Martin C. Steward

Molecular and Functional Identification of a Ca2+ (Polyvalent Cation)-sensing Receptor in Rat Pancreas

The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3 −secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3 − secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.

Distribution of aquaporin water channels AQP1 and AQP5 in the ductal system of the human pancreas

Background: The exocrine pancreas secretes large volumes of isotonic fluid, most of which originates from the ductal system. The role of aquaporin (AQP) water channels in this process is unknown. Methods: Expression and localisation of known AQP isoforms was examined in normal human pancreas, pancreatic adenocarcinoma, and pancreatic cell lines of ductal origin (Capan-1, Capan-2, and HPAF) using reverse transcriptase-polymerase chain reaction and immunohistochemistry. Results: Messenger RNAs for AQP1, -3, -4, -5, and -8 were detected in normal pancreas and in pancreatic adenocarcinoma. The cell lines expressed AQP3, -4, and -5 but lacked AQP1 and AQP8. Immunohistochemistry of normal pancreas revealed that AQP1 is strongly expressed in centroacinar cells and in both the apical and basolateral domains of intercalated and intralobular duct epithelia. AQP1 expression declined with distance along the small interlobular ducts and was not detectable in larger interlobular ducts. AQP3 and AQP4 were not detectable by immunohistochemistry. AQP5 was observed at the apical membrane of intercalated duct cells and also in duct associated mucoid glands. AQP8 was confined to the apical pole of acinar cells. Both AQP1 and AQP5 were colocalised with cystic fibrosis transmembrane conductance regulator (CFTR) at the apical membrane of intercalated duct cells. Conclusions: AQP1 and AQP5 are strongly expressed in the intercalated ducts of the human pancreas. Their distribution correlates closely with that of CFTR, a marker of ductal electrolyte secretion. This suggests that fluid secretion is concentrated in the terminal branches of the ductal tree and that both AQP1 and AQP5 may play a significant role.

Fluid secretion in interlobular ducts isolated from guinea‐pig pancreas

1 Pancreatic HCO3− and fluid secretion were studied by monitoring luminal pH (pHL) and luminal volume simultaneously in interlobular duct segments isolated from guinea‐pig pancreas. The secretory rate and HCO3− flux were estimated from fluorescence images obtained following microinjection of BCECF‐dextran (70 kDa, 20 μM) into the duct lumen. 2 Ducts filled initially with a Cl−‐rich solution swelled steadily (2.0 nl min−1 mm−2) when HCO3−/CO2 was introduced, and the luminal pH increased to 8.08. When Cl− was replaced by glucuronate, spontaneous fluid secretion was reduced by 75 %, and pHL did not rise above 7.3. 3 Cl−‐dependent spontaneous secretion was largely blocked by luminal H2DIDS (500 μM). We conclude that, in unstimulated ducts, HCO3− transport across the luminal membrane is probably mediated by Cl−‐HCO3− exchange. 4 Secretin (10 nM) and forskolin (1 μM) both stimulated HCO3− and fluid secretion. The final value of pHL (8.4) and the increase in secretory rate (1.5 nl min−1 mm−2) after secretin stimulation were unaffected by substitution of Cl−. 5 The Cl−‐independent component of secretin‐evoked secretion was not affected by luminal H2DIDS. This suggests that a Cl−‐independent mechanism provides the main pathway for luminal HCO3− transport in secretin‐stimulated ducts. 6 Ducts filled initially with a HCO3−‐rich fluid (125 mM HCO3−, 23 mM Cl−) secreted a Cl−‐rich fluid while unstimulated. This became HCO3−‐rich when secretin was applied. 7 Addition of H2DIDS and MIA (10 μM) to the bath reduced the secretory rate by 56 and 18 %, respectively. Applied together they completely blocked fluid secretion. We conclude that basolateral HCO3− transport is mediated mainly by Na+‐HCO3− cotransport rather than by Na+‐H+ exchange.

CFTR Functions as a Bicarbonate Channel in Pancreatic Duct Cells

Pancreatic duct epithelium secretes a HCO3−-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO3− permeability of CFTR provides a pathway for apical HCO3− efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO3− induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pHi) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K+ concentration. Apical HCO3− fluxes activated by cyclic AMP were independent of Cl− and luminal Na+, and substantially inhibited by the CFTR blocker, CFTRinh-172. Furthermore, comparable HCO3− fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (ΔF) mice. To estimate the HCO3− permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO3− and 24 mM Cl− in the presence of 5% CO2. From the changes in pHi, membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO3− across the apical membrane were determined and used to calculate the HCO3− permeability. Our estimate of ∼0.1 µm sec−1 for the apical HCO3− permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO3− secretion. This suggests that CFTR functions as a HCO3− channel in pancreatic duct cells, and that it provides a significant pathway for HCO3− transport across the apical membrane.

Luminal ATP stimulates fluid and HCO3− secretion in guinea‐pig pancreatic duct

1 The location of purinoceptors in the pancreatic duct and their role in regulating ductal secretion have been investigated by applying ATP and UTP to basolateral and luminal surfaces of pancreatic ducts isolated from the guinea‐pig pancreas. 2 Changes in intracellular Ca2+ concentration were measured by microfluorometry in microperfused interlobular duct segments. Fluid and HCO3− secretion were estimated by monitoring luminal pH and luminal volume in sealed duct segments microinjected with BCECF‐dextran. 3 Both ATP and UTP (1 μm) caused biphasic increases in intracellular Ca2+ concentration in pancreatic duct cells when applied to either the basolateral or luminal membrane. 4 Luminal application of both ATP and UTP evoked fluid and HCO3− secretion. The maximum response to 1 μm ATP or UTP was about 75 % of that evoked by secretin. By contrast, basolateral application of ATP or UTP inhibited spontaneous secretion by 52 % and 73 %, respectively, and secretin‐evoked secretion by 41 % and 38 %, respectively. 5 The data suggest that luminal nucleotides may act in an autocrine or paracrine fashion to enhance ductal secretion while basolateral nucleotides, perhaps released from nerve terminals, may have an inhibitory effect. The fact that both apical and basolateral purinoceptors elevate intracellular Ca2+, but that they have opposite effects on secretion, suggests that additional signalling pathways are involved.

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO3 − across their luminal membrane into a HCO3 −-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO3 − transport cannot be achieved by luminal Cl−/HCO3 − exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO3 − efflux, we have measured the intracellular potential (Vm) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl−-25 mM HCO3 − solution, a condition similar to the basal state, the resting potential was approximately −60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (∼5 mV) due to activation of electrogenic Na+-HCO3 − cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately −50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO3 − concentration to 125 mM caused a hyperpolarization (∼10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl− efflux across the luminal membrane is minimized by the depletion of intracellular Cl− and offset by the hyperpolarizing effects of Na+-HCO3 − cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO3 − that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO3 − to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO3 −.

CO2 permeability and bicarbonate transport in microperfused interlobular ducts isolated from guinea‐pig pancreas

1 Permeabilities of the luminal and basolateral membranes of pancreatic duct cells to CO2 and HCO3− were examined in interlobular duct segments isolated from guinea‐pig pancreas. Intracellular pH (pHi) was measured by microfluorometry in unstimulated, microperfused ducts loaded with the pH‐sensitive fluoroprobe 2′7′‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). 2 When HCO3−/CO2 was admitted to the bath, pHi decreased transiently as a result of CO2 diffusion and then increased to a higher value as a result of HCO3− uptake across the basolateral membrane by Na+‐HCO3− cotransport. 3 When HCO3−/CO2 was admitted to the lumen, pHi again decreased but no subsequent increase was observed, indicating that the luminal membrane was permeable to CO2 but did not allow HCO3− entry to the cells from the lumen. Only when the luminal HCO3− concentration was raised above 125 mm was HCO3− entry detected. The same was true of duct cells stimulated with forskolin. 4 Recovery of pHi from an acid load, induced by exposure to an NH4+ pulse, was dependent on basolateral but not luminal Na+ and could be blocked by basolateral application of methylisobutylamiloride and H2DIDS. This indicates that the Na+‐H+ exchangers and Na+‐HCO3− cotransporters are located exclusively at the basolateral membrane. 5 In the presence of HCO3−/CO2, substitution of basolateral Cl− with glucuronate caused larger increases in pHi than substitution of luminal Cl−. This suggests that the anion exchanger activity in the basolateral membrane is greater than that in the luminal membrane. 6 We conclude that the luminal and basolateral membranes are both freely permeable to CO2, but while the basolateral membrane has both uptake and efflux pathways for HCO3−, the luminal membrane presents a significant barrier to the re‐entry of secreted HCO3−, largely through the inhibition of the luminal anion exchanger by high luminal HCO3− concentrations.

Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea‐pig pancreatic ducts

1 HCO3− secretion was investigated in interlobular duct segments isolated from guinea‐pig pancreas using a semi‐quantitative fluorometric method. Secretagogue‐induced decreases in intracellular pH, following blockade of basolateral HCO3− uptake with a combination of amiloride and DIDS, were measured using the pH‐sensitive fluoroprobe BCECF. Apparent secretory HCO3− fluxes were calculated from the initial rate of intracellular acidification. 2 In the presence of HCO3−, stimulation with secretin (10 nm) or forskolin (5 μm) more than doubled the rate of intracellular acidification. This effect was abolished in the absence of HCO3−. It was also abolished in the presence of HCO3− when DIDS and NPPB were applied to the luminal membrane by microperfusion. We therefore conclude that the increase in acidification rate is a useful index of secretagogue‐induced HCO3− secretion across the luminal membrane. 3 Secretin, cholecystokinin (CCK) and bombesin each stimulated HCO3− secretion in a dose‐dependent fashion. They evoked comparable maximal responses at about 10 nm and the EC50 values were 0.5 nm for secretin, 0.2 nm for CCK and 30 pm for bombesin. Acetylcholine (ACh) was also effective, with a maximum effect at 10 μm. 4 The stimulatory effect of CCK was blocked completely by the CCK1 receptor antagonist devazepide but not by the CCK2 receptor antagonist L365,260. The CCK analogue JMV‐180 (Boc‐Tyr(SO3H)‐Nle‐Gly‐Trp‐Nle‐Asp‐phenylethyl ester), which is an agonist of the high‐affinity CCK1 receptor but an antagonist of the low‐affinity receptor, also stimulated HCO3− secretion but with a smaller maximal effect than CCK. JMV‐180 partially inhibited the response to a high concentration of CCK but not to a lower concentration, suggesting that both high‐ and low‐affinity states of the CCK1 receptor evoke HCO3− secretion. 5 The stimulatory effect of bombesin was blocked completely by the gastrin‐releasing peptide (GRP) receptor antagonist d‐Phe6‐bombesin(6‐13)‐methyl ester (BME) but not by the neuromedin B (NMB) receptor antagonist d−Nal−cyclo[Cys−Tyr−d−Trp−Orn−Val−Cys]−Nal−NH2 (BIM−23127). 6 Secretagogue‐evoked fluid secretion was also examined using video microscopy to measure the rate of swelling of ducts whose ends had sealed during overnight culture. Secretin, CCK, bombesin and ACh all evoked fluid secretion with maximal rates of approximately 0.6 nl min−1 mm−2, and with concentration dependences similar to those obtained for HCO3− secretion. 7 We conclude that CCK, bombesin and ACh stimulate the secretion of a HCO3−‐rich fluid by direct actions on the interlobular ducts of the guinea‐pig pancreas and that these responses are mediated by CCK1 receptors, GRP receptors and muscarinic cholinoceptors, respectively.

Basolateral anion transport mechanisms underlying fluid secretion by mouse, rat and guinea-pig pancreatic ducts.

Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO3− requirement for secretin‐evoked secretion in mouse, rat and guinea‐pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nm secretin and 5 μm forskolin in the absence of extracellular HCO3−. In guinea‐pig ducts, however, fluid secretion was totally dependent on HCO3−. Forskolin‐stimulated fluid secretion by mouse and rat ducts in the absence of HCO3− was dependent on extracellular Cl− and was completely inhibited by bumetanide (30 μm). It was therefore probably mediated by a basolateral Na+–K+–2Cl− cotransporter. In the presence of HCO3−, forskolin‐stimulated fluid secretion was reduced ∼40% by bumetanide, ∼50% by inhibitors of basolateral HCO3− uptake (3 μm EIPA and 500 μm H2DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin‐evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na+–H+ exchange and Na+–HCO3− cotransport mediating HCO3− uptake, and Na+–K+–2Cl− cotransport mediating Cl− uptake. The absence or inactivity of the Cl− uptake pathway in the guinea‐pig pancreatic ducts may help to account for the much higher concentrations of HCO3− secreted in this species.

Chloride transport in microperfused interlobular ducts isolated from guinea‐pig pancreas

Isolated interlobular ducts from the guinea‐pig pancreas secrete a HCO3−‐rich fluid in response to secretin. To determine the role of Cl− transporters in this process, intracellular Cl− concentration ([Cl−]i) was measured in ducts loaded with the Cl−‐sensitive fluoroprobe, 6‐methoxy‐N‐ethylquinolinium chloride (MEQ). [Cl−]i decreased when the luminal Cl− concentration was reduced. This effect was stimulated by forskolin, was not dependent on HCO3− and was not inhibited by application of the anion channel/transporter inhibitor H2DIDS to the luminal membrane. It is therefore attributed to a cAMP‐stimulated Cl− conductance, probably the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. [Cl−]i also decreased when the basolateral Cl− concentration was reduced. This effect was not stimulated by forskolin, was largely dependent on HCO3− and was inhibited by basolateral H2DIDS. It is therefore mediated mainly by Cl−/HCO3− exchange. With high Cl− and low HCO3− concentrations in the lumen, steady‐state [Cl−]i was 25‐35 mm in unstimulated cells. Stimulation with forskolin caused [Cl−]i to increase by approximately 4 mm due to activation of the luminal anion exchanger. With low Cl− and high HCO3− concentrations in the lumen to simulate physiological conditions, steady‐state [Cl−]i was 10–15 mm in unstimulated cells. Upon stimulation with forskolin, [Cl−]i fell to approximately 7 mm due to increased Cl− efflux via the luminal conductance. We conclude that, during stimulation under physiological conditions, [Cl−]i decreases to very low levels in guinea‐pig pancreatic duct cells, largely as a result of the limited capacity of the basolateral transporters for Cl− uptake. The resulting lack of competition from intracellular Cl− may therefore favour HCO3− secretion via anion conductances in the luminal membrane, possibly CFTR.