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Background/aim The aim of this study was to investigate the administration of cinnamon extract that is known to be effective in decreasing the high blood glucose and the distribution of NGF and Trk-A receptor in pancreas with immunohistochemistry way. Materials and methods The experimental groups were defined as control, sham, cinnamon, diabetes, and diabetes-cinnamon. At the end of the experiment, the pancreatic tissue samples were obtained for the rats. The hematoxylin-eosin and triple staining were used to examine histology. The immunohistochemical methods were performed on the sections of pancreatic tissue. In all groups, the body weight and fasting blood glucose obtained from the male and female rats and the values were statistically evaluated. Results The NGF immunoreactivity was observed in acinus, excretory pars, excretorius ducts, and islets of Langerhans for the pancreatic tissues of female and male rats in all groups. The Trk-A immunoreactivity was observed in acinus and islets of Langerhans for the pancreatic tissues of female and male rats in the control, sham, and cinnamon groups. Conclusion As a result, it was determined that the cinnamon, which is effective on blood glucose levels, has a positive effect on the NGF production in pancreas.

BACKGROUND: Acute pancreatitis (AP) is a widespread disease resulting from the inflammation of acinar cells in the pancreas. β-hydroxybutyrate (BHB) is a water-soluble main ketone body synthesized in the human liver. The purpose of this study was to examine the possible therapeutic effects of BHB in the experimentally-induced AP model in rats. METHODS: In our study, male rats were randomly allotted into 6 groups, as control (0.9% saline i.p.), BHB1 (200 mg/kg BHB i.p.), BHB2 (2 doses of 200 mg/kg BHB i.p.), AP (4 doses of 50 µg/kg cerulein i.p., 4 doses at 1 h intervals), AP+BHB1 and AP+BHB2 groups. In pancreatic tissue sections, immunohistochemistry staining and western blot analysis for the inflammasome complex (caspase-1, ASC, and NLRP3) and inflammation-associated proteins (TNF-α and NF-κB) and a histopathological examination were performed. The levels of lipase, amylase, interleukin (IL)-18 and IL-1β in serum were measured. RESULTS: Several pathological degenerations, including edema, inflammatory cell infiltration, acinus necrosis, and bleeding were observed in the AP group, while the histological architecture of the control and the sham BHB1 and BHB2 groups were regular. The AP-induced pathological changes were considerably alleviated in the AP+BHB1 and AP+BHB2 groups. In the AP group, a conspicuous increase in caspase-1, ASC, NLRP3, TNF-α, and NF-κB proteins, and in the levels of amylase, lipase, IL-18, and IL-1β were detected. BHB treatments after AP induction decreased those proteins to the level of control. CONCLUSIONS: We demonstrated that BHB has the potential to cure AP by suppressing the NLRP3 inflammasome and can be used in the treatment of many diseases which progress through the NLRP3 inflammasome.

Aim: The aim of this prospective animal study is to investigate the influence of multiple administrations of macrocyclic ionic (gadoteric acid) and linear nonionic (gadodiamide) gadolinium-based contrast agents (GBCA) on submandibular gland tissue (SGT) of the rats. Material and method: Twenty-four Sprague Dawley female rats were included the study. Group 1 was determined as a control group (n = 6). Group 2 was determined as saline group (n = 6). Group 3 was determined as Omniscan group (n = 6) and received only intraperitoneal (IP) 0.1 mmol (0.2 mL/kg)/kg gadodiamide for 8 days. Group 4 was determined as Dotarem group (n = 6) and received only IP 0.1 mmol (0.2 mL/kg)/mg/kg gadoteric acid daily for 8 days. On the 9th day of the administration, the rats were sedated with ketamine and xylazine through IP injection. The right SGT was removed after sedation. Histopathological and immunohistochemical changes in SGT were evaluated. Results: The SGT of the Omniscan and Dotarem groups decreased SGT acini surface area, and serous acinar cells number were observed. On the other hand, no pathology was observed. Mucous acinar cells’ caspase-3 positivity for the same markers in Omniscan and Dotarem sections was similar to the control group. However, Omniscan and Dotarem groups serous acinar cells were caspase-3 (+) staining. The intensity of serous acinar cells’ caspase-3 (+) for the same markers in Dotarem sections was similar to the Omniscan group. The results also revealed in the analysis of the mean area of the acinus area of the SGT; there were significantly decreased Dotarem group rats when compared to control rats (p < 0.05). Conclusion: We consider that numerical increased apoptosis results arise from repeated doses of GBCAs. Being aware of this effect of the contrast agent may have significance for the chronic sialo-adenitis patients group when used for recurrent contrasted MRI for diagnosis of diseases like MS which requires in follow-up. We should be aware about the frequently contrasted MRI in routine investigations. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.

We studied the efficiency of dihydroquercetin on the model of chronic nonbacterial inflammation of the prostatic gland in rats. It was found that administration of dihydroquercetin was followed by a significant decrease in the area of the connective tissue in the prostatic gland to initial levels, which attested to antifibrotic properties of this oxidant. Additionally, the substance prevented the development of atrophy of acinus epithelium. After administration of reference drug Prostamol Uno, only moderate antifibrotic effects were observed.

Background/Aims This study investigated the protection provided by gabexate mesylate thermo-sensitive in-situ gel (GMTI) against grade III pancreatic trauma in rats. Methods A grade III pancreatic trauma model with main pancreatic duct dividing was established, and the pancreas anatomical diagram, ascites, and serum biochemical indices, including amylase, lipase, C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), were examined. The pancreas was sliced and stained with hematoxylin eosin and subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results Ascites, serum amylase, lipase, CRP, IL-6, and TNF-α levels were significantly increased in the pancreas trauma (PT) groups with prolonged trauma time and were significantly decreased after GMTI treatment. The morphological structure of the pancreas was loose, the acinus was significantly damaged, the nuclei were irregular and hyperchromatic, and there was inflammatory cell invasion in the PT group compared to the control. After GMTI treatment, the morphological structure of the pancreas was restored, and the damaged acinus and inflammatory cell invasion were decreased compared to the PT group. Moreover, the cell apoptosis index was significantly increased in the PT group and restored to the same levels as the control group after GMTI treatment. Conclusions GMTI, a novel formulation and drug delivery method, exhibited specific effective protection against PT with acute pancreatitis therapy and has potential value as a minimally invasive adjuvant therapy for PT with acute pancreatitis.

The liver is a center of metabolic activity, including the metabolism of drugs, and consequently is prone to drug-induced liver injury. Failure to detect hepatotoxicity of drugs during their development will lead to the withdrawal of the drugs during clinical trials. To avoid such clinical and economic consequences, in vitro liver models that can precisely predict the toxicity of a drug during the pre-clinical phase is necessary. This review describes the different technologies that are used to develop in vitro liver models and the different approaches aimed at mimicking different functional aspects of the liver at the fundamental level. This involves mimicking of the functional and structural units like the sinusoid, the bile canalicular system, and the acinus.

The research material included 96 slaughter rabbit carcasses. Half of them came from the animals managed in small-scale backyard farming units where animals were fed a natural ingredient diet, while the other half was from rabbits kept under commercial production conditions and fed commercial rabbit pellets. The thigh and saddle muscle samples were collected from each carcass to establish a content of ubiquinone (CoQ10) and crude protein along with its collagen level. Determination of tissue coenzyme Q10 (UQ10) was carried out by high-performance liquid chromatography with some modification. Crude protein concentration was estimated using Kjeldahl procedure, while total collagen content by the method of Stegemann modified by Hurych-Chvapil, using hydrolysis according to Mohler and Volley. Ubiquinone level in slaughter rabbit tissue ranged between 76 and 127 μg/g tissue. The studies indicated that rabbit rearing system and muscle type are determinants of CoQ10 content. Meat of rabbits managed under the traditional backyard farming system exhibited higher CoQ10 concentration as compared to that determined in rabbits from the commercial rabbitry. Additionally, the CoQ10 level in the saddle was significantly higher than that in the thigh muscles, and the relationships was noted in both types of rabbit production systems. When the CoQ10 content was expressed per gram of fibrillar protein, there were not significant differences between saddle and thigh muscles. The correlation coefficient between ubiquinone and fibrillar protein averaged to 0.94. The studies also demonstrated a higher protein level in the saddle than in thigh muscles. However, no differences in protein concentration were reported in respect to the rabbit farming system. The protein composition in the saddle muscles, irrespective of a rabbit production system, revealed significantly lower collagen content compared to the proteins in thigh muscles. The obtained results and data from literature provide evidence that rabbit meat, especially from the traditional (organic) management system, is one of the best sources of animal protein and ubiquinone Q10.

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR ( P < 0.001 vs. WKY), and diabetes decreased this activity (∼50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Livers of four young, male, random-bred rats weighing 250 g were fixed in formalin and stained with Feulgen. In each section, 50 hepatocytes were randomly selected and their distance from the nearest terminal hepatic vein was measured with an eye-piece micrometer. Distance was expressed in two units: 1. micrometer and, 2. cell location, or the cell number separating a sampled cell from the terminal hepatic rim. The sections were scanned with an image cytometer. The tissue was magnified x 400 and digitized into 512 x 512 pixels. The software determined the nuclear boundary and estimated its area and optical absorbance, which was expressed in relative DNA units. One unit equals the absorbance of a diploid lymphocyte. Nuclear area and optical absorbance measurements were made up to 220 microns distance from the terminal hepatic vein which roughly covers zones 2 and 3. Previously we have shown that in zones 1 and 2 hepatocyte nucleus enlarges and becomes polyploid. The present study demonstrates that in zone-3 this trend is reversed, nuclear area and DNA absorbance decline. The average hepatocyte advances 2 microns daily. In zone-1 it accumulates daily 0.005 DNA absorbance units and in zone-3 it loses daily 0.24 DNA absorbance units. It is proposed that in zone-3 DNA leaves the intact cell in a physiological way. DNA accumulation and polyploid are regarded here as forms of gene amplification which may be reversed, so that under these circumstances amplified DNA may be "deamplified" and the excess DNA leaves the cell.

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca2+/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X7 receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.

Histone H2B phosphorylation tightly correlates with chromatin condensation during apoptosis. The caspase-cleaved acinus (apoptotic chromatin condensation inducer in the nucleus) provokes chromatin condensation in the nucleus, but the molecular mechanism accounting for this effect remains elusive. Here, we report that the active acinus p17 fragment initiates H2B phosphorylation and chromatin condensation by activating protein kinase C delta isoform (PKC-delta). We show that p17 binds to both Mst1 and PKC-delta, which is upregulated by apoptotic stimuli, enhancing their kinase activities. Acinus mutant susceptible to degradation elicits stronger chromatin condensation and higher H2B phosphorylation than wild-type acinus. Dominant-negative PKC-delta but not Mst1 robustly blocks acinus-initiated H2B phosphorylation. Surprisingly, depletion of Mst1 triggers caspase-3 activation, provoking H2B phosphorylation through activating PKC-delta. Further, acinus-elicited H2B phosphorylation and chromatin condensation are abrogated in PKC-delta-deficient mouse embryonic fibroblast cells and siRNA-knocked down PC12 cells. Thus, PKC-delta but not Mst1 acts as a physiological downstream kinase of acinus in promoting H2B phosphorylation and chromatin condensation.

Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16beta-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.

Although clusters of alveoli form the acinus, which is the most distal respiratory unit, it is not known whether interalveolar communication coordinates acinar surfactant secretion. To address this, we applied real-time digital imaging in conjunction with photo-excited Ca2+ uncaging in intact alveoli of the isolated, blood-perfused rat lung. We loaded alveolar cells with the Ca2+ cage o-nitrophenyl EGTA-AM (NP-EGTA-AM) together with the fluorophores, fluo 4, or LysoTracker green (LTG) to determine, respectively, the cytosolic Ca2+ concentration ([Ca2+]cyt) or type 2 cell secretion. To uncage Ca2+ from NP-EGTA, we exposed a region in a selected alveolus to high-intensity UV illumination. As a result, fluo 4 fluorescence increased, whereas LTG fluorescence decreased, in the photo-targeted region, indicating that uncaging both increased [Ca2+]cyt and induced secretion. Concomitantly, [Ca2+]cyt increases conducted from the uncaging site induced type 2 cell secretion in both the selected alveolus as well as in neighboring alveoli, indicating the presence of interalveolar communication. These conducted responses were inhibited by pretreating alveoli with the connexin43 (Cx43)-inhibiting peptides gap 26 and gap 27. However, although the conducted [Ca2+]cyt increase diminished with distance from the uncaging site, type 2 cell secretion rates were similar at all locations. We conclude that Cx43-dependent, interalveolar Ca2+ signals regulate type 2 cell secretion in adjacent alveoli. Such interalveolar communication might facilitate acinar coordination of alveolar function.

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.

Programmed cell death has been linked to AMPA-receptor-mediated excitotoxicity in pyramidal neurons of the hippocampus. The intent of this study was to investigate the roles of caspase-dependent and independent nuclear death-related factors in mediating AMPA-induced nuclear changes in PyNs by use of immunohistochemistry and transmission electron microscopy (TEM). Data indicate increases in the nuclear levels of caspase-activated acinus and DNase and Endonuclease G (a caspase-independent endonuclease) in CA1 and CA3 PyN nuclei with different temporal patterns following an AMPA-insult. Hoechst staining and TEM confirm AMPA-induced chromatin condensation. The presence of active acinus in nuclei suggests it mediates chromatin condensation. Interestingly, a DNA fragmentation labeling protocol showed that there was no chromatin cleavage up to 90 min after AMPA-insult. Overall, we conclude that: 1) AMPA-induced excitotoxicity increases nuclear immunoreactivity of pro-death enzymes from multiple programmed cell death pathways, 2) differential chromatin condensation patterns occur between CA1 and CA3, and 3) there is no chromatin cleavage within our experimental timeframe.

Olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated. This study aimed to clarify the mechanism of pancreatic adaptation to dietary fat. For this purpose, we examined whether dietary-induced modification of pancreatic membranes affects acinar cell function in response to the secretagogue acetylcholine (ACh). Weaning male Wistar rats were assigned to one of two experimental groups and fed for 8 weeks with a commercial chow (C) or a semisynthetic diet containing virgin olive oil as dietary fat (OO). The fatty acid composition of pancreatic plasma membranes was determined by gas-liquid chromatography. For assessment of secretory function, viable acini were incubated with ACh and amylase of supernatant was further assayed with a substrate reagent. Changes in cytosolic Ca(2+) concentration in response to ACh were measured by fura-2 AM fluorimetry. Compared to C rats, pancreatic cell membranes of OO rats had a higher level of monounsaturated fatty acids and a lower level of both saturated and polyunsaturated fatty acids, thus, reflecting the type of dietary fat given. Net amylase secretion in response to ACh was greatly enhanced after OO feeding, although this was not paralleled by enhancement of ACh-evoked Ca(2+) peak increases. In conclusion, chronic intake of diets that differ in the fat type influences not only the fatty acid composition of rat pancreatic membranes but also the responsiveness of acinar cells to ACh. This mechanism may be, at least in part, responsible for the adaptation of the exocrine pancreas to the type of fat available.

Akt promotes cell survival by phosphorylating and inhibiting components of the intrinsic cell death machinery. Akt translocates into the nucleus upon exposure of cells to survival factors, but little is known about its functions in the nucleus. Here, we show that acinus, a nuclear factor required for apoptotic chromatin condensation, is a direct target of Akt. We demonstrate that Akt phosphorylation of acinus on serine 422 and 573 results in its resistance to caspase cleavage in the nucleus and the inhibition of acinus-dependent chromatin condensation. Abolishing acinus phosphorylation by Akt through mutagenesis accelerates its proteolytic degradation and chromatin condensation. Acinus S422, 573D, a mutant mimicking phosphorylation, resists against apoptotic cleavage and prevents chromatin condensation. Knocking down of acinus substantially decreases chromatin condensation, and depletion of Akt provokes the apoptotic cleavage of acinus. Thus, Akt inhibits chromatin condensation during apoptosis by phosphorylating acinus in the nucleus, revealing a specific mechanism by which nuclear Akt promotes cell survival.

Cancer cachexia is a syndrome that causes profound metabolic disruption. Lipid metabolism in the liver is markedly affected. We investigated the effect of cachexia upon liver-acinus lipid-metabolism zonation in Walker 245 carcinosarcoma-bearing rats (TB). The expression of protein (by Western blotting) and mRNA (by semi-quantitative polymerase chain reaction) of the enzymes of the carnitine palmitoyltransferase system (CPT I and CPT II) and of liver fatty-acid-binding protein (L-FABP) was studied. Although no changes were found for these parameters, the maximal activities (by radioassay) of CPT I and II were reduced (P

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage ( Vm) and intracellular concentrations of Ca2+, Na+([Na+]c), Cl−, and H+(pHi) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 μM, 20 s), RSMGA showed strong delayed hyperpolarization ( Vh,ACh; −95 ± 1.8 mV) that was abolished by ouabain. In the HCO3−-free condition, the Vh,AChwas also blocked by bumetanide, a blocker of Na+-K+-2Cl−cotransporter (NKCC). In the presence of HCO3−(24 meq, bubbled with 5% CO2), however, the Vh,AChwas not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na+/H+exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na+]cwas totally blocked by bumetanide in the absence of HCO3−, but only by one-half in the presence of HCO3−. ACh induced a prominent acidification of pHiin the presence of HCO3−, and the acidification was further increased by EIPA treatment. Without HCO3−, an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pHiduring the application of NH4+(20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO3−. In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na+]c. The intracellular Na+-induced activation of electrogenic Na+/K+-ATPase causes Vh,ACh. The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO3−.

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca2+-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 ± 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2α, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.

The effects of glucagon on the subacinar distribution of hepatic transmembrane potentials were studied in the perfused fasted rat liver. The livers were perfused with a Krebs-Henseleit buffer, and membrane potentials of matched periportal and pericentral hepatocytes were determined using glass microelectrodes. Lactate- and pyruvate-induced glucose production and O2 uptake were potentiated by 10 �8 M glucagon. Twenty-five micromoles 8-bromoadenosine 3,5cyclic monophosphate (8-BrcAMP) exhibited stimulatory effects similar, in terms of glucose production and O2 uptake to those of glucagon. Octanol (0.1 and 0.5 mM) had no effect on glucose production but reversibly increased O2 uptake by 16% to 30% over all experiments. Under basal conditions (no exogenous substrate) hepatocyte membrane potentials averaged approximately �27 mV, and no gradients were found between periportal and pericentral hepatocytes. Addition of lactate and pyruvate produced hyperpolarization in all hepatocytes. However, there was a small but statistically significant gradient produced across the hepatic acinus in membrane potential, i.e., the hyperpolarization was higher in the periportal region compared with the pericentral region. Glucagon and 8-BrcAMP induced marked hyperpolarization in periportal and pericentral hepatocytes with no gradients across the acinus. Although no changes were found under basal and lactate plus pyruvate, 0.5 mM octanol induced heterogeneity of membrane potential during glucagon and 8-BrcAMP stimulation. Our findings suggest that glucagon-induced homogeneity of membrane potential may be mediated by increased gap junctional coupling. In addition, cAMP may be responsible for the increase in the intercellular communi- cation during glucagon stimulation. KEYWORDS—Gap junction, intercellular communication, membrane potential, glucagon, cAMP, octanol

In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl- channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl- secretion, the intracellular Cl- concentration ([Cl-]i) of RPAs was monitored using SPQ, a fluorescent dye quenchable by Cl-, and the effects of K+ channel blockers were examined. Also, the secretion of fluid and enzyme from the whole pancreas of rat was measured. The fluorescence of RPAs loaded with SPQ (FSPQ) was slightly increased by the application of ACh (ACh-Delta FSPQ), indicating net secretion of Cl-. However, the relative change of FSPQ normalized to the control fluorescence (F/F0) of RPAs was only about 20% of the effect observed in rat submandibular gland acinus. The ACh-Delta FSPQ of RPAs was not influenced by the pretreatment with 293B (20 micromol/L), a blocker of KCNQ-type K+ channels. Even the cocktail of K+ channel blockers (10 mmol/L TEA, 3 mmol/L Ba2+, 20 micromol/L 293B) exerted only minute inhibitory effects on ACh-Delta FSPQ in RPAs. In the vascularly perfused rat pancreas, the fluid and enzyme secretion induced by ACh was directly measured. 293B and HMR-1556, both specific blockers of KCNQ1 channel, did not block but even enhanced the secretion of fluid and amylase. These results suggest that the role of KCNQ1 channels may not be essential in the Ca2+-mediated Cl- secretion in rat pancreatic acini.

The present study aimed to elucidate the prenatal development of the rat palatine gland. Parasagittal 5 μm thick serial sections made from Wistar rats at embryonic days (E) 17 to 22 were stained with haematoxylin-eosin (HE), Alcian blue-Kernechtrot or immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) as a marker of proliferating cells. Additionally, three-dimensional images of developing glandular parenchyma were reconstructed from serial HE sections with a personal computer. At E 17, several thickenings of the palatal epithelium had appeared which thereafter became the epithelial cords. Branching and lumenization commenced at E 20, and immature acini were observed at E 21. Three-dimensional reconstruction showed that the proximal part of the epithelial cord differentiated into the duct, and the distal part of the epithelial cord differentiated into the acinus. In immunohistochemical staining, there were many BrdU-positive cells in the epithelial cords including thickenings of the palatal epithelium, ducts, and acini. The BrdU labeling index of the cells of the epithelial cord was the highest (statistically significant) of the three in the primitive palatine gland. In conclusion, during the development of the rat palatine gland, epithelial cords with very high proliferative activity arise from the palatal epithelium, and then the proximal part of the epithelial cord differentiates into the duct, and the distal part of the epithelial cord differentiates into the acinus. Proliferation of these glandular parenchyma contributes to the growth of the developing palatine gland.

SK4/IK1 encodes an intermediate conductance, Ca2+-activated K+ channel and fulfills a variety of physiological functions in excitable and nonexcitable cells. Although recent studies have provided evidence for the presence of SK4/IK1 channels in salivary acinar cells, the regulatory mechanisms and the physiological function of the channel remain unknown in these cells. Using molecular and electrophysiological techniques, we examined whether cytosolic ATP-dependent regulation of native SK4/IK1-like channel activity would involve endogenous cAMP-dependent protein kinase (PKA) in rat submandibular acinar (RSA) cells. Electrophysiological properties of tetraethylammonium (TEA) (10 mM)-insensitive, Ca2+-dependent K+ currents in macropatches excised from RSA cells matched those of whole cell currents recorded from human embryonic kidney-293 cells heterologously expressing rat SK4/IK1 (rSK4/IK1) cloned from RSA cells. In outside-out macropatches, activity of native SK4/IK1-like channels, defined as a charybdotoxin (100 nM)-blockable current in the presence of TEA (10 mM) in the bathing solution, ran down unless both ATP and Mg2+ were present in the pipette solution. The nonhydrolyzable ATP analog AMP-PNP failed to support the channel activity as ATP did. The addition of Rp-cAMPS (10 μM), a PKA inhibitor, to the pipette solution containing ATP/Mg2+ induced a rundown of the Ca2+-dependent K+ currents. Inclusion of cAMP (1 mM) into the pipette solution (1 μM free Ca2+) containing ATP/Mg2+ caused a gradual increase in the currents, the effect being pronounced for the currents induced by 0.1 μM free Ca2+. Forskolin (1 μM), an adenylyl cyclase activator, also increased the currents induced by 0.1 μM free Ca2+. In inside-out macropatches, cytosolic ATP/Mg2+ increased both the maximum current (proportional to the maximum channel activity) and Ca2+ sensitivity of current activation. Collectively, these results suggest that ATP-dependent regulation of native SK4/IK1-like channels, at least in part, is mediated by endogenous PKA in RSA cells.

A rat strain carrying the human c-Ha-ras protooncogene, established by our laboratory, is highly susceptible to mammary chemical carcinogens. The transgenic rats exhibit increased number of terminal endbuds (TEBs) at the tips of developing ducts in the mammary gland compared to non-transgenic littermates. Confocal microscopy revealed the level of active mitogen-activated protein kinase to be elevated in these TEBs, and a close correlation between their numbers and tumorigenic response initiated by 7,12-dimethylbenz[a]anthracene was confirmed. Single injections of N-methyl-N-nitrosourea into the transgenic rats caused mutations in codon 12 of human c-Ha-ras transgene in TEBs before tumor development, supporting the conclusion that these structures are the major targets of chemical carcinogens. In contrast, with spontaneous development of lesions, alveolar hyperplasia with elevated expression levels of rat and human c-Ha-ras protooncogenes is the first morphological alteration which becomes apparent. Some but not all hyperplastic alveolar nodules were found to harbor mutations in the transgene. The results indicate that elevated expression of c-Ha-ras protooncogene is sufficient in itself to cause a highly proliferative phenotype of mammary alveoli. Our data suggest that TEBs and acini are the major targets for chemical and sporadic carcinogenesis, respectively, in the mammary glands of human c-Ha-ras protooncogene transgenic rats.

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 μM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112–2117, 2002). Investigation of the effects of phorbol ester (12- o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 μM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

Although Ca2+-activated K+(KCa) channels distinct from maxi-K+channels have been suggested to contribute to muscarinically stimulated K+currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native KCacurrents in bovine parotid acinar (BPA) cells. Ca2+-dependent K+efflux from perfused bovine parotid tissues was not inhibited by a maxi-K+channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive KCaconductance, which was highly permeable to Rb+. In inside-out macropatches, TEA-insensitive Rb+currents were activated by Ca2+with half-maximal values of 0.4 μM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca2+-activated Rb+currents. In outside-out macropatches, Ca2+-activated Rb+currents were inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native KCacurrents in BPA cells.

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Organ function requires coordinated multicellular activities, which may require proper control of cell signalling dynamics at the supracellular level. By using high-speed confocal microscopy, we studied how calcium signalling is organised in the dissociated rat parotid gland. Salivary gland function is accomplished primarily by the compartmentalized epithelial domains, acini and ducts, the former involved in the production of primary saliva and the latter involved in its modification. Upon muscarinic stimulation with carbachol, both domains showed an increase in intracellular free calcium concentration([Ca2+]i) with distinctive spatiotemporal kinetics, as indicated by the fluo-3 fluorescence. Acini responded initially, and the ducts followed with a time lag of more than 0.3 second. Cells comprising an acinus responded synchronously, whereas those in the ducts responded heterogeneously with respect to the latency period, magnitude of response and the requirement of extracellular calcium to raise [Ca2+]i. ATP also elicited a non-synchronous [Ca2+]i response in the duct domain, under a pattern different from that of carbachol. The synchronous oscillations seen in the acinar domain were made asynchronous by octanol, an agent known to inhibit gap-junction function. Accordingly, a gap junction component, connexin 32, was immunolocalised predominantly between the acinar cells. Moreover, expression of the type 2 inositol (1,4,5)-trisphosphate receptor[Ins(1,4,5)P3R] was homogeneous in the acinar domain but heterogeneous in the duct domain. Together, these data suggest that the calcium signalling system in salivary glands is constructed specifically according to the tissue architecture.

Pancreatic acinar cells depend on the integrity of the cytoskeleton for regulated secretion. Stimulation of isolated rat pancreatic acini with the secretagogue CCK serves as a model for human acute edematous pancreatitis. It induces the breakdown of the actin filament system (F-actin) with the consecutive inhibition of secretion and premature activation of digestive enzymes. However, the mechanisms that regulate F-actin breakdown are largely unknown. Plectin is a versatile cytolinker protein regulating F-actin dynamics in fibroblasts. It was recently demonstrated that plectin is a substrate of caspase 8. In pancreatic acinar cells, plectin strongly colocalizes with apical and basolateral F-actin. Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase 8, followed by degradation of plectin that in turn precedes the F-actin breakdown. Inhibition of caspase 8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion. Thus we propose that the caspase 8-mediated degradation of plectin represents a critical biochemical event during CCK-induced secretory blockade and cell injury.

Considered to be an etiologic factor of acute pancreatitis, hypersecretion of pancreatic juice and digestive enzymes is often associated with hyperbilirubinemia. We explored the intracellular mechanisms through which bilirubin affects pancreatic exocrine secretory function by examining the effect of bilirubin on isolated rat pancreatic acini. Bilirubin stimulated amylase release in a concentration- and time-dependent manner, significantly increasing amylase release at concentrations >5 mg/100 ml and after 15 min of incubation. Coincubation of bilirubin with vasoactive intestinal polypeptide, 8-bromo-cAMP, or A-23187 had a synergistic effect on amylase release, whereas coincubation with CCK-8, carbamylcholine, or 12- O-tetradecanoylphorbol 13-acetate had an additive effect. Bilirubin did not affect acinar cAMP content or Ca2+efflux. Intracellular Ca2+pool depletion had no influence on bilirubin-evoked amylase release. The protein kinase C (PKC) inhibitors staurosporine and calphostin C partially but significantly inhibited bilirubin-stimulated amylase release, whereas the PKA inhibitor H-89 did not. The tyrosine kinase (TK) inhibitor genistein, phospholipase A2(PLA2) inhibitor indoxam, and PLC inhibitor U-73122 also inhibited amylase release. Bilirubin significantly translocated PKC activity from the cytosol to the membrane fraction and activated TK in cytosol and membrane fractions. These results indicate that bilirubin stimulates amylase release by activating PKC and TK in rat pancreatic acini and that PLC and PLA2partly mediate this process.

Aims The effect of different modes of thiol depletion on pancreatic exocrine secretory function and potential mechanisms of interference with the secretory process in pancreatic acinar cells were investigated. Methodology After incubation with three thiol-modulating agents (L-buthionine sulfoximine, ethacrynic acid, and diamide) for 30 minutes, caerulein-stimulated amylase release and cholecystokinin (CCK) receptor binding characteristics were assessed in isolated rat pancreatic acini. The level of thiol groups (glutathione and protein thiols) and cytosolic-free calcium were measured in pancreatic acinar cells. Results All three thiol-modulating agents decreased caerulein (10(-10)M)-stimulated amylase release and the level of pancreatic acinar glutathione in a dose-dependent fashion without a marked increase in cell damage. Diamide also diminished the level of protein thiols. Ethacrynic acid and diamide, but not L-buthionine sulfoximine, inhibited the caerulein (10(-9)M)-induced Ca(2+) mobilization in pancreatic acinar cells. None of the three thiol-modulating agents altered the CCK receptor binding characteristics. Conclusion The present findings strongly suggest an important role of glutathione in the secretory process in pancreatic acinar cells and in the secretory blockade observed in acute pancreatitis. A decrease in caerulein-induced Ca(2+) mobilization might participate in the inhibition of amylase release by some oxidative agents, but it is not the prominent cause in general.

The structure of the gas exchange region of the human lung (the pulmonary acinus) undergoes profound change in the first few years of life. In this paper, we investigate numerically how the change in alveolar shape with time affects the rate of nanoparticle deposition deep in the lung during postnatal development. As human infant data is unavailable, we use a rat model of lung development. The process of postnatal lung development in the rat is remarkably similar to that of the human, and the structure of the rat acinus is indistinguishable from that of the human acinus. The current numerical predictions support our group's recent in vivo findings, which were also obtained by using growing rat lung models, that nanoparticle deposition in infants is strongly affected by the change in the structure of the pulmonary acinus. In humans, this major structural change occurs over the first 2 yr of life. Our current predictions would suggest that human infants at the age of ∼2 yr might be most at risk to the harmful effects of air pollution. Our results also suggest that dose estimates for inhalation therapies using nanoparticles, based on fully developed adult lungs with simple body weight scaling, are likely to overestimate deposition by up to 55% for newborns and underestimate deposition by up to 17% for 2-yr-old infants.

The pulmonary acinus is a gas exchange unit distal to the terminal bronchioles. A model of its structure is important for the computational investigation of mechanical phenomena at the acinus level. We propose a mathematical model of a heterogeneous acinus structure composed of alveoli of irregular sizes, shapes, and locations. The alveoli coalesce into an intricately branched ductal tree, which meets the space-filling requirement of the acinus structure. Our model uses Voronoi tessellation to generate an assemblage of the alveolar or ductal airspace, and Delaunay tessellation and simulated annealing for the ductal tree structure. The modeling condition is based on average acinar and alveolar volume characteristics from published experimental information. By applying this modeling technique to the acinus of healthy mature rats, we demonstrate that the proposed acinus structure model reproduces the available experimental information. In the model, the shape and size of alveoli and the length, generation, tortuosity, and branching angle of the ductal paths are distributed in several ranges. This approach provides a platform for investigating the heterogeneous nature of the acinus structure and its relationship with mechanical phenomena at the acinus level. Display Omitted We propose a mathematical model of a heterogeneous acinus structure.The model uses Voronoi and Delaunay tessellations and simulated annealing.The model presents the alveolar and ductal structure distributions.Applying the modeling technique to rat acinus demonstrates the availability.The approach provides a platform for investigating heterogeneous nature of acinus.

INTRODUCTION Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined. AIMS To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted. METHODOLOGY AND RESULTS High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin-phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE-stimulated amylase release from adenovirus-cytomegalovirus (AdCMV)-SNAP-23 or AdCMV-SNAP-25-infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE-evoked enzyme secretion from AdCMV-SNAP-23deltaC8- and AdCMV-SNAP-25(1-197)-infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV-SNAP-25AC8- or AdCMV-SNAP-25(1-197)-infected acini were co-infected with wild-type SNAP-23 or SNAP-25. CONCLUSION From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.

Background & Aims: The present study was undertaken to evaluate the role of serine proteases in regulating digestive enzyme secretion in pancreatic acinar cells. Methods: Isolated acini were stimulated by various secretagogues in the presence or absence of cell-permeant serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride and Nα-p-tosyl-L-phenylalanine chloromethyl ketone. F-actin distribution was studied after staining with rhodamine phalloidin. Results: Both cell-permeant serine protease inhibitors blocked amylase secretion in response to secretagogues that use calcium as a second messenger (e.g., cerulein, carbamylcholine, and bombesin) but not to those that use adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger (e.g., secretin and vasoactive intestinal polypeptide). Incubation of the acini with these inhibitors also resulted in a dramatic redistribution of the F-actin cytoskeleton. This redistribution was energy dependent. Similar redistribution of F-actin from the apical to the basolateral region was also observed when acini were incubated with a supramaximally stimulating concentration of cerulein, which is known to inhibit secretion. Conclusions: These results suggest that a serine protease activity is essential for maintaining the normal apical F-actin distribution; its inhibition redistributes F-actin from the apical to the basolateral region and blocks secretion induced by secretagogues that act via calcium. cAMP reverses the F-actin redistribution and hence cAMP-mediated secretion is not affected. GASTROENTEROLOGY 2001;120:1818-1827

Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-κB inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-κB in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.

Neutrophil infiltration into the pancreas is a key event in pancreatitis. Here we show that intercellular adhesion molecule-1 (ICAM-1), which regulates neutrophil adhesion, is present on rat pancreatic acinar cells, is upregulated by a hormone (cerulein) and mediates direct binding of neutrophils to acinar cells. ICAM-1 was upregulated in pancreas of rats with experimental pancreatitis induced by supramaximal doses of cerulein. Furthermore, cerulein time and dose dependently stimulated expression of ICAM-1 mRNA and protein in isolated pancreatic acinar cells. Inhibitory analysis showed that activation of transcription factor nuclear factor-κB (NF-κB) was involved in ICAM-1 upregulation by cerulein, but NF-κB did not mediate basal expression of ICAM-1 mRNA in acinar cells. With an adhesion assay, we found that neutrophils bind to isolated pancreatic acinar cells and that cerulein upregulates the extent of adhesion. Neutralizing ICAM-1 antibody blocked neutrophil binding to both control and cerulein-stimulated acinar cells, suggesting ICAM-1 involvement in this adhesion. Thus the acinar cell is capable of targeting neutrophils to its surface, a process that may be important for inflammatory and cell death responses in pancreatitis and other pancreatic disorders.

Aims/hypothesis. In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated.¶Methods. Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR.¶Results. During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeobox-containing transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a pan-neuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models.¶Conclusion/interpretation. Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation. [Diabetologia (2000) 43: 907–914]

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca2+-ATPase thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca2+-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca2+-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

Differences in particle deposition patterns between human and rat lungs may be attributed primarily to their differences in breathing patterns and airway morphology. Heterogeneity of lung structure is expected to impact acinar particle deposition in the rat. Two different morphometric models of the rat lung were used to compute particle deposition in the acinar airways: the multiple-path lung (MPL) model (Anjilvel and Asgharian, 1995, Fundam. Appl. Toxicol. 28, 41-50) with a fixed airway geometry, and the stochastic lung (SL) model (Koblinger and Hofmann, 1988, Anat. Rec. 221, 533-539) with a randomly selected branching structure. In the MPL model, identical acini with a symmetric subtree (Yeh et aL, 1979, Anat. Rec. 195, 483-492) were attached to each terminal bronchiole, while the respiratory airways in the SL model are represented by an asymmetric stochastic subtree derived from morphometric data on the Sprague-Dawley rat (Koblinger et al., 1995, J. Aerosol. Med. 8, 7-19). In addition to the original MPL and SL models, a hybrid lung model was also used, based on the MPL bronchial tree and the SL acinar structure. Total and regional deposition was calculated for a wide range of particle sizes under quiet and heavy breathing conditions. While mean total bronchial and acinar deposition fractions were similar for the three models, the SL and hybrid models predicted a substantial variation in particle deposition among different acini. The variances of acinar deposition in the MPL model were consistently much smaller than those for the SL and the hybrid lung model. The similarity of acinar deposition variations in the two latter models and their independence on the breathing pattern suggests that the heterogeneity of the acinar airway structure is primarily responsible for the heterogeneity of acinar particle deposition.

Pancreatic caerulein-induced activation of c-Jun NH2-terminal kinase (JNK) has been reported, and JNK has been proposed as a mediator during induction of hyperstimulated pancreatitis. CEP-1347 has recently been described as a specific JNK inhibitor. We tested whether CEP-1347 inhibits caerulein-induced pancreatic JNK activation in isolated acini and in vivo. CEP-1347 dose dependently inhibited acinar caerulein-induced JNK activation with nearly complete inhibition at 2 μM but had no effect on digestive enzyme release. For in vivo studies, rats were pretreated with CEP-1347 before caerulein hyperstimulation. For assessment of JNK activation and histological alterations, animals were killed 30 min or 2 and 4 h after caerulein hyperstimulation, respectively. Pancreatic wet weight, serum enzyme levels, and pancreatic activity of p38 and extracellular signal-regulated kinase (ERK) were also determined. Caerulein hyperstimulation strongly activated JNK, p38, and ERK. CEP-1347 pretreatment dose dependently reduced caerulein-induced pancreatic JNK activation without p38 or ERK inhibition. JNK inhibition also reduced pancreatic edema formation and reduced histological severity of pancreatitis. Thus we show that CEP-1347 inhibits JNK activation in vivo and ameliorates caerulein-induced pancreatitis.

Three-month-old female Wistar rats were fed with 20% alcohol in their drinking fluid over 6-17 mo using an interrupted feeding regimen. At different times, pancreatic acini were isolated by mild collagenase digestion. The concentrations of inositol-1,4,5-trisphosphate (1,4,5-IP3) were determined by a specific radioreceptor assay, before and at different times after stimulation with varying concentrations of CCK-8. CCK-induced dynamics of cytoplasmic calcium ([Ca2+]c) was investigated in acinar cells by confocal laser raster microscopy. Acinar alpha-amylase (Aml) secretion was measured as enzyme activity in the medium compared to the total activity in the suspension.In 12-13-mo-old rats, the CCK-stimulated 1,4,5-IP3 formation in acini was found to be decreased compared to young rats (age 4 mo). In rats of the same age fed with ethanol from the age of 3 mo on, 1,4,5-IP3 concentrations in acini were higher and reached values comparable to those in young rats. Correspondingly, the CCK-induced [Ca2+]c dynamics in acini isolated from 9-mo-old rats was impaired compared to that of young rats but normal in aged, chronically alcohol-fed rats. Aml secretion under CCK stimulation, however, which was decreased in aged rats, was additionally impaired after alcohol feeding.Chronic alcohol feeding modifies 1,4,5-IP3 formation, the [Ca2+]c dynamics of, and the Aml secretion of rat pancreatic acini in response to CCK stimulation. Obviously, the age-related impairment of 1,4,5-IP3 formation and [Ca2+]c dynamics is improved. In contrast, the decrease in Aml secretion of acini isolated from aged rats is more pronounced after long-term alcohol-feeding.

Salivary glands secrete K+and[Formula: see text] and reabsorb Na+and Cl−, but the identity of transporters involved in[Formula: see text] transport remains unclear. We investigated localization of Cl−/[Formula: see text] exchanger isoform AE2 and of Na+-[Formula: see text] cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at ∼160 and 180 kDa for AE2 and ∼130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of [Formula: see text] by rat salivary duct cells than previously believed.

Background: Prostatic epithelial cells organize functional acinus structures under epithelial– extracellular matrix and epithelial–stromal cell interactions. Recently, the adipose tissue, which surrounds and even exists within the prostate, has been suggested to affect the differentiation and proliferation of some cell types. Therefore, tissue fragments, which consist mainly of epithelial and fibromuscular stromal cells, were cultured in three-dimensional collagen gel matrix culture with adipocytes. Methods: Tissue fragments of rat dorsal prostate, including both epithelial and fibromuscular stromal components, were cultured in collagen gel with or without adipocytes. Epithelial cell differentiation was evaluated with the reconstruction of acinus-like structures and with immuno-histochemistry of rat dorsal prostate-specific proteins, dorsal protein-1 and probasin. The proliferation was examined by uridine uptake. Results: Under coculture of the fragments and adipocytes, epithelial cells reconstructed more differentiated acinus-like structures surrounded by fibromuscular stromal cells than tissue fragment culture without adipocytes. Dorsal protein-1 and probasin expressions of epithelial cells in this coculture system were the same as in rat prostate in vivo. In the coculture, epithelial cells had a higher proliferation activity. Conclusion: These results indicate that adipocytes promote proliferation and differentiation of prostatic epithelial cells. Our new culture model with adipocytes suggests the importance of cell–cell interactions, including those of epithelial cells and adipocytes.

The periportal (PP) and perivenous (PV) zones of the liver acinus differ in enzyme complements and capacities for cholesterol and bile acid synthesis and other metabolic processes. The aim of this investigation was to determine the acinar distribution of the catalytic activity of the enzymes governing the formation and hydrolysis of cholesteryl esters using PP and PV hepatocytes from normal or cholestyramine-fed rats. The hepatocyte subpopulations were isolated by centrifugal elutriation, characterized according to the distribution pattern of a number of cell parameters and marker enzymes, and assayed for acyl-CoA: cholesterol acyltransferase (ACAT) and lysosomal, cytosolic and microsomal cholesteryl ester hydrolase (CEH). In normally fed rats, no zonation was found in the activity of lysosomal CEH and ACAT, and the activity of both cytosolic and microsomal CEH zonated toward the PV zone of the acinus. Concentrations of free and esterified cholesterol in homogenates, cytosol, and microsomes of PP and PV cells were, however, similar. Cholestyramine raised significantly the PV/PP ratio of ACAT because of an exclusive PP reduction of activity and abolished the heterogeneity in microsomal CEH because of a greater inhibitory PV response, whereas the PV dominance of cytosolic CEH and the homogeneous distribution of lysosomal CEH were unaffected. These results demonstrated homogeneity within the liver acinus for the enzymatic degradation of endocyted lipoprotein-derived cholesteryl esters, a structural zonation of the cytosolic CEH and a dynamic zonation of ACAT and the microsomal CEH, with a PV dominance of the enzymatic capacity for the degradation of stored cholesteryl esters in normal livers.

High speed laser confocal microscopy (8 ms/image) was applied to the dissociated parotid acini as a model to study Ca2+ signaling mechanisms in non-excitable exocrine secretory cells. Immunofluorescence microscopy showed the localization of IP3 receptor type 2 along the apical membrane region. Muscarinic stimulation with carbachol evoked a rise in [Ca2+]i that was initiated from apical region and propagated into basal region as Ca2+ waves. This was most clearly observed when extracellular Ca2+ was omitted. Carbachol also triggered the abrupt increase of [Ca2+]i simultaneously at both basal and apical regions in many acini. Within an acinus, each cell responded synchronously. The present results suggest that one Ca2+ initiation site in the rat parotid acinar cell is apical region, corresponding to the localization of IP3 receptors. Another Ca2+ initiation site is basal region, which seems to be related to Ca2+ entry from extracellular medium and/or Ca2+ release from basally located organelles such as nuclei and endoplasmic reticulum.

The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.