In many species the pancreatic duct epithelium secretes HCO3- ions at a concentration of around 140 mM by a mechanism that is only partially understood. We know that HCO3- uptake at the basolateral membrane is achieved by Na+-HCO3- cotransport and also by a H+-ATPase and Na+/H+ exchanger operating together with carbonic anhydrase. At the apical membrane, the secretion of moderate concentrations of HCO3- can be explained by the parallel activity of a Cl-/HCO3- exchanger and a Cl- conductance, either the cystic fibrosis transmembrane conductance regulator (CFTR) or a Ca2+-activated Cl- channel (CaCC). However, the sustained secretion of HCO3- into a HCO- -rich luminal fluid cannot be explained by conventional Cl-/HCO3- exchange. HCO3- efflux across the apical membrane is an electrogenic process that is facilitated by the depletion of intracellular Cl-, but it remains to be seen whether it is mediated predominantly by CFTR or by an electrogenic SLC26 anion exchanger.

1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]1) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI).2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4C1 pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pHi was Na+ dependent, abolished by 0-2 mm amiloride and by 10 am N-methyl-N-isobutylamiloride and was therefore attributed to Na+-H+ exchange.3. In the presence of HC03-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0 5 mm H2DIDS and unaffected by depletion of intracellular Cl-and was therefore attributed to Na+-HCO03 cotransport.4. Stimulation with 10 nm secretin did not cause a significant change in pHi despite a significant increase in HC03-efflux. However, in the presence of secretin, addition of 0 5 mm H2DIDS caused a decline in pHi that was three times more rapid than that obtained with 0-2 mm amiloride.5. In secretin-stimulated ducts, Na+ uptake increased when HC03-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mm H2DIDS. 6. We conclude that Na+-HCO -cotransport contributes approximately 75% of the HC03-taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HC03-efflux at the luminal membrane and electrogenic

Aquaporin (AQP) water channels are expressed in a variety of fluid-transporting epithelia and are likely to play a significant role in salivary secretion. Our aim was to identify and localize the aquaporins expressed in human salivary glands. Total RNA was extracted from human parotid, submandibular, sublingual, and labial glands and from human brain. Expression of aquaporin mRNA was assessed by RT-PCR using specific primers for human AQP1, AQP3, AQP4, and AQP5. All four aquaporins were detected by RT-PCR in all of the glands, and the sequences were confirmed after further amplification with nested primers. Cleaned PCR products were then used as (32)P-labeled cDNA probes in a semiquantitative Northern blot analysis using glyceraldehyde-3-phosphate dehydrogenase as reference. Only AQP1, AQP3, and AQP5 mRNAs were present at significant levels. AQP localization was determined by immunohistochemistry on paraffin sections using affinity-purified primary antibodies and peroxidase-linked secondary antibodies. Each salivary gland type showed a broadly similar staining pattern: AQP1 was localized to the capillary endothelium and myoepithelial cells; AQP3 was present in the basolateral membranes of both mucous and serous acinar cells; AQP4 was not detected; and AQP5 was expressed in the luminal and canalicular membranes of both types of acinar cell. We conclude that AQP3 and AQP5 together may provide a pathway for transcellular osmotic water flow in the formation of the primary saliva.

SUMMARY1. The role of extracellular and intracellular Ca2+ in pancreatic enzyme secretion has been assessed by correlating the exchange of 45Ca with amylase secretion in the isolated uncinate pancreas of baby rats.2. The rate coefficient of 45Ca efflux from pre-loaded glands declined continually (indicating that 45Ca is retained in several different pools) and probably reflects changes in the concentration of cytoplasmic free KCa, which is determined by the rate at which 45Ca is released from intracellular organelles into the cytoplasm.3. The rate coefficient of45Ca release was not influenced by extracellular Ca2+ or Mg&+ concentrations.4. Cholecystokinin-pancreozymin (CCK-PZ) and acetylcholine accelerated the release of both 45Ca and amylase in a dose-dependent fashion, even when extracellular Ca2+ was reduced to 0.1 mm, but did not affect the initial rate of KCa uptake by the tissue.5. In Ca2+-free media (containing 0.5 mM-EGTA) basal amylase secretion slowly declined and stimulated secretion was virtually abolished, but the accelerated release of 45Ca was maintained.6. These observations indicate that natural stimuli of pancreatic enzyme secretion alter 45Ca distribution in the cell by a process which is independent of extracellular Ca2+ and which is associated with amylase secretion provided that the plasma membrane has not been depleted of R. M. CASE AND T. CLAUSEN substitution also increased 45Ca uptake. Thus, under special conditions, secretion may be stimulated when increased amounts of Ca2+ are made available from extracellular sources.9. Hyperosmolarity (known to increase 45Ca release in muscle) also accelerated 45Ca release and amylase secretion.10. 2,4-Dinitrophenol markedly accelerated 45Ca efflux but did not stimulate amylase secretion, indicating that a rise in cytoplasmic Ca2+ will not initiate secretion if energy metabolism is impaired.11. CCK-PZ slightly increased the rate coefficient of 42K release, indicating a changed membrane permeability.12. The stimulatory effects of CCK-PZ and acetylcholine were suppressed during Na+-substitution by Li+, suggesting that the Na+ concentration gradient across the membrane is important in secretion.13. It is concluded that the primary action of CCK-PZ and acetylcholine may be to increase the influx of Na+ into the cell by changing membrane permeability. This in turn is responsible for the release of Ca2+ from intracellular stores (probably endoplasmic reticulum), leading to a rise in Ca2+ concentration close to the structures involved in enzyme secretion. Secretion then follows provided that ATP is available and the plasma membrane is not depleted of Ca2+.

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1. Pancreatic HCOצ 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 HCOצ flux were estimated from fluorescence images obtained following microinjection of BCECF-dextran (70 kDa, 20 ìÒ) into the duct lumen. 2. Ducts filled initially with a Cl¦-rich solution swelled steadily (2·0 nl min¢ mm¦Â) when HCOצÏCOµ 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 HµDIDS (500 ìÒ). We conclude that, in unstimulated ducts, HCOצ transport across the luminal membrane is probably mediated by Cl¦-HCOצ exchange. 4. Secretin (10 nÒ) and forskolin (1 ìÒ) both stimulated HCOצ and fluid secretion. The final value of pHL (8·4) and the increase in secretory rate (1·5 nl min¢ mm¦Â) after secretin stimulation were unaffected by substitution of Cl¦. 5. The Cl¦-independent component of secretin-evoked secretion was not affected by luminalHµDIDS. This suggests that a Cl¦-independent mechanism provides the main pathway for luminal HCOצ transport in secretin-stimulated ducts. 6. Ducts filled initially with a HCOצ-rich fluid (125 mÒ HCOצ, 23 mÒ Cl¦) secreted a Cl¦-rich fluid while unstimulated. This became HCOצ-rich when secretin was applied. 7. Addition of HµDIDS and MIA (10 ìÒ) to the bath reduced the secretory rate by 56 and 18%, respectively. Applied together they completely blocked fluid secretion. We conclude that basolateral HCOצ transport is mediated mainly by Na¤-HCOצ cotransport rather than by Na¤-H¤ exchange.7803

SUMMARY After an outline description of pancreatic structure and function, and a more detailed account of acinar cell morphology, this review traces the pathway of amino acids as they are taken up by the acinar cell, incorporated into digestive hydrolases, transported through the cell and finally discharged from the cell, and considers the mechanisms by which these steps are controlled. At all stages comparisons are made with other secretory cells. The use of radioautography and cell‐fractionation techniques in determining this pathway in the pancreas are described. The route and kinetics of the process in pancreas are compared with those in other cells. Amino‐acid entry is by an active mechanism. However the intracellular pool of accumulated amino acids may not be used directly in protein synthesis. Selection of amino acids for incorporation into proteins may occur whilst they are associated with carrier systems within the plasma membrane. There is no convincing evidence that amino‐acid entry can be influenced by the pancreatic secretagogues, cholecystokin‐pancreazymin (CCK‐PZ) or acetylcholine. Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and the nascent proteins vectorially transferred across the ER membrane into the ER cisternae. All messenger RNA molecules which are templates for secretory proteins appear to possess an initial sequence of codons whose translation produces a ‘signal’ sequence of amino acids. This signal sequence somehow triggers attachment of the ribosomes to the ER, thereby automatically determining that the final translation product is destined for the ER cisternae. The effects of CCK‐PZ and acetylcholine on pancreatic protein synthesis are controversial. Whereas stimulation can be observed in vivo, this has not been convincingly demonstrated in vitro. I conclude that while CCK‐PZ and acetylcholine may accelerate protein synthesis, the physiological significance of this effect remains to be clarified. Long‐term stimulation can modify pancreatic enzyme synthesis and this, together with other factors, may be the means of dietary adaptation by the gland. Newly synthesized proteins travel from the ER cisternae via the peripheral Golgi components to the Golgi cisternae. Transport from ER to Golgi cisternae may occur by a vesicle shuttle service or by direct tubular connexions. Although sustained stimulation with CCK‐PZ analogues can accelerate this intracellular transport step, pancreatic secretagogues have not yet been shown to accelerate transport under physiological conditions. The Golgi complex has a number of functions including: glycosylation and, where appropriate, sulphation of glycoprotein and mucopolysaccharide components of the zymogen granules (ZG) and granule membranes; sequestration of divalent cations which bind to secretory proteins; the formation of condensing vacuoles (CV) from the inner Golgi cisternae. Aggregation of proteins occurs passively within CV so as to form osmotically inert complexes, thereby reducing internal osmotic activity and causing water to diffuse out. This condensation imparts a gel‐like consistency to the mature ZG so formed. Discharge of ZG occurs by a process of exocytosis involving fusion of the ZG membrane with the apical plasma membrane, release of the ZG contents, and retrieval of the ZG membrane from the plasma membrane by endocytotic mechanisms. The mechanisms responsible for migration of ZG towards the cell apex and for exocytosis remain unknown but may involve the participation of microtubules and/or microfilaments. Although there is a small, basal discharge of ZG at all times, stimulation with CCK‐PZ or acetylcholine greatly accelerates the process. The basic tenet of the secretory mechanism summarized above is that, following synthesis, secretory proteins are confined within an intracellular organelle at all times. This ‘segregation’ hypothesis has been challenged by the ‘equilibrium’ hypothesis in which secretory proteins are suggested to move across cellular membranes and are therefore at equilibrium within the various compartments of the cell. While many of the observations on which the equilibrium hypothesis are based are tenuous, some others cannot readily be explained by the segregation model. Proponents of the equilibrium hypothesis therefore suggest that preferential release of individual hydrolases from ZG occurs, followed by their separate transport across the apical cell membrane. The claims of this alternative model are discussed. In the final section are discussed the intracellular mechanisms by which CCK‐PZ and acetylcholine act on the acinar cell to cause discharge. The overall membrane perturbations brought about by CCK‐PZ and acetylcholine appear to be the same and include cell depolarization, and perhaps increased phospholipid turnover. Both events may be related to an altered membrane permeability to cations. CCK‐PZ, but not acetylcholine, will activate adenylate cyclase, but cyclic AMP does not appear to be involved in regulating enzyme discharge. Instead, Ca2+ is the major intracellular second messenger. However, rather than increase Ca2+ uptake into the cell, CCK‐PZ and acetylcholine appear to raise the intracellular Ca2+ concentration by causing release of Ca2+ from intracellular stores. The mechanism by which they do this, and the role of Ca2+ in the discharge process remain unknown.

Manchester M13 9PT, UK 1. The transport of HC03-across the luminal membrane of pancreatic duct cells was studied by monitoring the luminal pH of isolated guinea-pig interlobular ducts after microinjection of an extracellular fluoroprobe, the dextran conjugate of 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-dextran). Luminal Cl-concentration was also measured by microfluorometry following microinjection of the dextran conjugates of 6-methoxy-N-(4-aminoalkyl)quinolinium bromide (ABQ-dextran) and Cl-NERF (Cl-NERF-dextran).2. When HC03-/C02 was admitted to the bath, a transient acidification of the duct lumen was observed, followed by a marked alkalinization. The latter was abolished when the luminal CF-concentration was reduced to 25-35 mm by replacement with glucuronate and may, therefore, be attributed to Cl--HC03-exchange at the luminal membrane.3. Secretin, forskolin and acetylcholine stimulated HC03-secretion into the lumen even when the luminal Cl-concentration was reduced to approximately 7 mm. Furthermore, agonistevoked HC03-secretion was not inhibited by luminal glibenclamide, dihydro-4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (H2DIDS) or 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). These observations are not easily reconciled with HC03-transport across the luminal membrane being mediated by Cl--HC03-exchange in parallel with a Clconductance.4. Agonist-stimulated HC03-secretion was blocked by omitting Nae from the bath but not by addition of N-methyl-N-isobutylamiloride (MIA) or bafilomycin A1. This supports our previous conclusion that HC03-entry into duct cells from the extracellular fluid requires Nae but is not dependent on Na+-H+ exchange or vacuolar-type H+-ATPase activity. 5. The three actions of secretin on guinea-pig pancreatic duct cells described in this and the accompanying paper -stimulation of a relatively Cl--insensitive luminal HC03-efflux pathway, stimulation of basolateral Nae-HC03-cotransport, and lack of effect on intracellular pH -require the current model of pancreatic HC03-secretion to be modified.