Guinea Pig Pancreas Research Articles

Electrogenic bicarbonate transport across the luminal membrane in interlobular ducts isolated from guinea-pig pancreas

Electrogenic bicarbonate transport across the luminal membrane in interlobular ducts isolated from guinea-pig pancreas

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 HCO(3)(-) 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 microm forskolin in the absence of extracellular HCO(3)(-). In guinea-pig ducts, however, fluid secretion was totally dependent on HCO(3)(-). Forskolin-stimulated fluid secretion by mouse and rat ducts in the absence of HCO(3)(-) was dependent on extracellular Cl(-) and was completely inhibited by bumetanide (30 microm). It was therefore probably mediated by a basolateral Na(+)-K(+)-2Cl(-) cotransporter. In the presence of HCO(3)(-), forskolin-stimulated fluid secretion was reduced approximately 40% by bumetanide, approximately 50% by inhibitors of basolateral HCO(3)(-) uptake (3 microm EIPA and 500 microm H(2)DIDS), 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(+)-HCO(3)(-) cotransport mediating HCO(3)(-) 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 HCO(3)(-) secreted in this species.

Ethanol induces fluid hypersecretion from guinea-pig pancreatic duct cells

Ethanol is the leading cause of pancreatitis; however, its cellular effects are poorly understood. We examined the direct effects of ethanol in the concentration range 0.1-30 mM, i.e. relevant to usual levels of drinking, on fluid secretion from guinea-pig pancreatic duct cells. Fluid secretion was continuously measured by monitoring the luminal volume of interlobular duct segments isolated from the guinea-pig pancreas. [Ca2+]i was estimated by microfluorometry in duct cells loaded with fura-2. Ethanol at 0.3-30 mM significantly augmented fluid secretion stimulated by physiological (1 pM) or pharmacological (1 nM) concentrations of secretin. It augmented dibutyryl cAMP-stimulated fluid secretion but failed to affect spontaneous or acethylcholine-stimulated secretion. Ethanol at 1 mM shifted the secretin concentration-fluid secretion response curve upwards and raised the maximal secretory response significantly by 41%. In secretin-stimulated ducts, 1 mM ethanol induced a transient increase in [Ca2+]i that was dependent on the presence of extracellular Ca2+. Ethanol failed to augment secretin-stimulated secretion from ducts pretreated with an intracellular Ca2+ buffer (BAPTA) or a protein kinase A inhibitor (H89). In conclusion, low concentrations of ethanol directly augment pancreatic ductal fluid secretion stimulated by physiological and pharmacological concentrations of secretin, and this appears to be mediated by the activation of both the intracellular cAMP pathway and Ca2+ mobilization.

Substance P inhibits bicarbonate secretion from guinea pig pancreatic ducts by modulating an anion exchanger.

The stimulatory pathways controlling HCO3- secretion by the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, which may play an important role in the physiological control of the pancreas. The aim of this study was to investigate the cellular mechanism by which substance P (SP) inhibits pancreatic ductal HCO3- secretion. Small intra/interlobular ducts were isolated from the pancreas of guinea pigs. During overnight culture the ducts seal to form a closed sac. Transmembrane HCO3- fluxes were calculated from changes in intracellular pH (measured using the pH-sensitive dye BCECF) and the buffering capacity of the cells. We found that secretin can stimulate HCO3- secretion in guinea pig pancreatic ducts about fivefold and that this effect could be totally blocked by SP. The inhibitory effect of SP was relieved by spantide, an SP receptor antagonist. SP had no effect on the activity of basolateral Na+-HCO3- cotransporters and Na+/H+ exchangers. However, the peptide did inhibit a Cl--dependent HCO3- efflux (secretory) mechanism, most probably the Cl-/HCO3 exchanger on the apical membrane of the duct cell.

Post-natal maturation of acinar cells of the guinea pig pancreas: an ultrastructural morphometric study.

The morphological maturation of the acinar cells of the guinea pig pancreas during post-natal development was characterized morphometrically by determining the intracytoplasmic accumulation of rough endoplasmic reticulum (RER) and zymogen granules. The following results were obtained for the period analysed, i.e., from 2 to 70 days of post-natal life: (a) the acinar cell volume increased by 210% (P < 0.01); (b) the mostly cisternal RER occupied more than 30% of the cytoplasm at any age studied and their total volume and surface in the cell were increased by 300 and 534% (P < 0.01), respectively; (c) maturation in the morphological pattern of the RER was observed; (d) the mean number of zymogen granules per cell increased from 261 at 2 days to 422 at 70 days (P < 0.01), while their mean diameter increased from 0.52 to 0.94 micron (P < 0.01) during the same period; (e) these increases in granule number and size were responsible for a 500% (P < 0.01) increase in total volume from 2 to 70 days and for a 304% increase (P < 0.01) in total surface from 2 to 35 days; (f) the RER and the zymogen granules together occupied 44, 54, 55 and 57% of the cytoplasm at 2, 14, 35 and 70 days of age, respectively. We conclude that although the pancreatic acinar cells of the guinea pig are morphologically well differentiated at 2 days of age, with the cytoplasm already showing a large amount of RER and zymogen granules, they are still immature. Morphological maturation of the acinar cell occurs during the first months of post-natal life and is characterized by a substantial gain in cell volume and intracytoplasmic accumulation of RER and zymogen granules, which significantly increase of both their absolute volume and total surface, with a higher growth rate being observed during the period from 2 to 14 days of post-natal life.

HISTOCHEMICAL DETECTION OF PANCREATIC TISSUE IN VITAMIN C‐ADMINISTERED GUINEA PIGS

The aim of this study was to investigate the effects of fasting (F) periods and vitamin C supplementation on the serum C‐peptide levels and endocrine pancreas of guinea pigs. Vitamin C supplementation (a single intraperitoneal dose of 500 mg/kg−1) was given at the beginning of the fasting period. The animals were divided into seven groups (n=6 each) as follows: Group I (untreated group) was given food and water ad libitum; Group II, III and IV were acute (24 h, 48 h) and chronic F (120 h) respectively; while Groups V, VI and VII were acute and chronic F with supplementary vitamin C (VitC + F24 h, VitC + F48 h and VitC + F120 h, respectively). Serum C‐peptide levels were measured and pancreatic tissues were collected for immunohistochemical evaluation. Serum C‐peptide levels in the VitC + F24 and VitC + F48 groups were higher than in the F24 and F48 groups (P&lt;0.05). Immunohistochemical examination of the endocrine pancreas confirmed that vitamin C administration in fasting may indirectly increase glucagon and insulin secretions.

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

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

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.

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa ( Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.

5-hydroxytryptamine strongly inhibits fluid secretion in guinea pig pancreatic duct cells.

We studied the distribution of 5-hydroxytryptamine- (5-HT-) containing cells in the guinea pig pancreas and examined the effects of 5-HT on fluid secretion by interlobular pancreatic ducts. The 5-HT-immunoreactive cells with morphological characteristics of enterochromaffin (EC) cells were scattered throughout the duct system and were enriched in islets of Langerhans. The fluid secretory rate in the isolated interlobular ducts was measured by videomicroscopy. Basolateral applications of 5-HT strongly but reversibly reduced HCO(3)-dependent, as well as secretin- and acetylcholine- (ACh-) stimulated, fluid secretion, whereas 5-HT applied into the lumen had no such effects. Secretin-stimulated fluid secretion could be inhibited by a 5-HT(3) receptor agonist, but not by agonists of the 5-HT(1), 5-HT(2), or 5-HT(4) receptors. Under the stimulation with secretin, 5-HT decreased the intracellular pH (pH(i)) and reduced the rate of pH(i) recovery after acid loading with NH(4)(+), suggesting that 5-HT inhibits the intracellular accumulation of HCO3(-). The elevation of intraductal pressure in vivo reduced secretin-stimulated fluid secretion, an effect that could be attenuated by a 5-HT(3) receptor antagonist. Thus, 5-HT, acting through basolateral 5-HT(3) receptors, strongly inhibits spontaneous, secretin-, and ACh-stimulated fluid secretion by guinea pig pancreatic ducts. 5-HT released from pancreatic ductal EC cells on elevation of the intraductal pressure may regulate fluid secretion of neighboring duct cells in a paracrine fashion.

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.

Ethanol Combined with Cocaine Inhibits Amylase Release in Guinea PIG Pancreatic Lobules

Concurrent ingestion of alcohol and cocaine is a common occurrence in cocaine-dependent individuals. Cocaethylene is a pharmacologically active metabolite of cocaine that is formed in the liver in the presence of ethanol. The effects of ethanol combined with cocaine on the exocrine pancreas are not known. We studied the effect of ethanol and cocaine, alone or in combination, and cocaethylene on amylase release from isolated lobules of the guinea pig pancreas. Incubation of lobules with ethanol plus cocaine produced a more evident reduction of amylase release than each drug alone. An even larger reduction was observed with cocaethylene. HPLC analysis of incubation medium showed that no cocaethylene was formed in vitro in the presence of ethanol and cocaine. It is concluded that cocaethylene could strongly contribute to inhibition of exocrine pancreatic secretion in individuals who coadminister alcohol with cocaine.

Pancreatic lymphatic system in rodents.

The lymphatic network of the pancreas has been little investigated and recent studies have provided contrasting data. This research is aimed to supply the morphologic basis to outline the involvement of the lymphatic system in pancreatic pathology. Guinea pigs, rats, and mice were anesthetized with ether and sacrificed with the same anesthetic. Pieces of pancreas were processed for transmission electron microscopy. Semithin sections were observed by light microscopy and, after positive identification by transmission electron microscopy, lymphatics were followed with long series of consecutive sections to define their distribution. Lymphatics were detected in the pancreas of all the animals both in the inter and the intralobular sites. Closer relations with the exocrine parenchyma (ducts and acini) were observed in guinea pig pancreas. Remarkably, interesting relationships between lymphatics and endocrine tissue were observed in all the animals. Overall, however, the lymphatic network of rat pancreas was less develop and preferentially associated with blood vessels. The distribution of the pancreatic lymphatic network appears consistent with an active role in pancreatic pathology.

Ethanol potentiales secretin-stimulated fluid secretion in interlobular ducts isolated from guinea-pig pancreas

Ethanol potentiales secretin-stimulated fluid secretion in interlobular ducts isolated from guinea-pig pancreas

Ethanol potentiales secretin-stimulated fluid secretion in interlobular ducts isolated from guinea-pig pancreas

Ethanol potentiales secretin-stimulated fluid secretion in interlobular ducts isolated from guinea-pig pancreas

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.

2,7-Dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1,4]diazonine as a new template for the design of CCK(2) receptor antagonists.

A novel series of nonpeptide CCK(2) receptor antagonists has been prepared, in which 2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (5) was used as a chemical template. This uncommon ring system was obtained in a highly substituted form and in high yield by ozonolysis of the enamine bond of 1,2,3,4-tetrahydro-9H-pyrido[3, 4-b]indole derivatives (4), in which the configuration of the substituents was established stereoselectively via the Pictet-Spengler reaction. Further structural manipulation was guided by molecular modeling through comparison of fieldpoint-based structures of candidate compounds with a selected low-energy conformation of the representative CCK(2) receptor antagonist 5-[[[(1S)-[[(3, 5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyl]amino]carbonyl]-6- [[( 1-adamantylmethyl)amino]carbonyl]indole (JB93182 (3)). By this approach compounds such as (3R, 5S)-4-acetyl-3-(1-adamantyl)methyl-1-(2-chlorobenzyl)-5-carboxymet hyl aminocarbonyl-2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (32) were prepared. Compound 32 behaved as a competitive CCK(2) receptor antagonist in vitro as judged by its inhibition of pentagastrin-stimulated acid secretion in an isolated, lumen-perfused, immature rat stomach assay (pK(B) = 6.74 +/- 0.27) and by its displacement of [(125)I]CCK-8S from CCK(2) sites in mouse cortical homogenates (pK(i) = 6.99 +/- 0.05). Compound 32 was 100-fold selective for CCK(2) over CCK(1) receptors based on the affinity estimate obtained in a guinea pig pancreas radioligand binding assay (pK(i) = 5.0).

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.

Arginine vasopressin inhibits fluid secretion in guinea pig pancreatic duct cells.

The effects of arginine vasopressin (AVP) on pancreatic ductal secretion were studied in guinea pigs. In the isolated vascularly perfused pancreas, AVP reduced secretin-stimulated fluid secretion and increased the vascular resistance when the perfusion rate was held constant. In the isolated interlobular duct segments, AVP inhibited secretin-stimulated fluid secretion, indicating the direct inhibitory action of AVP on the duct cells. AVP affected neither the basal nor the secretin-induced cAMP productions, suggesting that AVP inhibits the fluid secretion at a point distal to the production of cAMP. AVP increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence of extracellular Ca(2+). When [Ca(2+)](i) was elevated by the application of thapsigargin, AVP caused a rapid decrease in [Ca(2+)](i). AVP seems to activate both Ca(2+) release from intracellular stores and Ca(2+) efflux across the plasma membrane, but its relation to the inhibition of fluid secretion remains to be clarified. It is concluded that AVP directly inhibits secretin-stimulated ductal fluid secretion in the guinea pig pancreas.

Colocalization of ChAT, DβH and NADPH-d in the pancreatic neurons of the newborn guinea pig

Choline acetyltransferase (ChAT) as a rate-limiting enzyme in the biosynthetic pathway of acetylcholine is thought to be present in all cholinergic neurons. However, its immunoreactivity has not been successfully applied to the study of cholinergic neurons in the pancreas. In a previous study in the pancreas of newborn guinea pig we reported the colocalization of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS) with various neuropeptides as well as dopamine-beta-hydroxylase (DbetaH), the enzyme responsible for converting dopamine to noradrenaline. Whether NADPH-d is colocalized with ChAT in the pancreatic neurons is not known. Also it would be interesting to find out whether noradrenaline and acetylcholine could be colocalized in the same pancreatic neurons. In the present study, a method for triple labelling of ChAT, DbetaH and NADPH-d was used to answer the above questions. Colocalization of ChAT, DbetaH and NADPH-d was constantly demonstrated in the same neurons in the same sections. It is concluded that some of the pancreatic neurons may utilize more than one neurotransmitter such as nitric oxide (NO), acetylcholine and noradrenaline to achieve their function. The possible cotransmission of acetylcholine and noradrenaline was extremely intriguing, and its mechanism and significance needs to be further investigated.