Organic osmolytes increase expression of specific tight junction proteins in skin and alter barrier function in keratinocytes.

BACKGROUND: The epidermal barrier is important for water conservation, failure of which is evident in dry-skin conditions. Barrier function is fulfilled by the stratum corneum, tight junctions (TJs, which control extracellular water) and keratinocyte mechanisms, such as organic osmolyte transport, which regulate intracellular water homeostasis. Organic osmolyte transport by keratinocytes is largely unexplored and nothing is known regarding how cellular and extracellular mechanisms of water conservation may interact. OBJECTIVES: We aimed to characterize osmolyte transporters in skin and keratinocytes, and, using transporter inhibitors, to investigate whether osmolytes can modify TJs. Such modification would suggest a possible link between intracellular and extracellular mechanisms of water regulation in skin. METHODS: Immunostaining and quantitative polymerase chain reaction of organic osmolyte-treated organ-cultured skin were used to identify changes to organic osmolyte transporters, and TJ protein and gene expression. TJ functional assays were performed on organic osmolyte-treated primary human keratinocytes in culture. RESULTS: Immunostaining demonstrated the expression of transporters for betaine, taurine and myo-inositol in transporter-specific patterns. Treatment of human skin with either betaine or taurine increased the expression of claudin-1, claudin-4 and occludin. Osmolyte transporter inhibition abolished this response. Betaine and taurine increased TJ function in primary human keratinocytes in vitro. CONCLUSIONS: Treatment of skin with organic osmolytes modulates TJ structure and function, which could contribute to the epidermal barrier. This emphasizes a role for organic osmolytes beyond the maintenance of intracellular osmolarity. This could be harnessed to enhance topical therapies for diseases characterized by skin barrier dysfunction.

Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model.

Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized, presumably by HCO3 (-)ions transported from ameloblasts into the developing enamel matrix. Ameloblasts express a number of transporters and channels known to be involved in HCO3 (-)transport in other epithelia. However, to date, there is no functional evidence for HCO3 (-)transport in these cells. To address questions related to HCO3 (-)export from ameloblasts, we have developed a polarized 2-dimensional culture system for HAT-7 cells, a rat cell line of ameloblast origin. HAT-7 cells were seeded onto Transwell permeable filters. Transepithelial resistance was measured as a function of time, and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. Intracellular pH regulation and HCO3 (-)transport were assessed by microfluorometry. HAT-7 cells formed epithelial layers with measureable transepithelial resistance on Transwell permeable supports and expressed claudin-1, claudin-4, and claudin-8-key proteins for tight junction formation. Transport proteins previously described in maturation ameloblasts were also present in HAT-7 cells. Microfluorometry showed that the HAT-7 cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 (-)uptake, which was sensitive to Na(+)withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 (-)transport showed a marked increase in response to Ca(2+)- and cAMP-mobilizing stimuli. Collectively, 2-dimensional HAT-7 cell cultures on permeable supports 1) form tight junctions, 2) express typical tight junction proteins and electrolyte transporters, 3) are functionally polarized, and 4) can accumulate HCO3 (-)ions from the basolateral side and secrete them at the apical membrane. These studies provide evidence for a regulated, vectorial, basolateral-to-apical bicarbonate transport in polarized HAT-7 cells. We therefore propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts.

Vectorial bicarbonate transport by Par-C10 salivary cells.

Salivary glands produce a HCO(3)(-)-rich fluid that is important for the neutral milieu in the upper gastrointestinal tract. The molecular mechanism of this secretion is poorly understood. Par-C10, an immortalized rat parotid acinar line, has been shown to secrete Cl(-)- in response to Ca(2+-)-mobilizing stimuli. Our aim was to assess the capacity of polarized monolayers of Par-C10 cells to transport and secrete HCO(3)(-)-. Transepithelial electrolyte movement was evaluated by short-circuit current measurements. Intracellular pH (pH(i)) was measured by microfluorometry in cells loaded with BCECF. Monolayers of Par-C10 cells, grown on Transwell membranes, developed high transepithelial resistance and exhibited vectorial anion secretion which was activated by both ATP and forskolin. The currents were partially inhibited by bumetanide and by withdrawal of HCO(3)(-) indicating the dependence of ion movements on NKCC and on HCO(3)(-) ions, respectively. In HCO(3)(-)-free solutions the recovery of pH(i) from acid loading was abolished by EIPA. In the presence of HCO(3)(-) there was a strong EIPA-insensitive recovery from acid loading which was inhibited by H(2)DIDS. ATP and forskolin stimulated HCO(3)(-) efflux from the cells. Furthermore, HCl(-) withdrawal experiments showed the presence of DNDS-sensitive basolateral anion exchange. In conclusion Par-C10 cells achieve transepithelial transport that is sensitive to both intracellular Ca(2+)- and cAMP-dependent stimulation. We identified Na(+)/H(+) exchange, Na(+)-HCO(3)(-) cotransport and anion exchange at the basolateral side of the cells as being involved in intracellular pH regulation and vectorial HCO(3)(-) secretion. This cell line offers a good model for further studies to understand the molecular mechanisms of salivary HCO(3)(-) secretion.

The role of aquaporin water channels in fluid secretion by the exocrine pancreas.

The mammalian exocrine pancreas secretes a near-isosmotic fluid over a wide osmolarity range. The role of aquaporin (AQP) water channels in this process is now becoming clearer. AQP8 water channels, which were initially cloned from rat pancreas, are expressed at the apical membrane of pancreatic acinar cells and contribute to their osmotic permeability. However, the acinar cells secrete relatively little fluid and there is no obvious defect in pancreatic function in AQP8 knockout mice. Most of the fluid secreted by the pancreas is generated by ductal epithelial cells, which comprise only a small fraction of the gland mass. In the human pancreas, secretion occurs mainly in the intercalated ducts, where the epithelial cells express abundant AQP1 and AQP5 at the apical membrane and AQP1 alone at the basolateral membrane. In the rat and mouse, fluid secretion occurs mainly in the interlobular ducts where AQP1 and AQP5 are again co-localized at the apical membrane but appear to be expressed at relatively low levels. Nonetheless, the transepithelial osmotic permeability of rat interlobular ducts is sufficient to support near-isosmotic fluid secretion at observed rates. Furthermore, apical, but not basolateral, application of Hg(2+) significantly reduces the transepithelial osmotic permeability, suggesting that apical AQP1 and AQP5 may contribute significantly to fluid secretion. The apparently normal fluid output of the pancreas in AQP1 knockout mice may reflect the presence of AQP5 at the apical membrane.

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.

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

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) 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 HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) 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 HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 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+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

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.

Expression of a sodium bicarbonate cotransporter in human parotid salivary glands.

The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

The paracellular component of water flow in the rat submandibular salivary gland.

1. The pathway of water flow during salivary secretion by the isolated, perfused rat submandibular gland was examined using a family of homologous radiodextran molecules as probes of paracellular fluid transfer. 2. The secretion/perfusate ratio (S/P) of the secreted probes versus molecular radius during fluid secretion evoked by ACh could be resolved into two components: one that fitted a free-diffusion (Stokes-Einstein) curve and indicated diffusion through large channels, and a convective component that was linearly related to radius. 3. The convective component had a cut-off point at 0.5 nm (5 A) radius and an S/P intercept of near 1.0 at the radius of water, which indicates that most of the volume flow was paracellular. 4. The nature of such a paracellular flow is discussed together with the possible integration of this volume flow with the cellular transport of ions, resulting in an isotonic primary secretion from the gland.

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 microM) 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 microM. 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 x min(-1) x 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.

Identification and localization of aquaporin water channels in human salivary glands.

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.

Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas.

Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.

Pancreatic ductal bicarbonate secretion: past, present and future.

The pancreatic duct epithelium in the guinea-pig and many other species secretes HCO(3)(-) at concentrations approaching 150 mM. This cannot be explained by conventional models based upon HCO(3)(-) secretion via an anion exchanger at the luminal membrane because: 1) under these conditions, the Cl(-) and HCO(3)(-) concentration gradients would favour HCO(3)(-) reabsorption rather than secretion, and 2) the luminal anion exchanger appears to be inhibited by luminal HCO(3)(-) concentrations of 125 mM or more. There may, however, be a sufficiently large electrochemical gradient to drive HCO(3)(-) secretion across the luminal membrane via an anion conductance. In contrast to earlier studies on rat ducts, the membrane potential E(m) in guinea-pig duct cells does not depolarise appreciably upon stimulation with secretagogues but remains constant at about -60 mV. Consequently, even with 125 mM or more HCO(3)(-) in the lumen and an estimated 20 mM in the cytoplasm, the electrochemical gradient for HCO(3)(-) will still favour secretion to the lumen. Under the same conditions, the intracellular Cl(-) concentration drops to very low levels (approximately 7 mM) presumably because, although Cl(-) may leave freely through the cystic fibrosis transmembrane conductance regulator (CFTR) channels in the luminal membrane, there is no major pathway for Cl(-) uptake across the basolateral membrane. Consequently a HCO(3)(-)-rich secretion may arise as a result of the lack of competition from intracellular Cl(-) for efflux via the anion conductances at the luminal membrane. Whether CFTR, or another anion conductance, provides such a pathway for HCO(3)(-) remains to be seen.

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

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). 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. 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. 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. 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. 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.

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 microM) 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 microM 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.

Expression of aquaporin 1 (AQP1) water channels in human labial salivary glands.

Aquaporin (AQP) water channels are widely expressed in the membranes of fluid-transporting epithelia. Despite the fact that salivary glands are the site of considerable water movement, relatively little is known about the role of aquaporins in human salivary glands. We have examined the expression of AQP1 in human parotid, sublingual and labial salivary glands. Total RNA was extracted from glandular tissue obtained from surgery or biopsy. The presence of AQP1 mRNA was demonstrated in each of the three glands by RT-PCR using primers specifically designed for human AQP1. The PCR product from the labial gland RNA was further amplified with nested primers and the sequence confirmed by automated fluorescent DNA sequencing. The cleaned first PCR product from these glands was then used as a 32P-labelled hybridization probe in a Northern analysis which confirmed the presence of significant amounts of AQP1 transcript in all three glands. AQP1 expression was also demonstrated in cryosections of human labial glands by immunohistochemistry using peroxidase-linked antibodies. Antibody labelling was most prominent in the capillaries but was also evident in the basal regions of the labial gland acini, and may therefore be associated with the serous demilunes which are believed to be a significant site of fluid movement.

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.

Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells.

We used the Ca2+-sensitive fluorescent dye fura 2, together with measurements of intracellular D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to assess the inhibitory effects of caffeine on signal transduction via G protein-coupled receptor pathways in isolated rat mandibular salivary acinar cells. ACh, norepinephrine (NE), and substance P (SP) all evoked substantial increases in the intracellular free Ca2+ concentration ([Ca2+]i). Responses to ACh and NE were markedly inhibited by prior application of 20 mM caffeine. The inhibitory effect of caffeine was not reproduced by phosphodiesterase inhibition with IBMX or addition of cell-permeant dibutyryl cAMP. In contrast to the ACh and NE responses, the [Ca2+]i response to SP was unaffected by caffeine. Despite this, SP and ACh appeared to mobilize Ca2+ from a common intracellular pool. Measurements of agonist-induced changes in Ins(1,4,5)P3 levels confirmed that caffeine inhibited the stimulus-response coupling pathway at a point before Ins(1,4,5)P3 generation. Caffeine did not, however, inhibit [Ca2+]i responses evoked by direct activation of G proteins with 40 mM F-. These data show that caffeine inhibits G protein-coupled signal transduction in these cells at some element that is common to the muscarinic and alpha-adrenergic signaling pathways but is not shared by the SP signaling pathway. We suggest that this element might be a specific structural motif on the G protein-coupled muscarinic and alpha-adrenergic receptors.

Regulation of cell volume and diffusion of intracellular water in salivary acinar cells.

The regulation of acinar cell volume and the properties of intracellular water were investigated in perfused rat mandibular salivary glands by proton nuclear magnetic resonance (NMR) spectroscopy. Using an inversion-recovery pulse sequence, and an extracellular relaxation reagent (10 mM Gd-DTPA) to suppress the proton NMR signal from extracellular water, acinar cell volume (intracellular water content) in unstimulated glands was shown to depend upon Cl- uptake by basolateral Na+-K+-2Cl- cotransport. Muscarinic and beta-adrenoceptor stimulation induced shrinkage and swelling respectively. In pulsed-field-gradient NMR experiments, the diffusion coefficient of intracellular water was found to be more than an order of magnitude smaller than that of extracellular water. Using this intrinsic difference in diffusivity between the two compartments, cell volume regulation was investigated in intact, perfused glands in the absence of relaxation reagents. Using both NMR techniques, acinar cells in perfused glands were observed to behave like simple osmometers in response to anisosmotic media, and did not show the volume regulatory responses described in dissociated acinar cells.