R. Maynard Case

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.

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.

Duodenal enteroendocrine I-cells contain mRNA transcripts encoding key endocannabinoid and fatty acid receptors.

Enteroendocrine cells have a critical role in regulation of appetite and energy balance. I-cells are a subtype of enteroendocrine cells localized in duodenum that release cholecystokinin in response to ingested fat and amino-acids. Despite their potentially pivotal role in nutrient sensing and feeding behaviour, native I-cells have previously been difficult to isolate and study. Here we describe a robust protocol for the isolation and characterization of native duodenal I-cells and additionally, using semi-quantitative RT-PCR we determined that mouse duodenal I-cells contain mRNA transcripts encoding key fatty acid and endocannabinoid receptors including the long chain fatty acid receptors GPR40/FFAR1, GPR120/O3FAR1; short chain fatty acid receptors GPR41/FFAR3 and GPR43/FFAR2; the oleoylethanolamide receptor GPR119 and the classic endocannabinoid receptor CB1. These data suggest that I-cells sense a wide range of gut lumen nutrients and also have the capacity to respond to signals of fatty-acid derivatives or endocannabinoid peptides.

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.

Cell Physiology of Pancreatic Ducts

Pancreatic ducts are unique in that they can secrete a juice containing near isotonic sodium bicarbonate. This alkaline fluid washes digestives enzymes (secreted by acinar cells) down the ductal tree into the duodenum and also helps neutralize acid chyme that enters the duodenum from the stomach. The key importance of ductal secretion to pancreatic function is illustrated by the inherited disease cystic fibrosis where failure of the ducts to secrete bicarbonate and fluid leads to destruction of the gland. This chapter discusses the patterns of pancreatic electrolyte secretion, the structural basis of secretion, recent advances in studying duct cell physiology, the mechanisms of ductal bicarbonate secretion, and its regulation under physiological and pathological conditions. How recent advances in understanding the duct cell may contribute to the treatment of pancreatic diseases such as cystic fibrosis and acute pancreatitis in the future is also considered.

Calcium concentration and amylase secretion in guinea pig pancreatic acini: Interactions between carbachol, cholecystokinin octapeptide and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on amylase secretion and cytoplasmic free calcium concentration ([Ca2+]i) were investigated in dispersed guinea pig pancreatic acini. Carbachol evoked dose-dependent increases in amylase secretion and [Ca2+]i with half-maximal responses at 2.5 and 5 microM, respectively. Carbachol-induced calcium transients could be blocked by atropine. In the presence of a maximal effective dose of carbachol, cholecystokinin octapeptide caused no further increase in [Ca2+]i, suggesting that both agonists act on the same pool of trigger calcium. TPA (10(-9)-10(-6) M) stimulated amylase secretion with no change in [Ca2+]i. Maximum amylase secretion occurred at 0.5 microM TPA. Preincubation of acini in the presence of TPA resulted in a time- and dose-dependent inhibition (IC50 = 30 nM) of the carbachol-induced rise in [Ca2+]i, the maximal effect being observed within 3 min. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate was ineffective in inhibiting the carbachol-stimulated rise in [Ca2+]i. These findings suggest that, in addition to stimulating amylase secretion, probably through protein kinase C, TPA may also exert a negative feedback control over secretagogue-induced calcium transients.

Vectorial bicarbonate transport by Capan-1 cells: a model for human pancreatic ductal secretion

Human pancreatic ducts secrete a bicarbonate-rich fluid but our knowledge of the secretory process is based mainly on studies of animal models. Our aim was to determine whether the HCO₃^- transport mechanisms in a human ductal cell line are similar to those previously identified in guinea-pig pancreatic ducts. Intracellular pH was measured by microfluorometry in Capan-1 cell monolayers grown on permeable filters and loaded with BCECF. Epithelial polarization was assessed by immunolocalization of occludin. Expression of mRNA for key electrolyte transporters and receptors was evaluated by RT-PCR. Capan-1 cells grown on permeable supports formed confluent, polarized monolayers with well developed tight junctions. The recovery of pH_i from an acid load, induced by a short NH₄⁺ pulse, was mediated by Na⁺-dependent transporters located exclusively at the basolateral membrane. One was independent of HCO₃^- and blocked by EIPA (probably NHE1) while the other was HCO₃^--dependent and blocked by H₂DIDS (probably pNBC1). Changes in pH_i following blockade of basolateral HCO₃^- accumulation confirmed that the cells achieve vectorial HCO₃^- secretion. Dose-dependent increases in HCO₃^- secretion were observed in response to stimulation of both secretin and VPAC receptors. ATP and UTP applied to the apical membrane stimulated HCO₃^- secretion but were inhibitory when applied to the basolateral membrane. HCO₃^- secretion in guinea-pig ducts and Capan-1 cell monolayers share many common features, suggesting that the latter is an excellent model for studies of human pancreatic HCO₃^- secretion.

Is the rat pancreas an appropriate model of the human pancreas

During my lifetime in pancreatic research, rat and mouse have largely replaced dog and cat in experimental studies. However, as this review clearly demonstrates, the anatomy, physiology and molecular cell biology of the rat pancreas (and also probably the mouse pancreas) differ substantially from those in humans. Indeed, they differ more in rat/mouse than any other common laboratory species. These differences may be irrelevant if one is using the pancreas as a generic model in which to study, say, acinar cell exocytosis or signalling. But if one is interested in more specific aspects of human pancreatic function, especially ductal function, in health and disease, in my opinion the simple answer to the question posed by the title of this article is no: other species are more appropriate.

Effects of cholera toxin on cyclic adenosine 3′,5′-monophosphate concentration and secretory processes in the exocrine pancreas

1. The effect of purified cholera toxin on secretory processes of exocrine pancreas has been studied in the isolated, saline-perfused cat pancreas and in incubated pieces of rat pancreas. 2. The toxin evoked a biphasic secretory response from the perfused cat pancreas. An initial small phase, which began within minutes of toxin application, was an artefact due to the presence of NaN3 in the cholera toxin preparation as supplied; it could be entirely reproduced by NaN3 at the concentration expected during toxin stimulation. A second, sustained phase of secretion, due to the action of the toxin proper, began within 30-60 min, increasing in magnitude for many hours and persisting in the absence of toxin. It was accompanied by a parellel rise in tissue cyclic AMP concentration, and could be potentiated by theophylline. 3. The composition of the secretion stimulated by cholera toxin resembled that evoked by secretin; e.g. it contained a high concentration of bicarbonate and only basal amounts of digestive enzymes. 4. Similarly, cholera toxin did not stimulate enzyme secretion by incubated rat pancreas, despite large rises in tissue cyclic AMP concentration. 5. Because cholera toxin has thus far been shown to have no other effect than that of stimulating adenylate cyclase, these observations support the conclusion that cyclic AMP does mediate the electrolyte secretory response of the pancreas to secretin, but offers no evidence that cyclic AMP plays a similar role in the regulation of pancreatic enzyme secretion stimulated by cholecystokinin-pancreozymin or acetylcholine.