Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-alpha nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulfide

The present study investigated the mechanism of mouse pancreatic acinar cell apoptosis induced by H₂S in an in vitro system, using isolated pancreatic acini. Treatment of pancreatic acini with 10 µM NaHS (a donor of H₂S) for 3 h caused phosphatidylserine externalization as shown by annexin V binding, an indicator of early stages of apoptosis. This treatment also resulted in the activation of the caspase cascade and major changes at the mitochondrial level. Caspase-3, -8, and -9 activities were stimulated by H₂S treatment. Treatment with inhibitors of caspase-3, -8, and -9 significantly inhibited H₂S-induced phosphatidylserine externalization as shown by reduced annexin V staining. The mitochondrial membrane potential was collapsed in H₂S-treated acini as evidenced by fluorescence microscopy and quantitative analysis. Furthermore, the treatment of acini with H₂S caused the release of cytochrome c by the mitochondria. To investigate the mechanism underlying pancreatic acinar cell apoptosis, we also characterized the protein expression of a range of molecules that are each known to influence the apoptotic pathway. Among proapoptotic proteins, Bax expression was activated in H₂S-treated cells but not Bid, and the antiapoptotic proteins Bcl-X_L and Bcl-2 did not show any activation in pancreatic acinar cell apoptosis. The death effector domain-containing protein Flip is downregulated in H₂S-treated acini. These results demonstrate the induction of pancreatic acinar cell apoptosis in vitro by H₂S and the involvement of both mitochondrial and death receptor pathways in the process of apoptosis. caspase; H₂S; mitochondria; Bcl-2; Flip     

Effects of cycloheximide and tunicamycin on cell surface expression of pancreatic muscarinic acetylcholine receptors

The importance of glycosylation in cell surface expression of muscarinic receptors in cultured guinea pig pancreatic acini was investigated. Recovery of the muscarinic receptor population after carbachol-induced down regulation was blocked by cycloheximide but not by tunicamycin, although tunicamycin reduced [3H]mannose incorporation into acinar macromolecules by up to 90%. Tunicamycin treatment also failed to alter carbachol stimulation of amylase secretion from cultured acini. These results indicate that glycosylation of the glandular subtype of muscarinic receptor in the pancreatic acinar cell is not necessary for its insertion in the plasma membrane or for its functional activity.     

Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.     

Muscarinic acetylcholine receptor structure in acinar cells of mammalian exocrine glands

Characterization of muscarinic acetylcholine receptors in acinar cells from rat pancreas and lacrimal and parotid glands was achieved by binding of the reversible muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the specific alkylating reagent [3H]propylbenzilylcholine mustard (PrBCM) to intact acini or dispersed acinar cells. Binding studies with [3H]QNB showed that acinar cells from pancreas contain 26,400, from parotid 21,400, and from lacrimal gland 25,700 binding sites/cell. To assess molecular size of the receptor in each gland, acini were prepared by digestion with purified collagenase and singly dispersed acinar cells were prepared by a combination of digestion with crude collagenase, hyaluronidase, and alpha-chymotrypsin and divalent cation chelation using EDTA. Muscarinic receptors on acini or dispersed cells were covalently labeled with 5 nM [3H]PrBCM, solubilized directly in hot sodium dodecyl sulfate buffer, and resolved by polyacrylamide gel electrophoresis. When solubilized acini were electrophoresed, a major labeled peak was observed on gels along with a smaller peak of lower apparent molecular weight. For pancreatic acini, the apparent molecular weights of these peaks were 117,600 and 85,700; for parotid acini, 104,800 and 74,500; and for lacrimal acini, 87,200 and 63,100. Addition of muscarinic antagonists to the labeling medium abolished both peaks. When dispersed acinar cells were labeled, the larger peak was eliminated, and all radioactivity was concentrated in a single peak: 87,600 for pancreas, 78,000 for parotid gland, and 62,800 for lacrimal gland. Digestion of prelabeled acini with the mixture of enzymes used to produce dispersed acinar cells similarly shifted all radioactivity into this second peak. Limited digestion of acini or dispersed cells with 1 mg/ml of papain resulted in the disappearance of these higher molecular weight peaks and the appearance of a broad peak at Mr = 40,000. Cells of nonepithelial origin, IM-9 lymphocytes and NG108 neuroblastoma X glioma hybrids, also were labeled with [3H]PrBCM and electrophoresed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat.

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.     

Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.     

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos.     

A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell

The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.     

Carbonic anhydrase isozyme VI in rat lacrimal gland

Using monoclonal antibody specific to rat carbonic anhydrase isozyme VI (CA VI), the isozyme was localized in the lacrimal gland. A minority of acini (less than 10% of the total) contained a few immunoreactive acinar cells. Enzyme histochemistry indicated that the CA VI-positive cells were the only cells possessing CA in the lacrimal acini. In the acinar cells, the reaction product for CA VI was distributed in the secretory granules and cytosol between secretory granules. Except for mitochondrial enzyme (CA V) activity, the intracellular distribution of enzyme activity was similar to that of CA VI immunoreactivity, suggesting that rat lacrimal acinar cells contain only CA VI and CA V. CA VI in the secretory granules was discharged into the acinar lumen and is considered to carry out its function on the surface of the conjunctiva and cornea. The cytosolic CA VI may function in situ and be involved in electrolyte and water secretion by the acinar cells. Polyclonal antibody to rat erythrocyte CA (CA I and CA II) stained only the interlobular ducts. In contrast, all the ductal elements exhibited CA enzyme activity. This discrepancy between immunohistochemistry and enzyme histochemistry suggests the presence of CA isozyme(s) other than CA I, CA II and CA VI in the lacrimal duct.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

Clonal and territorial development of the pancreas as revealed by eGFP-labelled mouse chimeras

The clonal structure of the pancreas was analysed in neonatal and adult mouse chimeras in which one partner displayed cell patches expressing green fluorescent protein (eGFP). Coherent growth during pancreatic histogenesis was suggested by the presence of large eGFP-labelled acinar clusters rather than a scattered distribution of individual labelled acinar cells. The adult chimeric pancreas contained monophenotypic acini, whereas surprisingly 5% of acini in neonates were polyclonal. Monophenotypic acini presumably arose by coherent expansion leading to large 3D patches and may not be monoclonal. Islets of Langerhans were oligoclonal at both ages investigated. The proportion of eGFP positive cells within islets did not correlate with that of the surrounding acinar tissue indicating clonal independence of islets from their neighbourhood. The patterns observed argue against a secondary contribution of blood-borne progenitor/stem cells to the acinar compartment during tissue turnover. The different clonal origins of acini and islets are integrated into a model of pancreatic histogenesis.     

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.     

Ultrastructural and immunoelectron microscopic studies on infiltrating mononuclear cells in lymphocytic submandibulitis in NOD mice

The ultrastructural relations of the infiltrating mononuclear cells to the parenchymal tissues were studied in the submandibular gland of the female non-obese diabetic (NOD) mouse. In addition, the phenotype of mononuclear cells infiltrating the submandibular gland has been determined by light and electron microscopy by using monoclonal antibodies against T-cell subsets (Thy1.2, Lyt1, Lyt2). Ultrastructurally, lymphoid cells were frequently observed around and in the acini and ducts. Some of the lymphoid cells observed in the acini and ducts were irregular in shape and sometimes sent spike-like projections into acinar and ductal cells. Immunohistochemical study demonstrated that Thy1.2+ cells were predominant among the infiltrating cells, and the majority of these infiltrating T-cells were composed of Lyt1+ cells with a small proportion of Lyt2+ cells. By immunoelectron microscopy, lymphocytes carrying Thy1.2, Lyt1 or Lyt2 antigen were identified, as is demonstrated by an electron-dense reaction product on the entire cell surface, and these immunopositive cells were frequently observed around and in the acini and ducts. Some of the Thy1.2+, Lyt1+ or Lyt2+ cells observed in the acini and ducts demonstrated a close contact with acinar and ductal cells and both Lyt1+ and Lyt2+ cells sent spike-like projections into them. Occasionally, a partial degeneration of acinar cell adjacent to the invading lymphocytes was observed. These observations suggest that T-lymphocytes are involved in the direct destruction of acinar and ductal cells in the NOD mouse submandibular glands.     

Agonist-induced changes in cell shape during regulated secretion in rat pancreatic acini

The actin cytoskeleton plays an important role in the mediation of exocytosis and the determination of cell shape. Experimentally induced changes in cell shape have been shown to affect stimulated secretion in pancreatic acini. In this study, we have examined whether physiologic agonists induce changes in acinar cell shape to modulate secretion. Computer-enhanced video microscopy, immunofluorescence confocal microscopy, and quantitative Western blotting were used to study cell shape changes and cytoskeletal dynamics in rat pancreatic acini. Amylase assays were performed to study the effect of the actin-myosin cytoskeletal antagonists latrunculin A, BDM, and ML-9 on secretion. We found that pancreatic acini underwent a prominent and reversible shape change in response to the physiologic secretory agonist cholecystokinin. This was accompanied by an apical activation of myosin II as well as a basolateral redistribution of both actin and myosin II. Cytoskeletal antagonists inhibited this shape change and attenuated stimulated amylase secretion. Therefore, in addition to acting as a barrier at the apex, the actin-myosin cytoskeleton may also function to modulate cell shape to further regulate stimulated secretion.     

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation

Summary This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.     

The ultrastructure of MCF-10A acini

MCF-10A human mammary epithelial cells cultured inside reconstituted basement membrane form acini that resemble the acinar structures of mammary lobules. This three-dimensional culture system has been used for identifying and characterizing the signal transduction pathways controlling cell proliferation and death, and for studying their disregulation in malignant progression. We have compared the ultrastructure of MCF-10A acini, MCF-10A cells grown in monolayer, and the acinar structures of human breast lobules. The tissue architecture of MCF-10A acini was formed by hemidesmosomes connected to a basement membrane and by abundant desmosomes between acinar cells. Intermediate filaments that joined into large and abundant filament bundles connected hemidesmosomes and desmosomes to sites at the nuclear surface. Fewer and thinner bundles of filaments were observed in monolayer MCF-10A cells and even fewer in breast tissue. Tight junctions were observed between cells in breast tissue but missing in MCF-10A acini. The cytoplasm of MCF-10A acinar cells had a polar organization similar to that observed in breast tissue, with centrosomes and the Golgi apparatus on the apical side of the nucleus. MCF-10A acinar nuclei had an irregular, frequently invaginated surface and had a single nucleolus. The distribution of heterochromatin was similar to that in the epithelial cells of breast tissue. The nuclei of monolayer MCF-10A cells had multiple nucleoli, a more regular profile, and less heterochromatin. Electron microscopy has the resolution required to survey features of MCF-10A cell and acinus architecture that may change with manipulations designed to induce malignant phenotypes.     

The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.