Acinar Model Research Articles

Aerosol bolus dispersion in acinar airways—influence of gravity and airway asymmetry

The aerosol bolus technique can be used to estimate the degree of convective mixing in the lung; however, contributions of different lung compartments to measured dispersion cannot be differentiated unambiguously. To estimate dispersion in the distal lung, we studied the effect of gravity and airway asymmetry on the dispersion of 1 μm-diameter particle boluses in three-dimensional computational models of the lung periphery, ranging from a single alveolar sac to four-generation (g4) structures of bifurcating airways that deformed homogeneously during breathing. Boluses were introduced at the beginning of a 2-s inhalation, immediately followed by a 3-s exhalation. Dispersion was estimated by the half-width of the exhaled bolus. Dispersion was significantly affected by the spatial orientation of the models in normal gravity and was less in zero gravity than in normal gravity. Dispersion was strongly correlated with model volume in both normal and zero gravity. Predicted pulmonary dispersion based on a symmetric g4 acinar model was 391 ml and 238 ml under normal and zero gravity, respectively. These results accounted for a significant amount of dispersion measured experimentally. In zero gravity, predicted dispersion in a highly asymmetric model accounted for ∼20% of that obtained in a symmetric model with comparable volume and number of alveolated branches, whereas normal gravity dispersions were comparable in both models. These results suggest that gravitational sedimentation and not geometrical asymmetry is the dominant factor in aerosol dispersion in the lung periphery.

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin™ 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.

Functional role of an islet transcription factor, INSM1/IA‐1, on pancreatic acinar cell trans‐differentiation

In this study, the functional role of INSM1 is examined with an AR42J acinar cell model for trans-differentiation into insulin-positive cells. Islet transcription factors (ITFs: INSM1, Pdx-1, and NeuroD1) are over-expressed in AR42J cells using adenoviral vectors. Addition of Ad-INSM1 alone or the combination of three ITFs to the AR42J cells triggers cellular trans-differentiation. Ectopic expression of INSM1 directly induces insulin, Pax6, and Nkx6.1 expression, whereas Pdx-1 and NeuroD1 were slightly suppressed by INSM1. Addition of Pdx-1 and NeuroD1 with INSM1 further enhances endocrine trans-differentiation by increasing both the numbers and intensity of the insulin-positive cells with simultaneous activation of ITFs, Ngn3 and MafA. INSM1 expression alone partially inhibits dexamethasone-induced exocrine amylase expression. The combination of the three ITFs completely inhibits amylase expression and concomitantly induces greater acinar cell trans-differentiation into endocrine cells. Also, addition of the three ITFs promotes EGF and TGFβ receptors expression. Stimulation by the three ITFs along with the EGF/TGFβ growth factors strongly promotes insulin gene expression. The combination of the three ITFs and EGF/TGFβ growth factors with the primary cultured pancreatic acini also facilitates exocrine to endocrine cell differentiation. Taken together, both the AR42J cell line and the primary cultured mouse acinar cells support INSM1 induced acini trans-differentiation model.

Live pancreatic acinar imaging of exocytosis using syncollin-pHluorin

In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.

Stochastic Morphometric Model of the Balb/c Mouse Lung

The laboratory mouse is often used as a human surrogate in aerosol inhalation studies. Morphometric data on the tracheobronchial geometry of three in situ lung casts of the Balb/c mouse lung produced by the Air Pollution Health Effects Laboratory were analyzed in terms of probability density functions and correlations among the different airway parameters. The results of this statistical analysis reveal significant differences in diameters and branching angles between major and minor progeny branching off from the same parent airway at a given airway bifurcation. Number of bronchial airways generations along a given path, expressed by the termination probability, branching angles, and daughter-to-parent diameter ratios indicate that the location of an airway with defined linear airway dimensions within the lung is more appropriately identified by its diameter (or its parent diameter) than by an assigned generation number. We, therefore, recommend classifying the mouse lung airways by their diameters and not by generation numbers, consistent with our previous analysis of the rather monopodial structure of the rat lung (Koblinger et al., J Aerosol Med 1995;8:7–19; Koblinger and Hofmann, J Aerosol Med 1995;8:21–32). Because of lack of corresponding information on respiratory airways, a partly stochastic symmetric acinar airway model was attached to the tracheobronchial model, in which the number of acinar airways along a given path was randomly selected from a measured acinar volume distribution. The computed distributions of the geometric airway parameters and their correlations will be used for random pathway selection of inhaled particles in subsequent Monte Carlo deposition calculations.

A Spontaneous Acinar Cell Carcinoma Model for Monitoring Progression of Pancreatic Lesions and Response to Treatment Through Noninvasive Bioluminescence Imaging

We have generated an EL1-luc/TAg transgenic mouse model that develops spontaneous and bioluminescent acinar cell carcinomas. We applied this model to noninvasively monitor tumor development and drug response. EL1-luc/TAg transgenic mice of 11 weeks of age were treated with rapamycin (5 mg/kg, i.p.) or vehicle for 6 to 12 weeks. Tumor development was monitored through bioluminescence imaging and necropsy at the study end point. EL1-luc/TAg transgenic mice showed pancreas-specific bioluminescence signal before tumor progression and produced increasing light emission from the onset of the pancreatic acinar cell carcinomas. The latency of tumor development ranged from 10 to >20 weeks of age in these mice. Progression of the primary acinar cell carcinoma was accompanied by emergence of metastatic lesions in the abdominal organs, including liver and gastrointestinal fat tissues. Rapamycin treatment suppressed tumor development. The EL1-luc/TAg mouse provides a noninvasive approach for monitoring spontaneous acinar cell carcinoma development and comprises a convenient tool for the evaluation of novel therapeutics against pancreatic cancers. Tumor growth suppression through inhibition of the mammalian target of rapamycin pathway further validates this model as clinically relevant.

Alveolar duct expansion greatly enhances aerosol deposition: a three-dimensional computational fluid dynamics study.

Obtaining in vivo data of particle transport in the human lung is often difficult, if not impossible. Computational fluid dynamics (CFD) can provide detailed information on aerosol transport in realistic airway geometries. This paper provides a review of the key CFD studies of aerosol transport in the acinar region of the human lung. It also describes the first ever three-dimensional model of a single fully alveolated duct with moving boundaries allowing for the cyclic expansion and contraction that occurs during breathing. Studies of intra-acinar aerosol transport performed in models with stationary walls (SWs) showed that flow patterns were influenced by the geometric characteristics of the alveolar aperture, the presence of the alveolar septa contributed to the penetration of the particles into the lung periphery and there were large inhomogeneities in deposition patterns within the acinar structure. Recent studies have now used acinar models with moving walls. In these cases, particles penetrate the alveolar cavities not only as a result of sedimentation and diffusion but also as a result of convective transport, resulting in a much higher deposition prediction than that in SW models. Thus, models that fail to incorporate alveolar wall motions probably underestimate aerosol deposition in the acinar region of the lung.

Cytopathology and Exocrine Dysfunction Induced in Ex Vivo Rabbit Lacrimal Gland Acinar Cell Models by Chronic Exposure to Histamine or Serotonin

Lacrimal immunohistopathology has diverse clinical presentations, suggesting that inflammatory mediators exert diverse influences. Chronic exposure to agonistic acetylcholine receptor autoantibodies has been studied previously; the present work addressed mediators that signal through other G protein-coupled receptors. Acinus-like structures and reconstituted acinar epithelial monolayers from rabbit lacrimal glands were exposed to varying concentrations of histamine or 5-hydroxytryptamine (5-HT) for 20 hours. Net and vectorial beta-hexosaminidase secretion, cytosolic Ca(2+) (Ca(i)) elevation, apical recruitment of p150(Glued), actin microfilament meshwork organization, and ultrastructure were assessed. Histamine and 5-HT acutely stimulated beta-hexosaminidase secretion at lower, but not higher, concentrations. Neither of them acutely elevated Ca(i) levels. Both recruited p150(Glued) at concentrations that failed to induce secretion. Chronic exposure to 10 mM histamine inhibited carbachol (CCh)-induced beta-hexosaminidase secretion and prevented the formation of continuous monolayers; 1 mM 5-HT partially inhibited secretion at the apical medium. Neither altered secretion to the basal medium. Chronic exposure to histamine or 5-HT partially decreased CCh induced Ca(i) elevations and p150(Glued) recruitment, even at concentrations that did not inhibit secretion. Both expanded acinar lumina and thickened microfilament meshworks, and both caused homotypic fusion of secretory vesicles and formation of aqueous vacuoles in the apical and basal cytoplasm. Chronic exposure to forskolin, which activates adenylyl cyclase, induced similar cytopathologic changes but impaired secretion modestly and only at the highest concentration tested. Inflammatory mediators that signal through G protein-coupled receptors cause acinar cell cytopathology and dose-dependent reductions of CCh-induced beta-hexosaminidase secretion. Although agonistic acetylcholine receptor autoantibodies may cause pervasive functional quiescence, inflammatory mediators may cause varying degrees of exocrine dysfunction.

Respiratory Flow Phenomena and Gravitational Deposition in a Three-Dimensional Space-Filling Model of the Pulmonary Acinar Tree

The inhalation of micron-sized aerosols into the lung's acinar region may be recognized as a possible health risk or a therapeutic tool. In an effort to develop a deeper understanding of the mechanisms responsible for acinar deposition, we have numerically simulated the transport of nondiffusing fine inhaled particles (1 mum and 3 microm in diameter) in two acinar models of varying complexity: (i) a simple alveolated duct and (ii) a space-filling asymmetrical acinar branching tree following the description of lung structure by Fung (1988, "A Model of the Lung Structure and Its Validation," J. Appl. Physiol., 64, pp. 2132-2141). Detailed particle trajectories and deposition efficiencies, as well as acinar flow structures, were investigated under different orientations of gravity, for tidal breathing motion in an average human adult. Trajectories and deposition efficiencies inside the alveolated duct are strongly related to gravity orientation. While the motion of larger particles (3 microm) is relatively insensitive to convective flows compared with the role of gravitational sedimentation, finer 1 microm aerosols may exhibit, in contrast, complex kinematics influenced by the coupling between (i) flow reversal due to oscillatory breathing, (ii) local alveolar flow structure, and (iii) streamline crossing due to gravity. These combined mechanisms may lead to twisting and undulating trajectories in the alveolus over multiple breathing cycles. The extension of our study to a space-filling acinar tree was well suited to investigate the influence of bulk kinematic interaction on aerosol transport between ductal and alveolar flows. We found the existence of intricate trajectories of fine 1 microm aerosols spanning over the entire acinar airway network, which cannot be captured by simple alveolar models. In contrast, heavier 3 microm aerosols yield trajectories characteristic of gravitational sedimentation, analogous to those observed in the simple alveolated duct. For both particle sizes, however, particle inhalation yields highly nonuniform deposition. While larger particles deposit within a single inhalation phase, finer 1 microm particles exhibit much longer residence times spanning multiple breathing cycles. With the ongoing development of more realistic models of the pulmonary acinus, we aim to capture some of the complex mechanisms leading to deposition of inhaled aerosols. Such models may lead to a better understanding toward the optimization of pulmonary drug delivery to target specific regions of the lung.

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.

Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-β and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122–E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; γ-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

Simulation of the apparent diffusion of helium-3 in the human acinus

Functional MRI of the lungs with hyperpolarized helium provides an index of apparent diffusion measured over several seconds (ADCsec) that is only 2% of its free diffusion in air (0.88 cm2/s). The potential of ADCsec to noninvasively assess in vivo lung structure of diseased lungs at the length scales corresponding to several seconds is critically dependent on the exact link between ADCsec and lung peripheral structure. To understand the intruigingly small ADCsec, numerical simulations of gas transport were performed in 1) a trumpet model, 2) a symmetrical, and 3) an asymmetrical multiple-branch-point model of the human acinus. For initial gas boluses in different locations of the acinar models, ADCsec was quantified as follows. At different time intervals, we computed a coefficient of variation (CoV) of the concentration distributions within each acinar model. The slope in the semilog plot of log(CoV) vs. time was proportional to the ADCsec generated by the internal model structure, provided that the outer model boundaries were similar across all models (i.e., similar cumulative cross section vs. average path length). The simulations revealed an ADCsec that amounted to approximately 1% of free diffusion in the trumpet model of the acinus, i.e., corresponding to free diffusion within the acinar geometric boundaries. Our simulations show that for initial conditions corresponding to those used in MRI experiments, intra-acinar branching introduces a dramatic diffusion delay, comparable to what is observed experimentally.

Stress distribution in a three dimensional, geometric alveolar sac under normal and emphysematous conditions

Pulmonary emphysema is usually the result of chronic exposure to cigarette smoke in at risk individuals. To investigate the hypothesis that lung damage in emphysema results from coincident weakening in the structural properties of the tissue and increased mechanical forces--as one explanation of the continued development of pulmonary emphysema after smoking cessation--we developed a three dimensional, geometric dodecahedron-based acinar model. Using the model numerical analysis of the stress distribution in normal conditions could be compared with those resulting in emphysematous conditions. Finite element analysis was used to evaluate the model at a number of lung inflation levels, using quasi-static loading of the alveolar pressure. When internal alveolar pressure was increased along with the adjustment of the material properties to represent a weakening of one wall in the acinus, increased stress resulted at the perimeters of the weakened area. In particular this increased stress was localized at the junction points of the internal alveolar septa. It was also found that under the proposed simulated emphysematous conditions, a significant disruption in the stress distribution within the acinus model occurred at low, rather than high, lung volumes. This is supportive of the physiological observation that destruction of the diseased tissue can occur under less stress than those existing in the normal state.

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of beta-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of beta-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.

Agonist-Induced Vesiculation of the Golgi Apparatus in Pancreatic Acinar Cells

The pancreatic acinar cell is known to regulate exocytosis, total protein synthesis, and secretory protein transport in response to a secretory stimulus. Whether secretory vesicle formation also is regulated is unclear. In this study, we determined whether agonist stimulation induces morphologic alterations in the acinar cell Golgi apparatus, and we evaluated the role of the vesicle severing protein dynamin. Changes in Golgi structural integrity by examining the distribution of various Golgi and TGN lipid and protein markers in live and fixed cells on stimulation with cholecystokinin were noted in a primary pancreatic acinar cell model. Multiple dynamin reagents were used to examine the distribution and function of this molecular pinchase in resting and stimulated cells. Regulated secretion in acinar cells induced (1) marked fragmentation of the trans-Golgi network (TGN) that corresponded temporally with an increase in cytoplasmic calcium whereas pre-TGN compartments of the Golgi and regions of the TGN involved in the generation of constitutively trafficking vesicles were unaffected by agonist, and (2) significant recruitment of dynamin to the acinar cell Golgi apparatus that appeared to potentiate fragmentation of the TGN. These results suggest that the TGN is a dynamic organelle that fragments in response to cholecystokinin stimulation, a process that may contribute to zymogen granule formation.

Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses [Mircheff, A.K., 2003. Sjögren's syndrome as failed local immunohomeostasis: prospects for cell-based therapy. Ocular Surface 1, 160–179; Mircheff, A.K., Hamm-Alvarez, S.F., Kaslow, H.R., Schechter, J.E., Trousdale, M.D., 2004. Epithelial cells in lymphocyte regulation in the lacrimal gland. In: Zierhut, M. (Ed.), Immunology of the Ocular Surface and the Lacrimal Glands. Swets & Zeitlinger, Amsterdam; in press]. Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjögren's syndrome [Qian, L., Wang, Y., Xie, J., Rose, C.M., Yang, T., Nakamura, T., Sandberg, M., Zeng, H., Schechter, J.E., Chow, R.H., Hamm-Alvarez, S.F., Mircheff, A.K., 2003. Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist. Scand. J. Immunol. 58, 550–565]. Chronic MAChR stimulation also elicits changes in the compartmental distribution of β-hexosaminidase, a product that normally is dually targeted into the lysosomal pathway and the regulated apical secretory pathway. Here, we use subcellular fractionation analyses to further explore the nature of the stimulation-induced traffic changes and to identify effectors that might mediate this change. Overnight stimulation of primary cultured rabbit lacrimal gland acinar cells with 10 μ m carbachol (CCh) significantly decreased the abundance of mature cathepsin B in the pre-lysosome and lysosome; decreased the abundance of preprocathepsin B in fractions containing the TGN and late endosome; increased the abundance of procathepsin B in fractions containing the basal-lateral membrane; and increased the accumulation of endocytosed [ 125I]-EGF in the recycling endosome. Alterations in distribution or abundance of traffic effectors included: increased abundances of rab5A and rab6 in the TGN; decreased overall abundance of γ-adaptin; remarkably increased relative abundance of membrane phase-associated actin; redistribution of cytoplasmic dynein from biosynthetic and proximal endocytic compartments to the lysosome; and redistribution of p150 Glued from the lysosome to biosynthetic or proximal endocytic compartments. We conclude that chronic MAChR stimulation blocks traffic from the early endosome and the TGN to the lysosome, causing lysosomal proteins to reflux to the TGN, endosomes, and basal-lateral membrane. These traffic alterations may be mediated through action on one or more of the effectors noted.

Acinar primary cell culture from the digestive gland of Pecten maximus (L.): an original model for ecotoxicological purposes

The purpose of this study was to establish an original primary acinar cell culture model for the mollusc bivalve Pecten maximus (L.), and to define its values and limits for subsequent ecotoxicological applications. To prevent microbial contaminations occurring frequently in invertebrate cell cultures, a perfusion of the stomach-digestive gland complex was performed in situ using a sterile salt solution containing broad-range antibiotics. Digestive acini were isolated using a pronase enzyme that was removed by several washings of the acinar suspension, after which their viability and functionality were determined by three different assays: fluorescein diacetate (FDA) de-esterification, 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT) reduction and neutral red (NR) incorporation describing de-esterification, mitochondrial dehydrogenase and lysosome activity, respectively. The kinetic conditions for these assays were defined beforehand. The results showed that digestive acini could be maintained in vitro both cytologically and functionally for at least 96 h, which is sufficient for many ecotoxicological applications. Preliminary contamination assays, according to the function studied (cell esterases, mitochondrial respiration, lysosomal incorporation), indicated that polycyclic aromatic hydrocarbons had a negative effect on the survival of acini in vitro.

Effect of Herbal Medicine Keishi-To (TJ-45) and Its Components on Rat Pancreatic Acinar Cell Injuries in vivo and in vitro

Background: In an attempt to clarify the mechanism of the effect of a herbal medicine, Saiko-keishi-to (TJ-10), which is a combination of Keishi-to (TJ-45) and Sho-saiko-to (TJ-9), we investigated the effects of these two herbal medicines and their components on pancreatic acinar cell injury models in vivo and in vitro. Methods: Four-week-old male WBN/Kob rats were fed an MB-3 pellet diet containing herbal medicine (TJ-9, TJ-10 and TJ-45). Expressions of pancreatitis-associated protein (PAP) and manganese superoxide dismutase (Mn-SOD) were analyzed with a reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The herbal medicines and two of their components, Keihi (Cinnamomi cortex) and Shakuyaku (Paeoniae radix alba), were tested in vitro using an arginine-treated rat pancreatic acinar AR4-2J cell injury model. The inducible nitric oxide synthase (iNOS) was assayed in in vitro experiments. Results: TJ-45-treated WBN/Kob rats showed no evidence of pancreatitis whereas there were pathological changes of chronic pancreatitis in TJ-9-treated WBN/Kob rats. PAP was not expressed and Mn-SOD expression was increased in the TJ-10-, and TJ-45-treated rats. The herbal medicines and two components suppressed PAP mRNA expression and enhanced Mn-SOD and iNOS mRNA expression in arginine-treated AR4-2J cells. Conclusion: These results suggest that the herbal medicine TJ-45 is effective for chronic pancreatitis caused by pancreatic ischemia.

Smoking and pancreatic disorders.

Cigarette smoking is a major risk factor for pancreatic cancer and probably pancreatitis. It has been reported that patients with chronic pancreatitis are 16 times more likely to develop pancreatic cancer than normal individuals. Nicotine, a major component of tobacco and an addicted drug of abuse in humans, appears to play a role in the aetiology of both pancreatic cancer and pancreatitis. This review discusses major published reports pertaining to the influence of nicotine on these diseases in humans and the probable effects of nicotine on the pathophysiology of the pancreas in animal models. A hypothetical pancreatic acinar cell model showing multiple signal transduction pathways is presented relating to possible mechanisms of action of nicotine on the pathophysiology of the pancreas.

A three-dimensional model of the human pulmonary acinus

A three-dimensional (3-D) model of the human pulmonary acinus, a gas exchange unit, is constructed with a labyrinthine algorithm generating branching ducts that fill a given space completely. Branching down to the third respiratory bronchioles is generated with the proposed algorithm. A subacinus, a region supplied by the last respiratory bronchiole, is approximated to be a set of cubic cells with a side dimension of 0.5 mm. The labyrinthine algorithm is used to determine a pathway through all cells only once, except at branching points with the smallest path lengths. In choosing each step of a pathway, random variables are used. Resulting labyrinths have equal mean path lengths and equal surface areas of inner walls. An alveolus can be generated by attaching alveolar septa, 0.25 mm long and 0.1 mm wide, to the inner walls. Total alveolar surface area and numbers of alveolar ducts, alveolar sacs, and alveoli in our 3-D acinar model are in good accordance with those reported in the literature.