Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

Multiple effects of Na+ removal on pancreatic secretion in vitro

Summary The effects of removing Na⁺ from the incubation medium on basal and secretagogue induced zymogen release by pancreatic fragments and isolated pancreatic acini were studied by both morphological evaluation and measurement of amylase release. In both fragments and isolated acini, removal of Na⁺ led to an increased basal secretion of zymogen granule contents from acinar cells via exocytosis; secretory material, however, accumulated in acinar and ductular lumina as a result of the lack of fluid secretion necessary to wash out the enzymes. In studies with fragments, after Na⁺ removal there was no significant increase in amylase release into the medium; isolated acini, in contrast, showed an increased amylase release consistent with the shorter distance from the acinar lumen to the bathing medium. Stimulation with either bethanechol or caerulein led to a further depletion of zymogen granules in both preparations; in the absence of Na⁺ secretory product accumulated in intracellular lakes as well as in duct lumens. The hypothesis that Na⁺ influx is important in stimulus-secretion coupling to release intracellular Ca²⁺ was directly tested by measuring ⁴⁵Ca²⁺ efflux. No effect of removing Na⁺ on ⁴⁵Ca²⁺ efflux was seen. It was concluded, therefore, that while Na⁺ is essential for pancreatic fluid secretion, it is not necessary for the secretion of zymogen granule contents into acinar lumina.Supported by NIH grant GM-19998 from the United States Public Health Service     

Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.     

Impairment of pancreatic acinar function by reserpine in vivo and in vitro

Summary Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired pancreatic secretion, and decreased pancreatic content of amylase. The mechanisms of altered acinar function and decreased amylase content in both CF and the reserpine-treated rat are unknown. To examine this alteration, the rate of [³H]phenylalanine (phe) incorporation into cellular protein was determined in pancreatic acinar cells after reserpine treatment of rats in vivo (7 d) and of cells in vitro (1 to 24 h). Acinar cells isolated from control, chronic reserpine-treated, and pair-fed rats were incubated in vitro with 0, 30, 50, or 100 μM reserpine. Reserpine treatment in vitro for 24 h of acinar cells from control rats significantly decreased amylase activity (20 to 70%), an effect similar to that of reserpine treatment in vivo. In vivo, reserpine treatment decreased [³H]phe incorporation (disintegrations per minute per milligram protein) 56% in freshly isolated cells, but did not alter intracellular specific activity (disintegrations per minute per nanomole phe, SA) of [³H]phe. Reserpine treatment (30 and 50 μM) in vitro for 1 h also decreased [³H]phe incorporation by freshly isolated cells from control (53 to 85%) and pair-fed (40 to 68%) rats. Reserpine treatment for 24 h in vitro significantly decreased [³H]phe incorporation by cells from control (82 and 98%), pair-fed (80 and 95%), and chronic reserpine-treated (90 and 97%) rats as compared with cells from respective in vivo treatments cultured with no reserpine. In vitro reserpine treatment also decreased the intracellular SA of [³H]phe in freshly isolated cells from control (14 and 36%) and pair-fed (35 and 39%) rats and in cultured cells from control (11 and 86%), pair-fed (60 and 88%), and chronic reserpine-treated (49 and 76%) rats. However, these alterations of SA by reserpine did not account for the decreased incorporation of [³H]phe into acinar protein, which remained significantly lower (70 to 88%) when expressed as total phe incorporation. These results suggest (a) that reserpine acts directly on acinar cells to alter function and (b) that the ability of the pancreas to synthesize digestive enzymes may be impaired in this model of cystic fibrosis. This study was supported in part by the Cystic Fibrosis Foundation, Bethesda, MD.     

Phenotypic Modulation of Hamster Acinar Cells by Culture in Collagen Matrix

The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells. Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen. Four groups were established: Medium 1—5% NuSerum + basic medium (basic MEDIUM = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2—10% NuSerum + basic medium. Medium 3—Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:—Medium 3 supplemented with soybean trypsin inhibitor. Freshly isolated acinar cells were retrieved morphologically intact. In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture. Immunostaining for amylase was observed at the apical pole of the cells. The remaining cells showed variable degrees of degeneration. In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated. A few cystic structures were also observed. Positive immunostaining for amylase was limited to the cells with a normal histological appearance. The cells grown in Media 3 and 4 had similar courses of morphological changes. After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells. Some amylase-positive immunoreactive cells were integral components of the cystic wall. Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4. Uptake of [³H]thymidine did not show any significant differences between the media. It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells. This study shows that the acinar cell phenotype can be maintainedin vitrofor more than 1 month. This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.     

Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.     

GTPγs对柴胡皂甙(I)刺激胰腺腺泡酶分泌的影响

为了解柴胡皂甙(I)[SA(I)]刺激大鼠胰腺腺泡酶分泌的信号传导通路,研究了GTPγs对SA(I)剌激通透腺泡细胞酶分泌的影响.用SLO通透细胞的同时,加入GTPγs在15min期间能诱发酶分泌,10'mol·L-1GTPγs有最大促泌效应.GTPγs浓度依赖性的增强SA(I)促酶分泌作用,l0-7 mol·L-1 GTPγs导致10-5mol·L-1 SA(I)刺激酶分泌量增加到1.6倍.用SLO预通透腺泡10min后,加入GTPγs使SA(I)刺激酶分泌的量-效曲线左移,SA(I)的EC50从2 0×10-5mol·L-1减小到1 0×10-5mol·L-1.以上结果提示,SA(I)活化受体偶联的G蛋白包括在其刺激酶分泌的信号传导通路中.     

Effects of secretin and caerulein on enzymes of cultured pancreatic acinar cells

Summary We examined the effects of secretin (0 to 200 nM) and caerulein (0 to 100 nM) on rat pancreatic acinar cells cultured 0 to 48 h in serum-free medium. The effects of 100 nM secretin with 1 nM caerulein were also studied because secretin may potentiate the effects of caerulein. Cellular and media (secreted) lipase and amylase were analyzed as were cellular DNA and protein content. Cellular lipase and amylase activities significantly decreased (P<0.0001) over time in all treatment groups, whereas media amylase and lipase significantly increased (P<0.0001). Neither secretin nor caerulein affected cellular lipase or media amylase. However, secretin significantly increased (P<0.04) and caerulein tended to increase (P<0.08) media lipase in a dose-dependent manner. At 12 h, 10 nM secretin maximally increased media lipase (58%), suggesting that cultured acinar cells remain responsive to secretin in vitro. Caerulein, at all concentrations, significantly decreased (P<0.001) cellular amylase but exhibited a dose-dependent effect only at 24 h when 100 nM caerulein maximally decreased cellular amylase (34%). Secretin (100 nM) did not alter these effects of caerulein. These results support the proposed role of caerulein in the regulation of amylase but not a direct role of secretin in the regulation of lipase. This study was supported in part by grant RO1 DK32690 from the National Institutes of Health, Bethesda, MD.     

Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular mileu within minutes. The accessability to these markers seemed independent of the ionised Ca²⁺ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10^?9 M Ca²⁺, but typically 4% was released when the Ca²⁺ level was subsequently raised to 10^?5M levels, the EC₅₀ for Ca²⁺ being 2 μM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca²⁺ concentration. No amylase was released at 10^?7M Ca²⁺ when permeable cells were exposed to cyclic 3′,5′-AMP or cyclic 3′,5′-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca²⁺ in the presence of the phorbol ester 12-O-tetradecanoyl phorbol 13 acetate. These results suggest that when “functionally normal” isolated acinar cells are rendered permeable, Ca²⁺ — but not cyclic nucleotides — acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.     

In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells

Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.     

Pancreatic acinar cell necrosis with intact storage of digestive enzymes in selenomethionine treated rats

Morphological and biochemical changes were observed in the pancreas and serum of rats after the intraperitoneal administration of selenomethionine, sodium selenite and methionine. Selenomethionine caused rapidly developing acinar cell necrosis. The first pathological changes were mitochondrial swelling and flocculent densities, and dilatation of cisternae of the endoplasmic reticulum. Zymogen granules appeared disrupted only in disintegrated acinar cells. Signs of autodigestive pancreatic inflammation with fat necrosis, elevation of pancreatic phospholipase A2 and serum amylase activities, as well as pulmonary oedema were present. Sodium selenite caused similar histologic changes to those produced by selenomethionine, but no changes were seen after methionine administration. Destruction of pancreatic acinar cells by an intraductal oleic acid injection that resulted in exocrine atrophy did not prevent systemic selenomethionine toxicity. Our results show that selenomethionine causes pancreatic acinar cell necrosis and that intracellular transport and storage of digestive enzymes is not primarily altered by this chemical.     

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Summary The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.Supported by NIH grant GM-19998 from the United States Public Health Service. — We are indebted to Dr. John Heuser for advice throughout this project and assistance in the preparation of rapidly frozen acini, and to M. Lee for technical assistance     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in isolated pancreatic acini of guinea pigs and rats

In this study, we investigated the immunocytochemical distribution of NK-1 and NK-3 tachykinin receptors in guinea pig and rat isolated pancreatic acini. In dispersed acinar cells from guinea pig, immunofluorescence staining detected similar densities of NK-1 and NK-3 receptors; conversely, rat acinar cells expressed NK-1 receptors more strongly than NK-3 receptors. In line with previous functional studies, these immunocytochemical findings suggest that guinea pig NK-1 and NK-3 receptors and rat NK-1 receptors alone play a direct stimulatory role in the basal pancreatic acinar amylase release.     

Inhibition of CCK or carbachol-stimulated amylase release by nicotine

This study was undertaken to investigate the mechanisms of action of nicotine on receptor mediated enzyme secretion in isolated rat pancreatic acini. Acinar cells were isolated from untreated and nicotine treated rats by collagenase digestion and differential centrifugation. Cells from the untreated animals were incubated with either varying concentrations of nicotine (range 10 microM to 30 mM) or with a fixed dose of 10 mM nicotine with varying concentrations of carbachol(10nM to 100 microM). Cells from the nicotine treated animals(16 weeks in drinking water) were incubated with either a fixed dose of CCK-8(10(-10) M) or carbachol(10(-5) M). All incubations were conducted at 37 C for 30 min. Amylase released in the media was measured by spectrophotometry. In pancreatic acinar cells isolated from control rats, amylase release stimulated by carbachol was inhibited by nicotine. Acinar cells isolated from rats treated with nicotine at nicotine concentrations of 1.23 mM also showed significant inhibition of amylase release in response to CCK-8 and carbachol compared to their identical controls. Nicotine induced inhibition curves of amylase release stimulated by carbachol were non-parallel suggesting that the effect of nicotine on acinar cells is regulated by mechanisms other than carbachol receptors. Nicotine may have a direct inhibitory effect on the intracellular mechanisms of pancreatic enzyme secretion. We conclude that the mechanism by which nicotine inhibits pancreatic enzyme secretion is complex.     

Mouse pancreatic acinar/ductlar tissue gives rise to epithelial cultures that are morphologically,biochemically, and functionally indistinguishable from interlobular duct cell cultures

Summary Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.