Secretin dissipates red acridine orange fluorescence from pancreatic duct epithelium

This study was undertaken to elucidate whether duct cells in the pancreas contain acidic cytoplasmic compartments regulated by secretin. Microdissected pancreatic ducts from pigs were examined by acridine orange (AO) and 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein/tetraacetoxymethy l ester (BCECF/AM) epifluorescence microscopy. Estimated cytoplasmic pH using BCECF fluorescence was 7.43 +/- 0.04 and was not changed by altering CO2 tension in the incubation medium. The epithelium of acridine orange incubated peripheral interlobular pancreatic ducts exhibited green and red fluorescence; the colour depending on the experimental conditions. Red epithelial fluorescence was seen in resting pancreatic ducts and was greatly accentuated by raising CO2 in the incubation medium from 5.5 to 10 kPa. The red fluorescence was abolished by secretin, or following incubation with chloroquine or NH4Cl or the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or carbonyl cyanide m-chlorophenylhydrazone (CCCP), leaving uniform green fluorescence. These findings suggest that pancreatic duct cells contain CO2-dependent acidic compartments which vanish during secretin stimulation and which may be cytoplasmic tubulovesicles.     

Exocrine ductalpCO2 in the rabbit pancreas

An improvedpCO₂ microelectrode has been evaluated and used to investigate whether a significant barrier to diffusion of CO₂ exists in the rabbit pancreas. The results of this study show the improved Carter and CaflischpCO₂ microelectrode to be an accurate and reliable tool for measuring pancreatic venous and ductalpCO₂. The similarities betweenpCO₂ values from the pancreatic ducts and small pancreatic veins suggest that there is no barrier to CO₂ diffusion between small veins and exocrine ducts in the rabbit pancreas, and that ductalpCO₂ is probably strongly influenced by the CO₂ tension of the small pancreatic blood vessels.     

Effect of bicarbonate on potassium conductance of isolated perfused rat pancreatic ducts

The aim of this study was to investigate the role of the K⁺ conductance in unstimulated and stimulated pancreatic ducts and to see how it is affected by provision of exogenous HCO ₃ ^– /CO₂. For this purpose we have applied electrophysiological techniques to perfused pancreatic ducts, which were dissected from rat pancreas. The basolateral membrane potential PD_bl of unstimulated duct cells was between –60mV and –70mV, and the cells had a relatively large K⁺ conductance in the basolateral membrane as demonstrated by (a) 20–22 mV depolarization of PD_bl in response to increase in bath K⁺ concentration from 5 mmol/l to 20mmol/l and (b) the effect of a K⁺ channel blocker, Ba²⁺ (5 mmol/l), which depolarized PD_bl by 30–40mV. These effects on unstimulated ducts were relatively independent of bath HCO ₃ ^– /CO₂. The luminal membrane seemed to have no significant K⁺ conductance. Upon stimulation with secretin or dibutyryl cyclic AMP, PD_bl depolarized to about –35 mV in the presence of HCO ₃ ^– /CO₂. Notably, the K⁺ conductance in the stimulated ducts was now only apparent in the presence of exogenous HCO ₃ ^– /CO₂ in the bath solutions. Upon addition of Ba₂₊, PD_bl depolarized by 13±1 mV (n=7), the fractional resistance of the basolateral membrane, FR_bl increased from 0.66 to 0.78 (n=6), the specific transepithelial resistance, R _te, increased from 52±13 cm² to 59±15 cm² (n=11), and the whole-cell input resistance, R _c, measured with double-barrelled electrodes, increased from 20 M to 26 M (n=3). These results are consistent with Ba²⁺ inhibition of the K⁺ conductance. Following removal of exogenous HCO ₃ ^– /CO₂ in the same ducts, stimulation led to a larger depolarization on PD_bl to about –25 mV, and Ba²⁺ had a smaller effect on PD_bl and no significant effect on the resistances. The individual resistances in the duct epithelium were estimated from equivalent circuit analysis. The luminal membrane resistance, R ₁ decreased from about 2000 cm² to 80 cm² upon stimulation. The basolateral membrane resistance, R _bl, remained at 90–120 cm², and the paracellular shunt resistance, R _s, at 50–80 cm². Ba²⁺ increased R _bl of stimulated ducts to about 200 cm², an effect present only if the ducts were provided with exogenous HCO ₃ ^– /CO₂. Taken together, the present results indicate that the basolateral K⁺ conductance of pancreatic ducts is sensitive to exogenous HCO ₃ ^– /CO₂, i.e. without HCO ₃ ^– /CO₂ the conductance becomes very low although the ducts are undergoing stimulation.A preliminary report of the present study has been presented at the XXXI International Congress of Physiological Sciences, Helsiniki, Finland, July 1989     

Properties of the luminal membrane of isolated perfused rat pancreatic ducts

The aim of the present study was to investigate by what transport mechanism does HCO ₃ ^– cross the luminal membrane of pancreatic duct cells, and how do the cells respond to stimulation with dibytyryl cyclic AMP (db-cAMP). For this purpose a newly developed preparation of isolated and perfused intra-and interlobular ducts of rat pancreas was used. Responses of the epithelium to inhibitors and agonists were monitored by electrophysiological techniques. Addition of HCO ₃ ^– /CO₂ to the bath side of nonstimulated ducts depolarized the PD across the basolateral membrane (PD_bl) by about 9mV, as also observed in a previous study [21]. This HCO ₃ ^– effect was abolished by Cl^– channel blockers or SITS infused into the lumen of the duct: i. e. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 10^–5 M) hyperpolarized PD_bl by 8.2±1.6 mV (n=13); 3,5-dichlorodiphenylamine-2-carboxylic acid (DCl-DPC, 10^–5 M) hyperpolarized PD_bl by 10.3±1.7 mV (n=10); and SITS hyperpolarized PD_bl by 7.8±0.9 mV (n=4). Stimulation of the ducts with dbcAMP in the presence of bath HCO ₃ ^– /CO₂ resulted in depolarization of PD_bl, the ductal lumen became more negative and the fractional resistance of the luminal membrane decreased. Together with forskolin (10^–6 M), db-cAMP (10^–4 M) caused a fast depolarization of PD_bl by 33.8±2.5 mV (n=6). When db-cAMP (5×10^–4 M) was given alone in the presence of bath HCO ₃ ^– /CO₂, PD_bl depolarized by 25.3±4.2 mV (n=10). In the absence of exogenous HCO ₃ ^– , db-cAMP also depolarized PD_bl by 24.7±3.0 mV (n=10). The present data suggest that in the luminal membrane of pancreatic duct cells there is a Cl^– conductance in parallel with a Cl^–/HCO ₃ ^– antiport. Dibutyryl cyclic AMP increases the Cl^– conductance of the luminal membrane. Taking together our present results, and the recent data obtained for the basolateral membrane [21], a tentative model for pancreatic HCO ₃ ^– transport is proposed.Parts of this study have been presented at the Scandinavian Physiological Society Meeting in Copenhagen 1986 and 64th Meeting of the German Physiological Society in Homburg/Saar     

Secretagogue effects on intracellular calcium in pancreatic duct cells

Regulation of intracellular free calcium ([Ca²⁺]_i) in single epithelial duct cells of isolated rat and guinea pig pancreatic interlobular ducts by secretin, carbachol and cholecystokinin was studied by microspectrofluorometry using the Ca²⁺-sensitive, fluorescent probe Fura-2. Rat and guinea pig duct cells exhibited mean resting [Ca²⁺]_i of 84 nM and 61 nM, respectively, which increased by 50%–100% in response to carbachol stimulation, thus demonstrating the presence of physiologically responsive cholinergic receptors in pancreatic ducts of both species. The carbachol-induced increase in [Ca²⁺]_i involved both mobilization of Ca²⁺ from intracellular stores and stimulation of influx of extracellular Ca²⁺. In contrast, neither cholecystokinin nor secretin showed reproducible or sizeable increses in [Ca²⁺]_i. Both rat and guinea pig duct cells showed considerable resting Ca²⁺ permeability. Lowering or raising the extracellular [Ca²⁺]_i led, respectively, to a decrease or increase in the resting [Ca²⁺]_i. Application of Mn²⁺ resulted in a quenching of the fluorescence signal indicating its entry into the cell. The resting Ca²⁺ and Mn²⁺ permeability could be blocked by La³⁺ suggesting that it is mediated by a Ca²⁺ channel.     

Effect of vasoactive intestinal peptide,carbachol and other agonists on the membrane voltage of pancreatic duct cells

The regulation of pancreatic exocrine secretion involves hormonal, neural and neurohormonal components. Many agonists are known to be effective in pancreatic acinar cells, but less is known about the ducts. Therefore, we wanted to investigate the influence of various agonists on isolated perfused pancreatic ducts and, as a physiological response, we measured the basolateral membrane voltage of the duct cells (V _bl) with microelectrodes. Pancreatic ducts were dissected from pancreas of normal rats and bathed in a HCO₃ ^–-containing solution. Under control conditions, the average V _bl was between -50 and -70 mV. Vasoactive intestinal peptide (VIP) and carbachol (CCH) reversibly depolarized V _bl when applied to the bath. VIP (9×10^–9 mol/l) depolarized V _bl from -72±3 mV to -53±3 mV (n=20) and CCH (10^–5 mol/l) from -62±3 to -35±4mV (n=10). Furthermore, a decrease of the Cl^– concentration in the lumen led to an increase of VIP-induced depolarization of V _bl, suggesting that a luminal Cl^– conductance was increased. Cholecystokinin (CCK, 10^–10-10^–7 mol/l) and bombesin (10^–8, 10^–5 mol/l), which stimulate pancreatic exocrine secretion in acini or whole glands, showed no significant effect on V _bl of the duct cells tested in our preparation (n=7, 6). Neurotensin (10^–8 mol/l) had a marked depolarizing effect in two out of ten cases; V _bl depolarized from about -65 mV to-29 mV and the effect was reversible. Substance P (2×10^–7 mol/l), alone or in combination with secretin, had no effect on V _bl of the tested duct cells (n=11). We propose that the basolateral membrane of pancreatic duct cells possesses receptors for VIP, acetylcholine and neurotensin. CCK, bombesin and substance P had no detectable effects on V _bl of the duct cells tested, which could be due to the lack of corresponding receptors on these cells, or due to the absence of electrophysiologically detectable effects, in spite of receptor presence.Preliminary reports of the present study were presented at the 70th and 71st Meetings of the German Physiological Society, Germany, September 1991, March 1992     

Permeability properties of cell membranes and tight junctions of normal and cystic fibrosis sweat ducts

The transepithelial permeability properties to Na, K, and Cl in microperfused segments of human eccrine sweat ducts from normal (N) subjects and patients with cystic fibrosis (CF) were examined. Amiloride administered on the luminal surface caused the transepithelial potential (V _t ) of normal ducts to depolarize to 0 mV, but in the absence of Cl in the medium or in CF ducts, amiloride caused theV _t to significantly reverse electrical polarity from lumen negative to lumen positive with respect to the serosal bath. TheV _t responses to changes in Na concentration in the lumen and K concentration in the bath were similar in CF and N ducts and showed that the basolateral membrane of the duct is K permeable and the apical membrane (in the absence of an anion shunt) is an almost ideal Na electrode. TheV _t of N ducts was insensitive to 10-fold changes in luminal K and contraluminal Na solution concentrations. These responses show that in normal ducts, the apical membrane and tight junctions are relatively impermeable to K, and the basal membrane and tight junctions are relatively impermeable to Na. TheV _t was highly sensitive to Cl^– changes on either surface before or after ouabain inhibition in N ducts, but in every case were insensitive to Cl^– changes in CF ducts. By comparison to control ducts the cation selective properties of the CF duct are probably normal, but both cell membranes as well as the tight junctions of the CF duct are relatively impermeable to Cl. The present data are inconclusive as to whether the route of Cl movement across the N duct epithelium is trans- or paracellular.     

The development of the choledochus and pancreatic ducts in the gecko, Gymnodactylus kotschyi

Summary The development of the choledochus duct and pancreatic ducts in thirty-five embryos of the gecko, Gymnodactylus kotschyi, ranging from stages 20 to 34 (time of hatching), can be divided into four phases: First Phase: Four ducts enter the gut by separate portals. The first duct, posterior to the stomach, is from the ventral anlage of the ventral pancreas. The second is from the left anlage of the ventral pancreas. The third is the choledochus duct. The fourth duct to enter the gut posterior to the stomach is that of the dorsal pancreas. Second Phase: Three ducts enter the gut separately. The duct from the ventral anlage of the ventral pancreas persists, while the duct from the left anlage of the ventral pancreas disappears. The choledochus and dorsal pancreatic ducts enter the gut separately. Third Phase: Two ducts enter the gut separately. The choledochus and dorsal pancreatic ducts unite to enter the gut as a common duct. The ventral pancreatic duct enters the gut separately. Fourth Phase: One common duct results from the fusion of the ventral pancreatic duct with the common duct already formed from the dorsal pancreas and the choledochus duct.The material was collected from the Bosphorus region and killed inZenker's orBouin's B-3, and sectioned and stained in eitherDelafield's haematoxylin and eosin or borax-carmine and lichtgrün.

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Zusammenfassung Die Entwicklung des Ductus choledochus und Ductus pancreaticus in 35 Embryonen des Gecko (Gymnodactylus kotschyi) von Stufe 20–34 (Ausschlüpfung) kann man in 4 Zeitabschnitte einteilen: Erster Abschnitt. 4 Ausführungsgänge treten mit separaten Öffnungen in den Darm ein. Der erste Ductus, hinter dem Magen, kommt von der ventralen Anlage der Pankreasdrüse. Der zweite ist von der linken Anlage des ventralen Pankreas. Der dritte ist der Ductus choledochus. Der vierte, welcher hinter dem Magen in den Darm tritt, ist der des dorsalen Pankreas. Zweiter Abschnitt. 3 Ausführungsgänge treten gesondert in den Darm ein. Der Gang des ventralen Pankreas bleibt bestehen, während der Gang der linken Anlage des ventralen Pankreas verschwindet. Der Ductus choledochus und der Gang des dorsalen Pankreas treten gesondert in den Darm ein. Dritter Abschnitt. 2 Gänge treten gesondert in den Darm ein. Der Ductus choledochus und der dorsale Pankreasgang vereinigen sich zu einem Gang. Der Gang des ventralen Pankreas tritt gesondert in den Darm. Vierter Abschnitt. Der ventrale Gang vereinigt sich mit dem Gang, welcher aus der Fusion des Ductus choledochus mit dem Gang des dorsalen Pankreas hervorgegangen ist, zu einem einzigen Ductus.Das Material wurde in der Bosporusgegend gesammelt, inZenkers oderBouins B-3-Lösung fixiert und die Schnitte mitDelafields Hämatoxylin und Eosin oder mit Borax-Carmin und Lichtgrün gefärbt.

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Eight text-figures.     

Colchicine inhibits the effects of secretin on pancreatic duct cell tubulovesicles and HC03 secretion in the pig

Secretin stimulation clears the cytoplasm of intralobular pancreatic duct cells in pigs of tubulovesicles and causes these cells to secrete HCO₃^- into the pancreatic juice. To determine whether the clearance of cytoplasmic tubulovesicles involves the microtubule system and is important for initiation of HCO₃^- secretion, the effect of the microtubule poison colchicine on duct cell morphology and pancreatic HCO₃^- secretion was measured in anaesthetized pigs. Before colchicine, secretin reduced the density of tubulovesicles in the cytoplasm of pancreatic duct cells from 92 ± 8 U to 8 ± 2 U and initiated pancreatic secretion of 176 ± 21 μmol min^-1 HCO₃^-. After colchicine, secretin failed to lower duct cell tubulovesicle density and caused the secretion of only 77 ± 14μmol min^-1 HCO₃^-. By contrast, lumicolchicine, an isomer of colchicine that does not affect microtubules, did not inhibit pancreatic HCO3 secretion. Colchicine did not reduce carbonic anhydrase or Na,K-ATPase activities in in-vitro assays. The clearance of tubulovesicles from the cytoplasm of pancreatic duct cells therefore seems to be microtubule-dependent and important for the pancreatic HCO₃^- secretion.     

The impact of laparoscopic biopsy of pancreatic lymph nodes with helium and carbon dioxide on port site and liver metastasis in BOP-induced pancreatic cancer in hamster

The influence of pancreatic biopsy during laparoscopy with carbon dioxide (CO₂) and helium on the incidence of port site and liver metastasis in pancreatic carcinoma is still unknown. Ductal adenocarcinoma of the pancreas was induced in Syrian hamsters (n=30) by injection of N-nitrosobis-2-oxopropylamin (BOP, 10 mg/kg body weight/week) for 12 weeks. In week 13, hamsters were randomized in 3 groups (n=10): While in group 1 (gr. 1) a laparotomy and biopsy of pancreatic lymph nodes was performed, gr. 2 and gr. 3 underwent a laparoscopic biopsy either with CO₂ or helium. Therefore, one trocar was located in the left (biopsy) and the right abdominal wall (camera). In the 18th week all animals were sacrified and the incidence of abdominal wall, port site and liver metastases was histologically determined. While there were abdominal wall metastases after laparotomy in 10% (n=1), we observed trocar metastases in the CO₂ group in 20% (n=2). However, there were no trocar metastases in the helium group. The incidence of liver metastasis did not differ between the laparotomy and the helium group (20% vs 30%), but was increased in the CO₂ group (60%). Laparoscopic biopsy of pancreatic lymph nodes with CO₂ increased the incidence of port site and liver metastases in pancreatic cancer. The helium group was equal to the laparotomy group in this respect. Thus, staging laparoscopy with helium might become an alternative to explorative laparotomy in pancreatic cancer.     

Ducts isolated from the pancreas of CFTR-null mice secrete fluid

The pancreatic pathology in cystic fibrosis (CF) is normally attributed to the failure of ductal fluid secretion resulting from the lack of functional CF transmembrane conductance regulator (CFTR). However, murine models of CF show little or no pancreatic pathology. To resolve this dichotomy we analysed the transport mechanisms involved in fluid and electrolyte secretion by pancreatic ducts isolated from CFTR-null mice. Experiments were performed on cultured interlobular duct segments isolated from the pancreas of the Cftr^tm1Cam strain of CFTR-null mouse. Fluid secretion to the closed luminal space was measured by video microscopy. The secretory response of ducts isolated from CF mice to cAMP-elevating agonists forskolin and secretin was significantly reduced compared with wild type but not abolished. The Cl^?- and HCO ₃ ^? -dependent components of the ductal secretion were affected equally by the absence of CFTR. The secretory response to carbachol stimulation was unaltered in CF ducts. Loading the ductal cells with the Ca²⁺ chelator BAPTA completely abolished carbachol-evoked secretion, but did not affect forskolin-evoked secretion in CF or wild-type ducts. We conclude that pancreatic duct cells from CF mice can secrete a significant amount of water and electrolytes by a cAMP-stimulated mechanism that is independent of CFTR and cannot be ascribed to the activation of calcium-activated chloride channels.     

Effect of ATP,carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca²⁺ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca²⁺]_i, in perfused rat pancreatic ducts using the Ca²⁺-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, V _bl, of individual cells. The resting basal [Ca²⁺]_i was relatively high, corresponding to 263±28 nmol/l, and it decreased rapidly to 106±28 nmol/l after removal of Ca²⁺ from the bathing medium (n=31). Carbachol increased [Ca²⁺]_i in a concentration-dependent manner. At 10 mol/l the fura-2 fluorescence ratio increased by 0.49±0.06 (n=24), corresponding to an increase in [Ca²⁺]_i by 111±15 nmol/l (n=17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67±0.06 and 1.01±14 (n=46; 12), corresponding to a [Ca²⁺]_i increase of 136±22 nmol/l and 294±73 nmol/l respectively (n= 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized V _bl from –62±3 mV to-70±3 mV, an effect which was in some cases only transient (n=7). This effect of ATP was different from that of carbachol, which depolarized V_bl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of V _bl (n=4). Several other putative agonists of pancreatic HCO ₃ ^– secretion were also tested for their effects on [Ca²⁺]_i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 mol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca²⁺ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca²⁺]_i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.Preliminary reports of the present study have been presented at the 72nd Meeting of the German Physiological Society, March 1993     

The structure of the kidney from the freshwater teleost Carassius auratus

Summary The structure of the kidney of the crucian carp (Carassius auratus; a freshwater teleost, Cypriniformes) was studied by means of reconstruction from serial paraffin and semithin sections. In C. auratus, the Wolffian duct traverses the entire kidney. At various levels collecting ducts of different length and thickness join the Wolffian duct at right angles. Each collecting duct accepts a large number of connecting tubules, which are established by the joining of many nephrons. A regular pattern concerning the distribution of nephrons and the fusion of renal tubules is not apparent. Four segments have been distinguished in renal tubules; 1) proximal tubule, 2) distal tubule, 3) connecting tubule and 4) collecting duct. A neck and an intermediate segment are absent. The proximal tubule is established by proximal tubule cells which bear a brush border and have a conspicuous apical cytoplasmic rim containing few cell organelles, ciliated cells, mucous cells and dark cells. In the first part of the proximal tubule the brush border and the apical cytoplasmic rim of proximal tubule cells are well developed. Ciliated cells are interposed between proximal tubule cells, decreasing in number toward the end of this part. In the second part ciliated cells are absent and dark cells are numerous. In the third part the brush border and the apical cytoplasmic rim of proximal tubule cells are scarcely developed. Ciliated cells reapear and increase in number toward the distal tubule. The distal and connecting tubule are similar in epithelial structure. Connecting tubules are joined distal tubules and thus they belong to two or more nephrons. The main cells of distal and connecting tubules contain abundant mitochondria, but have no brush border. The connecting tubule becomes a collecting duct before joining the Wolffian duct. The main cells of collecting ducts are characterized by division of their cytoplasm into a dark apical half and a light basal half.     

Na+-H+ exchange is not important for pancreatic HCO-3 secretion in the pig

Veel , T., Villanger , O., Holthe , M.R., Cragoe jr ., E.J. & Reder , M.G. 1992. Na⁺-H exchange is not important for pancreatic HCO^-₃ secretion in the pig. Acta Physiol Scand 144 , 239–246. Received 13 September 1991, accepted 14 November 1991. ISSN 0001–6772. University of Oslo, Institute for Experimental Medical Research and Surgical Department, Ullevaal Hospital, Oslo, Norway. Pancreatic inter- and intralobular duct cells extrude H⁺-ions to interstitial fluid when they secrete HCO^-₃ to pancreatic juice. This study assesses the potential importance of Na⁺-H⁺-ion exchange for H⁺-ion extrusion and secretion of HCO^-₃ using the Na⁺-H⁺ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pH,) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H⁺-ion efflux was measured using a NH₄Cl prepulse, acid-loading technique. In HCO^-₃-free media, pH₁ recovery following acid loading was blocked by amiloride (10^-4 m) and hexamethylene-amiloride (10^-6 m) , demonstrating amiloride-and hexamethylene-amiloride-sensitive Na⁺-H⁺ exchange. However, 5 × 10^-6 M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO, secretion in vivo. Maximal H⁺-efflux through Na⁺-H⁺ exchange was 1.5 ± 0.2μmol min^-1 ml cell volume^-l, i.e. less than 1 % of estimated net H⁺-ion efflux during HCO^-₃ secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na⁺-H⁺ exchange is not important for secretin-dependent pancreatic HCO^-₃ secretion in the pig. Other mechanisms for H⁺ extrusion dominate.     

The mucosal folds at the pancreaticobiliary junction

Purpose

The structure and function of the mucosal folds in the terminal bile and pancreatic ducts and hepatopancreatic ampulla are poorly characterised. The distribution, muscularity, and innervation of these folds were investigated.

Methods

The pancreaticobiliary junction was excised from ten cadavers (five male, 66–90 years) and examined histologically by serially sectioning (4-μm thickness) along the length of the terminal bile and pancreatic ducts from the tip of the major duodenal papilla. Three surgical specimens (two male, 63–72 years) were also evaluated. Sections were stained with haematoxylin and eosin, anti-actin (smooth muscle), anti-S100 (innervation), and anti-cholecystokinin (CCK)-A receptor antibodies. ImageJ software was used to compare relative radial fold projection and semi-quantitatively assess the smooth muscle and nerve content. In one additional cadaver specimen, folds were examined by scanning electron microscopy.

Results

Mucosal folds in the terminal bile duct were arranged circumferentially in a lattice-like arrangement and were distributed over an average distance of 7.3 mm along the terminal bile duct compared to 4.2 mm along the pancreatic duct (P = 0.001), projected further into the lumen, and were more densely innervated than those in the terminal pancreatic duct. Folds in both ducts contained smooth muscle which was more prominent in folds nearest to the major duodenal papilla. Mucosal folds in cadaver and surgical specimens showed no evidence of CCK-A receptor immunoreactivity.

Conclusions

This study demonstrates that the mucosal folds of the terminal bile and pancreatic ducts contain muscle and nerve fibres, suggesting an active rather than purely passive function.     

Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts

 Extracellular adenosine 5’-triphosphate (ATP) has been described to act as a regulator in many cells and tissues, including epithelia, and in the gastrointestinal tract ATP is one of the substances involved in non-cholinergic non-adrenergic control. However, very little is known about the effect of ATP on pancreatic ducts, which normally secrete bicarbonate-rich fluid in response to secretin. Hence, the aim of our present study was to test the effect of ATP and other nucleotides on intracellular Ca²⁺ activity ([Ca²⁺]_i) of pancreatic ducts, and thereby get information about purinergic receptors that might play a role in the regulation of pancreatic bicarbonate transport. Native intralobular ducts were obtained from rat pancreas and [Ca²⁺]_i in 10–20 cells was measured using the fura-2 method. ATP (10^–4 mol/l) evoked a characteristic biphasic Ca²⁺ transient in duct cells. Nucleotides, used to classify the P₂ receptors, acted with the following potency on the peak Ca²⁺ in many ducts: uridine 5’-triphosphate (UTP) ≥ ATP >inosine 5’-triphosphate ≥ 2-methylthio-ATP > β,γ-methyl-ATP > adenosine. However, although the peak [Ca²⁺]_i responses to ATP and UTP were similar, the plateau [Ca²⁺]_i was nearly doubled with UTP. Moreover, in about one-third of the ducts studied, UTP had no effect on cell Ca²⁺, while the response to ATP was normal. In further experiments we found that removal of extracellular Mg²⁺ increased the peak [Ca²⁺]_i evoked in response to ATP. 2’&3’-O-(4-benzoylbenzoyl) ATP (BzATP) evoked a monophasic and slower increase in [Ca²⁺]_i, which was inhibited by removal of extracellular Ca²⁺, or by addition of 4, 4’-diisothiocyanatostilbene-2, 2’-disulphonic acid (DIDS). Taken together, our data indicate that there are two types of purinergic receptors on pancreatic ducts through which ATP can act. These are pharmacologically known as P_2U and P_2Z receptors and may correspond to P2Y₂ and P2X₇ receptors. Received: 2 June 1997 / Received after Revision: 10 January 1998 / Accepted: 28 January 1998     

Endocytosis of gold-labeled proteins and LDL byTrypanosoma cruzi

Endocytosis was studied at the ultrastructural level in different developmental forms ofTrypanosoma cruzi after incubation of the parasites in the presence of gold-labeled proteins (albumin-Au, peroxidase-Au and transferrin-Au) and low-density lipoprotein (LDL-Au). Epimastigote (culture) forms actively ingested LDL and proteins. Initially, gold particles were seen adhering only to the cytostome and inside the flagellar pocket. In parasites incubated at 4°C with transferrin-Au or peroxidase-Au, labeling was found only at these two sites, showing that receptor-mediated endocytosis occurs in both regions. In the cytoplasm, gold particles were seen only inside two different compartments: membrane-bound vesicles and reservosomes. Incubation of epimastigotes with acridine orange followed by fluorescence microscopy revealed intense orange staining, indicating that the reservosomes have an acidic pH. This staining was abolished after incubation of the parasites in the presence of ammonium chloride. These data confirm that this compartment is the site of accumulation of ingested lipids and proteins. Little intracellular labeling with transferrin-Au was found in in vitro — derived amastigotes and trypomastigotes (both lack reservosomes). However, although in amastigotes very few gold particles were seen bound to the cells, in trypomastigotes they were observed bound to the membrane that encloses the cell body, the flagellar pocket, and the flagellum, suggesting that the receptors are more abundant in this form.     

Changes in glucose metabolism and cyanide sensitivity in Schistosoma mansoni during development

Schistosoma mansoni was studied by biochemical and electrophysiological techniques to follow the physiological changes occurring during transformation in the mammalian host. Volume conducted electrical potentials and measurement of CO₂, evolution indicate that 3 h post-transformational schistosomula are highly sensitive to cyanide. By 24 h after transformation, evolution of CO₂ under control conditions is reducted by 77% from 3 h levels, while lactate excretion rises by 84%. Cyanide does not affect the frequency or magnitude of endogenous electrical transients, but does eliminate 83% of the already reduced levels of CO₂ evolved in 24 schistosomula. Electrophysiological analyses indicate that the timecourse of metabolic changes in skin- and mechanically transformed schistosomula are similar, and incubation of schistosomula in 200 μg ml^?1 puromycin does not alter the onset of cyanide insensitivity. The adult parasite evolves a low level of CO₂ which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 h after transformation; by 24 h, however, the parasites are metabolically similar to the adult stage, i.e., they depend on lactate fermentation for most of their energy requirements.     

Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas

Intracellular pH (pH_i) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectrofluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pH_i in glands perfused with a HCO ₃ ^– -free, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES)-buffered solution at 37°C was 7.36±0.01 (n=52) which is comparable with values obtained by ³¹P nuclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of the major phosphorus metabolites. Changes in pH in response to NH₄Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca²⁺ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.