Pancreatic duct secretion: experimental methods, ion transport mechanisms and regulation

The pancreatic ductal tree conveys enzymatic acinar products to the duodenumand secretes the fluid and ionic components of pancreatic juice. The physiology ofpancreatic duct cells has been widely studied, but many questions are still unansweredconcerning their mechanisms of ionic transport. Differences in the transportmechanisms operating in the ductal epithelium has been described both among differentspecies and in the different regions of the ductal tree. In this review we summarizethe methods developed to study pancreatic duct secretion both in vivo and invitro, the different mechanisms of ionic transport that have been reported to date inthe basolateral and luminal membranes of pancreatic ductal cells and the regulationof pancreatic duct secretion by nervous, endocrine and paracrine influences(AU)

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Pancreatic duct secretion: experimental methods, ion transport mechanisms and regulation.

The pancreatic ductal tree conveys enzymatic acinar products to the duodenum and secretes the fluid and ionic components of pancreatic juice. The physiology of pancreatic duct cells has been widely studied, but many questions are still unanswered concerning their mechanisms of ionic transport. Differences in the transport mechanisms operating in the ductal epithelium has been described both among different species and in the different regions of the ductal tree. In this review we summarize the methods developed to study pancreatic duct secretion both in vivo and in vitro, the different mechanisms of ionic transport that have been reported to date in the basolateral and luminal membranes of pancreatic ductal cells and the regulation of pancreatic duct secretion by nervous, endocrine and paracrine influences.

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.

Nitric oxide stimulates tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini.

Some of the effects of several oncogenes, integrins, growth factors, and neuropeptides are mediated by tyrosine phosphorylation of the non-receptor tyrosine kinase p125(FAK) and the cytoskeletal protein paxillin. We have demonstrated that different stimuli cause tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The aim of the present study was to determine whether exogenous NO activates this pathway. We demonstrate that in isolated rat pancreatic acini, a NO donor, sodium nitroprusside (SNP) stimulates, in a dose- and time-dependent way, tyrosine phosphorylation of p125(FAK) and paxillin. The same effects could be observed after incubating acini with 8-Br-cGMP. Moreover, the stimulation caused by SNP was completely abolished by two different guanylyl cyclase inhibitors, methylene blue, and LY-83583. These inhibitors also diminished unstimulated phosphorylation of p125(FAK) and paxillin. We conclude that in rat pancreatic acini exogenous NO causes p125(FAK) and paxillin tyrosine phosphorylation that is mediated by a guanylyl cyclase-dependent pathway.

Influence of chronic ethanol consumption on the muscarinic cholinergic control of rat pancreatic acinar cells.

There are a number of hypothetical explanations for the actions of ethanol on the exocrine pancreas; among them, the cholinergic hypothesis has received special attention. According to this hypothesis, chronic alcohol consumption induces alterations in the control of exocrine pancreatic function resulting in cholinergic hyperstimulation of pancreatic acinar cells and their muscarinic receptors. Our aim was to investigate the cholinergic control of pancreatic enzyme secretion and the number and affinity of muscarinic receptors in the pancreatic acinar cells of rats subjected to chronic ethanol ingestion. We also investigated whether a high-fibre diet modifies the actions of ethanol on these aspects of the exocrine pancreatic function. Four groups of rats received either a standard or a high fibre diet, and either water or 20% (v/v) ethanol. After 6 months of treatment, isolated pancreatic acini were used for the determination of carbachol-stimulated amylase secretion and for the analysis of muscarinic receptors, using 1-[N-methyl-3H]scopolamine as a radioligand. Neither chronic ethanol intake nor a high fibre diet caused any apparent alteration in pancreatic histology, neither did them modify plasmatic amylase levels. Chronic alcoholization resulted in a significant increase in the amylase released from pancreatic acini in response to carbachol stimulation, but it did not affect either the number or the affinity of pancreatic acinar muscarinic receptors. The actions of ethanol are not significantly modified by the simultaneous consumption of a high fibre diet.

Low bone density with normal bone turnover in ovariectomized and streptozotocin-induced diabetic rats.

Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 +/- 0.028 g, DO: 0.422 +/- 0.020 g) and BMDs (D: 0.171 +/- 0.006 g/cm2, DO: 0.174 +/- 0.006 g/cm2) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 +/- 0.024 g and BMD 0.258 +/- 0.004 g/cm2) and ovariectomized (O: BMC 0.640 +/- 0.044 g and BMD 0.240 +/- 0.009 g/cm2) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 +/- 16.8 ng/ml, PYD: 270.2 +/- 17.8 nM/mM) than those found in the control rats (BGP: 44.7 +/- 4.8 ng/ml, PYD: 165.6 +/- 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 +/- 14.6 ng/ml, PYD: 55.0 +/- 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 +/- 5.1 ng/ml, PYD: 146.7 +/- 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently "normal." Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone.

Impairment of intracellular calcium homoeostasis in the exocrine pancreas after caerulein-induced acute pancreatitis in the rat.

1. We have measured intracellular calcium concentrations in basal conditions and in response to cholecystokinin-octapeptide and acetylcholine in pancreatic acini isolated from rats with caerulein-induced acute pancreatitis and compared them with those in control rats. 2. We also measured amylase secretion in basal conditions and in response to cholecystokinin-octapeptide in both groups. 3. In pancreatic acini from rats with pancreatitis the basal intracellular calcium concentration was significantly increased (134.9 +/- 7.1 nmol/l compared with 71.8 +/- 2.9 nmol/l, P < 0.001). Moreover, the maximum values of intracellular calcium attained during the stimulation period were equivalent in acini from control and pancreatitic rats with no statistically significant differences. 4. In acini from control rats the differences between the resting levels of intracellular calcium and the maximum intracellular calcium values (delta[Ca2+]i) in response to several concentrations of cholecystokinin-octapeptide showed a clear dose-response relationship, with a half-maximal increase at 0.1 nmol/l and a maximal difference (delta[Ca2+]i = 259 +/- 50 nmol/l) at 1 nmol/l. In contrast, a right-shifted response, with a statistically significant smaller increase, was observed in acini from pancreatitic rats. 5. Basal amylase release was significantly higher in acini from rats with pancreatitis (11.7 +/- 1.0% of total compared with 5.9 +/- 1.1% of total, P < 0.001). In contrast, cholecystokinin-octapeptide and acetyl-choline-evoked amylase secretion was reduced by more than 85% in acini from pancreatitic rats. 6. In conclusion, calcium homoeostasis in pancreatic acinar cells from rats with caerulein-induced pancreatitis seems to be impaired. These results suggest excessive release of acinar free ionized calcium, or damage to the integrity of mechanisms that restore low resting levels of intracellular free ionized calcium, and the consequent calcium toxicity could be the key trigger in caerulein-induced acute pancreatitis.

Description of an automated method for the in vitro measurement of trypsinogen secretion from pancreatic segments.

The characterization of trypsinogen output from superfused pancreatic tissue by an automated spectrophotometric method is described. To test the method we investigated the time-course and the dose-response curve for acetylcholine-induced trypsinogen release from superfused pancreatic segments. We have demonstrated that this method allows the on-line detection and estimation of trypsinogen release with suitability, stability, and sensitivity.

Heterogeneous decrease of bone mineral density in the vertebral column of ovariectomized rats.

The long-term effect of ovariectomy on the loss of bone mineral density (BMD) was evaluated in rats with and without estrogen treatment; BMD was studied in the lumbar and caudal vertebrae, measured by DXA, to find how the losses of BMD occur in the axial skeleton. Seventy female Wistar rats of 3 months of age were divided into four groups as follows: group 1: control animals; group 2: ovariectomized animals; group 3: ovariectomized animals undergoing treatment with estrogen (0.25 mg/kg per week of 17-beta estradiol); group 4: ovariectomized rats undergoing estrogen treatment only during the last 3 months of the experimental period. No significant differences were found among the groups in regard to the BMD values of the caudal vertebrae at either 3 or 6 months. Likewise, in the lumbar vertebrae there were no significant differences among the groups after 3 months. However, at 6 months, a decrease in the BMDs of the ovariectomized animals with respect to the remaining groups was found: 226 +/- 11 mg/cm2 in the ovariectomized group; 262 +/- 14 mg/cm2 in the controls; 255 +/- 4 mg/cm2 in the rats receiving estrogen treatment for 6 months; and 259 +/- 5 mg/cm2 in the animals receiving estrogen for 3 months. The study also reveals the absence of differences in the bone mineral density between the ovariectomized and control rats when the former received estrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic pathways are involved in secretin and VIP release and the exocrine pancreatic response after intraduodenally perfused acetic and lactic acids in the rat.

The response of the exocrine pancreas to intraduodenal perfusion of acetic and lactic acids in normal and previously atropinized rats was studied. Secretin and vasoactive intestinal peptide (VIP) plasma levels in portal plasma were also measured. Intraduodenal perfusion of both acetic and lactic acids significantly stimulated flow rate (from 0.29 +/- 0.03 microliters/min to a maximum of 1.06 +/- 0.08 microliters/min after acetic and from 0.35 +/- 0.05 microliters/min to a maximum of 1.13 +/- 0.12 microliters/min after lactic acid perfusion) and protein output (from 11.16 +/- 2.33 micrograms/min to a maximum of 35.1 +/- 7.4 micrograms/min after acetic and from 8.98 +/- 0.95 micrograms/min to a maximum of 22.5 +/- 1.3 micrograms/min after lactic acid perfusion). Atropine treatment significantly inhibited pancreatic flow rate and protein output after acetic acid perfusion, but no inhibition of flow rate and a slight decrease in the protein output after lactic acid perfusion were seen. With respect to plasma peptide concentrations, significant increases in secretin and VIP levels were found after perfusion of both organic acids; atropine administration significantly decreased plasma secretin levels after acetic acid administration although it did not affect plasma VIP concentrations. By contrast, atropine significantly increased plasma secretin levels, but significantly lower values of plasma VIP concentrations were observed after lactic acid perfusion. Therefore, cholinergic mechanisms are involved in the release of secretin and VIP and different types of control of exocrine pancreatic secretion occur, depending on the features of the intraduodenal stimulant.

Effect of adenosine and adenosine agonists on amylase release from rat pancreatic lobules.

The effects of adenosine on the amylase secretion from rat pancreatic lobules have been studied. Adenosine induces a dose-dependent stimulation on amylase release, which is maximal at a concentration of 10(-4) M. It has been observed a clear inhibition of this secretory action when atropine was added whereas no amylase release was seen in isolated acini after adenosine. The effect of adenosine is completely blocked by the adenosine receptors antagonist theophylline (10(-4) M), but not by dipyridamole (10(-3) M), a drug that inhibits the transport of adenosine into the cell. The increase of amylase secretion induced by adenosine is inhibited by either the relatively selective A1 receptor antagonist PD116,948 (10(-6) M) and by the A2 receptor antagonist PD115,199 (10(-6) M). Significant increases of amylase release are observed after the relatively selective A1 receptor agonist R-PIA (10(-5) M) and after the relatively selective A2 receptor agonist NECA (10(-4) M). Finally, the effect of R-PIA is not modified by coincubation with PD115,199 and the effect of NECA is not affected by coincubation with PD116,948. These results suggest that the action of adenosine is mediated through the release of acetylcholine and probably by the simultaneous occupation of both A1 and A2 adenosine receptors, whereas the intracellular action of adenosine could be discarded.

Effects of acute intravenous ethanol on basal exocrine pancreatic secretion in rat: cholinergic involvement.

The effect of intravenous infusion of ethanol on the basal exocrine pancreatic secretion of anesthetized rats was studied. The cholinergic involvement on the actions of ethanol was also studied using previously atropinized animals. During the stimulation period, pancreatic flow rate was significantly increased by intravenous ethanol in both un-atropinized (199% compared with basal) and atropinized rats (195% compared with basal). Pancreatic protein output was also increased during ethanol administration in both groups of animals (171% and 165% compared with basal in, respectively, un-atropinized and atropinized rats). After the administration of ethanol, in the poststimulation period, pancreatic flow rate was further increased only in the atropinized group of rats (290% compared with basal), whose values were significantly higher than those of ethanol-treated un-atropinized animals (195% compared with basal). A similar profile of response was observed in pancreatic protein output. Since intravenous ethanol did not stimulate either secretin or VIP release to portal plasma, the present results point to a direct effect of this substance on the exocrine pancreas. Furthermore, atropine revealed the existence of an inhibitory cholinergic effect of ethanol on the exocrine pancreas. In summary, results show that the effect of intravenous ethanol on the basal exocrine pancreatic secretion is dual and antagonistic.

Nicotinic cholinergic influences in pancreatic secretion induced by intraduodenal alkaline and acid solutions in the rabbit.

1. The effect of hexamethonium on the exocrine pancreatic response to intraduodenal acidification and alkalinization, and the secretin and VIP release after these stimuli, was studied. 2. The hydroelectrolyte secretion after hydrochloric acid and sodium carbonate perfusion was reduced by hexamethonium treated (322 +/- 44% of maximum response in flow rate to sodium carbonate perfusion in untreated animals vs 140 +/- 12% in pretreated animals, and 252 +/- 19% of maximum response in flow rate to HCl in untreated animals vs 166 +/- 11% in pretreated animals). 3. However, hexamethonium has no effect on secretin plasma levels after either intraduodenal acidification or alkalinization. 4. On the contrary, the ganglion blocker significantly (P < 0.01) reduced plasma VIP levels in response to intraduodenal HCl (maximum response 320 +/- 74% in untreated vs 184 +/- 44% in hexamethonium-treated animals). 5. Plasma VIP levels showed a similar increase in both untreated (maximum response: 151 +/- 12%) and ganglion blocked animals (170 +/- 26%) in response to sodium carbonate. 6. These data suggest the existence of complex neural mechanisms in the exocrine pancreatic response to intraduodenal stimuli, these mechanisms being different depending on the intraduodenal stimulus.

Cerulein-induced acute pancreatitis in the rat. Study of pancreatic secretion and plasma VIP and secretin levels.

A study was made with different doses of cerulein (2, 4, 10 and 20 micrograms/kg) administered subcutaneously to rats by four injections at intervals of 1 hr; the aim of this work was to study exocrine pancreatic secretion of the rat under cerulein-induced acute pancreatitis, analyzing enzyme and hydroelectrolyte secretion of pancreatic juice. A further aim was to study the relationship between the dose of cerulein and the plasma levels of peptides controlling hydroelectrolyte secretion of the pancreas, like secretin and vasoactive intestinal peptide (VIP). At the lowest dose schedule, the amounts of total protein and enzymes (amylase and trypsin) in pancreatic juice decreased significantly, plasma amylase increased, and the pancreas became edematous. Higher doses magnified these effects. By contrast, ductular function (flow and HCO3-) was well preserved in cerulein-treated rats, and this was probably due to the significant increase in plasma levels of immunoreactive secretin whereas VIP levels were unchanged. The secretin released by treatment with cerulein is able to palliate the lack of flow from acinar origin that is affected in the process of acute pancreatitis, being a beneficial response to the cerulein treatment.

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.

Caerulein-induced acute pancreatitis in the rat. Pancreatic secretory response to cholecystokinin.

The response of pancreatic exocrine secretion to cholecystokinin (CCK), has been studied in experimental acute pancreatitis induced in rats by supramaximal doses of caerulein. Several doses of caerulein were used (4, 20 and 40 micrograms/Kg) and each one was administered by four subcutaneous injections over 3 h at hourly intervals. Pancreatic juice was collected 9 h after the first injection. The caerulein-treated animals showed a statistically significant increase in serum amylase levels. Secretory activity of ductular cells remained unchanged in all the caerulein-treated animals, but total protein and amylase secretion decreased significantly at all the caerulein doses used, both in resting conditions and under stimulation with CCK (1.25 micrograms/Kg/h). Despite this the acinar cells of rats treated with the lowest dose of caerulein retained a certain degree of secretory function since amylase activity in pancreatic juice was greater than in other groups of rats treated with higher doses of caerulein. Moreover, the percentage of increase observed in total protein and amylase in response to CCK respect to basal secretion is similar to that of the untreated animals. At higher doses (20 and 40 micrograms/Kg) the secretory capacity in response to CCK was inhibited. Therefore CCK administration in slight acute pancreatitis could be used as a therapy since it favours the secretion of pancreatic enzymes at percentual levels similar to those of the controls.

Exocrine pancreatic response to CCK in rats after long-term hydrocortisone administration.

The effects of prolonged administration of hydrocortisone (10 mg/kg/day) on exocrine pancreatic secretion were examined, analyzing the quantitative and qualitative variations in secretion at 7, 18 and 30 days of treatment. The weight of the pancreata was found to increase during the period of hydrocortisone treatment. After the 7th day of treatment the hormone significantly decreases basal exocrine pancreatic secretion, maintaining similar values up to the 30th day of treatment. Hydrocortisone enhances the pancreatic response to CCK since the percent increase in acinar secretion under stimulation compared to basal secretion surpassed control levels in all cases. However, the inhibitory effect on secretion shown by hydrocortisone opposes and surpasses the stimulatory effect of the secretagogue, secretion being reduced in the treated animals. Hydrocortisone especially affected the amylase fraction of the juice, to a larger extent inhibiting the isoenzyme A1, with an IEP of 8.5 when the animals were treated over 30 days; thus, a considerable reduction was observed in the amylase secreted both in resting conditions and under stimulation with CCK. There is a possibility that chronic treatment with glucocorticoids may sensitize the acinar cells, alter the composition of the pancreatic juice and inhibit secretion, effects that may involve pancreatic dysfunction.

Acinar cholecystokinin (CCK) receptors in the exocrine pancreas of the rat: effect of adrenalectomy.

The binding of [125I]BH-CCK-8 to membranes of acinar cells from rats at 6 and 21 days after adrenalectomy was studied. The optimum conditions of time and temperature were previously established as being 120 min and 30 degrees C. Under these conditions, the membranes of the adrenalectomized animals of both groups (6 and 21 days) bound more radioligand than those from control rats. However, a qualitative study of the binding showed that the affinities of binding were much lower; in particular, the high affinity receptors had a Kd of 0.94 +/- 0.33 nM in the controls and this was 14.9 +/- 1.29 nM in the 6-day adrenalectomized animals, although the maximum binding capacity did not vary significantly. However, in the case of the low affinity receptors, there was a gradual increase in the maximum binding capacity as the time after adrenalectomy progressed: 717 +/- 121, 1,987 +/- 183, and 10,175 +/- 862 fmol/mg for the control, 6-day, and 21-day adrenalectomized rats, respectively. In the latter situation, the high affinity receptors completely disappeared. These results, which coincide with a marked deficit in protein secretion already described in adrenalectomized rats, can be accounted for in terms of the possible negative cooperativity exerted by the low affinity receptors on the high affinity hormone-receptor complex, the protein secretion of the acinar cells normally mediated by the high affinity receptors becoming paralyzed.

Alpha-adrenergic influences on exocrine pancreatic secretion in the rabbit.

Action of phenylephrine (35 micrograms/Kg/min) alone or previously blocked by phentolamine (100 micrograms/Kg/min) on exocrine pancreatic secretion of anaesthetized rabbits has been studied, in basal state or under stimulation by secretin (1 C.U./Kg/h) or by the octapeptide of cholecystokinin (OP-CCK) (0.15 Ivy dog units/Kg/h). Phenylephrine increased arterial pressure. This effect was blocked by phentolamine. However no variations were seen in pancreatic blood flow in any of the experimental conditions assayed. Phenylephrine produced a secretin-like effect on hydroelectrolytic secretion in basal conditions. This action was maintained after the infusion of secretin but not after OP-CCK. This effect was not blocked by phentolamine. Phenylephrine increased protein secretion in the basal state, an action that was blocked by phentolamine. After secretin or OP-CCK stimulation phenylephrine did not increase protein secretion. It is concluded that phentolamine blocks the effects of phenylephrine on acinar cells but not on ductular cells.