will be low
(6 mM after the first NH4Cl pulse in
Fig. 4), and thus
HCO3- flux across the apical membrane should also be
low. Therefore, after acid loading, the initial rate of pHi
recovery will largely represent influx of base equivalents (i.e.,
HCO3- influx or H+ efflux) across the
basolateral membrane. Our finding that pHi recovery from an acid
load is dependent on the presence of HCO3- and
Na+, and is blocked by DIDS and amiloride, suggests that the
Na+-HCO3- cotransporter and
Na+/H+ exchanger are quantitatively the most important
transporters involved in this process. Similar results have been reported by
others (9). As expected,
neither SP nor secretin had any effect on the initial
J(B-) of recovery from an acid load, providing evidence
that these peptides do not directly modulate the activity of the basolateral
base-loading transporters. Secretin also has no significant effect on
pHi recovery from an acid load in rat pancreatic duct cells
(22).
Finally, we examined the effect of SP on the initial rate of pHi
recovery from an alkali load, induced by exposing the duct cells to 20 mM
NH4Cl. Recovery of pHi under these conditions was
dependent on the presence of HCO3- in the bathing
medium, suggesting that it results from HCO3- efflux
(i.e., secretion) out of the duct cell. Recovery from an alkali load was also
reduced by 50% in the absence of extracellular Cl-, indicating
that the recovery process consists of Cl--dependent and
Cl--independent HCO3- transport in about
equal measure.
Secretin increased -J(B-) after an alkaline load about threefold, consistent with -J(B-) representing HCO3- secretion. A modest 1.4-fold stimulatory effect of secretin on pHi recovery after an alkali load has also been reported in rat pancreatic duct cells (22). In contrast, secretin had no effect on -J(B-) when the experiments were performed in a Cl--free medium, indicating that the hormone activates a Cl--dependent HCO3- transport mechanism in the duct cell. The Cl- dependency of pancreatic HCO3- secretion from the whole gland in vitro has been known for some time (see Ref. 2 for a review). Similar results have been obtained with isolated rat ducts (4) and, more recently, in isolated microperfused guinea pig ducts (30). Both the basal -J(B-) and the secretin-stimulated -J(B-) were markedly inhibited by SP, and this inhibitory effect was reversed by spantide. Interestingly, SP had no inhibitory effect on the residual -J(B-) remaining in a Cl--free medium, suggesting that the peptide inhibits the Cl--dependent HCO3- efflux mechanism present in the duct cell. Candidate transporters for this Cl--dependent HCO3- efflux mechanism are the basolateral and apical anion exchangers and apical CFTR. Because SP inhibited HCO3- efflux in the presence of basolateral DIDS and amiloride in the inhibitor stop experiments, it seems unlikely that the basolateral anion exchanger could be involved (see above). We confirmed this by investigating the effects of SP on recovery from an alkaline load in the presence of DIDS. DIDS had no significant effect on the rate of -J(B-) under these conditions, indicating that pHi recovery after an alkali load cannot be mediated by either basolateral Cl-/HCO3- exchangers or the basolateral Na+-HCO3- cotransporter (working in reverse because of the elevated [HCO3-]i). Importantly, SP continued to inhibit -J(B-) in the presence of DIDS, confirming that the peptide does not affect basolateral HCO3- transport in the duct cell.
These data point to SP inhibiting the activity of either one or both of the apical HCO3- transporters in the duct cell, that is, the apical anion exchanger and CFTR. The fact that -J(B-) after an alkali load in the presence of secretin is largely Cl- dependent suggests that the apical anion exchanger is the transporter that is modulated by SP. We cannot completely exclude an effect on CFTR because this channel is known to conduct HCO3-, albeit less effectively than Cl- (PHCO3-/PCl- estimates vary between 0.1 and 0.5) (23). Moreover, we have recently shown that extracellular Cl- removal causes trans-inhibition of anion flux through CFTR, which might explain the fact that -J(B-) after an alkali load was Cl- dependent (23). However, in patch-clamp experiments on rat pancreatic duct cells, we were unable to detect a consistent inhibitory effect of SP on secretin-stimulated whole cell CFTR Cl- currents. Outward CFTR Cl- currents measured at -60 mV were 62 ± 10 pA/pF (n = 12 cells) in the presence of 10 nM secretin and 36 ± 13 pA/pF (n = 11 cells) in the presence of 10 nM secretin and 10 nM SP (P < 0.12, not significant) (Argent BE, McAlroy H, and Gray MA, unpublished observations). This observation suggests that neither HCO3- transport through CFTR nor modulation of the apical anion exchanger by changes in intracellular Cl- concentration (caused by changes in CFTR activity) can explain the inhibitory effect of SP. However, a regulatory role for CFTR in the effect of SP that is separate from its channel function remains a possibility. Interestingly, CFTR has recently been shown to activate luminal Cl-/HCO3- exchange in the submandibular and pancreatic ducts of mice by a mechanism that is unrelated to its Cl- channel function (16).
Molecular cloning has identified two anion exchangers families in mammalian cells, namely, SLC4 (formerly AE) and SLC26 families (for references, see Ref. 7). Expression of SLC4 isoforms in epithelial tissues is limited to the basolateral membrane (see Ref. 7). In contrast, SLC26 family members, nine of which have now been identified (19), have been shown to be expressed on the apical membrane of epithelial cells (see Ref. 7). Strong candidates for the apical anion exchanger in pancreatic duct cells are SLC26A3 (downregulated in adenoma, DRA) and SLC26A6 (putative anion transporter 1, PAT1). Both DRA (20) and PAT1 (7) have been shown to act functionally as Cl-/HCO3- exchangers and have been localized to the apical membrane of pancreatic duct cells using immunocytochemistry [PAT1 (18); DRA (7)]. There is mounting evidence that the expression and activity of SLC26 family can be regulated by CFTR, although the underlying mechanisms are unknown (5, 14, 16, 17).
The cellular signaling system that links SP receptor occupation to the apical anion exchanger remains to be determined. The undecapeptide binds to tachykinin receptors, which are seven transmembrane span receptors coupled to the Gq/G11 family of G proteins (13). Activation of tachykinin receptors leads to activation of phospholipase C, followed by the production of inositol 1,4,5-trisphosphate and diacylglycerol, which in turn lead to a rise in intracellular Ca2+ concentration and activation of protein kinase C (PKC), respectively (13). Previously, it has been reported that activation of PKC by phorbol 12,13-dibutyrate can inhibit fluid secretion by rat pancreatic ducts (6), and it is possible that SP exerts its effect on apical anion exchange via PKC-mediated phosphorylation.
In conclusion, we have shown that SP inhibits basal and secretin-stimulated HCO3- efflux from the pancreatic duct cell. Inhibition occurs by modulation of a Cl--dependent HCO3- transport process, most probably the SLC26 anion exchanger on the apical membrane of the duct cell. This will need to be confirmed in microperfusion experiments, which would allow the activity of the apical anion exchanger to be studied directly. It will also be necessary to establish the intracellular signaling system utilized by SP and whether functional CFTR is required for the action of SP.
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DISCLOSURES |
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Present address of P. Hegyi: First Department of Medicine, Albert Szent-Gyorgyi Medical University, PO Box 469, H-6701, Szeged, Hungary.
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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. Section 1734 solely to indicate this fact.
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