Myosin I is associated with zymogen granule membranes in the rat pancreatic acinar cell.

BACKGROUND & AIMS: The mechanisms whereby intracellular messengers mediate zymogen granule transport and exocytosis in the pancreatic acinar cell are not well defined. Electron microscopy has shown a periluminal network of actin in the acinar cell, suggesting a role for actin and myosin in the transport process. The possible involvement of two types of myosin in the secretory process was investigated, and their distribution in acinar cells was determined. METHODS: Antibodies specific to myosin I or to myosin II were used for immunocytochemistry and Western blot analysis. Ultrastructural studies were also performed. RESULTS: Western blot analysis showed that myosin I and myosin II were present in total pancreatic homogenate but that only myosin I was present on isolated zymogen granules and their membranes. By immunocytochemistry, myosin I was shown in the apical aspect of acinar cells colocalized with glycoprotein 2, a marker for zymogen granules, and actin. By immunocytochemistry, myosin I was also localized on isolated zymogen granules. CONCLUSIONS: The immunolocalization of myosin I to zymogen granule membranes and its close association with periluminal actin suggest that myosin I plays a direct role in the process of transport and exocytosis of zymogen granules in the pancreatic acinar cell.

The role of calcium in the regulation of protein synthesis in the exocrine pancreas.

The present study was designed to examine the role of Ca2+ in the regulation of digestive enzyme synthesis, to determine whether changes in intracellular Ca2+ stores or cytosolic Ca2+ caused the observed effects, and to establish the steps in the pathway of protein synthesis where the regulation occurs. Protein synthesis, polysome size, and the ratio of completed to nascent polypeptides were measured as a function of Ca2+ in the intracellular stores and the cytoplasm of pancreatic acinar cells. Rat acini and rabbit pancreatic lobules were incubated in media containing 1 mM CaCl2 with the following additives: cholecystokinin (CCK) octapeptide; the inhibitors of microsomal Ca2+ ATPase, thapsigargin (THP) and 2,5-di(tertbutyl)-hydroquinone (BHQ); the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA); an inhibitor of translational initiation, 7-methylguanosine 5'-triphosphate; and an inhibitor of translation elongation, cyclohexamide. THP and BHQ depleted intracellular pools of Ca2+ and caused a sustained elevation in cytosolic [Ca2+]. Under these conditions, the polysome size diminished, and the ratio of completed proteins increased twofold relative to nascent polypeptides despite an overall decrease in net protein synthesis (55.3 +/- 2.7% of control). These effects paralleled those caused by incubation with 1 nM CCK. Incubation of pancreatic acini with BAPTA plus THP or BHQ depleted the pool [Ca2+] without changing the cytosolic [Ca2+]. In addition, these agents decreased the net protein synthesis (30.1 +/- 3.6% compared to control) and polysome size and increased the ratio of completed to nascent polypeptides to 2:1. These results suggest that depletion of intracellular stores of Ca2+ without changes in cytosolic [Ca2+] decreases protein synthesis at translational initiation.

Proteolytic enzymes in the blood-feeding parasitic copepod, Phrixocephalus cincinnatus.

To understand digestive and invasive mechanisms employed by blood-feeding parasitic copepods, extracts of Phrixocephalus cincinnatus were assayed for specific proteolytic enzyme activity. Intact parasites were dissected into the 3 major body regions, cephalothorax (CT), genital segment (GS), and eggstrings (ES), and homogenized in ice-cold 1% Triton X-100 (v/v) in water. Protease activity in each body region was assayed using several synthetic fluorogenic peptide substrates. The greatest activity was detected when samples were incubated with carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin (CBZ-phe-arg-NHMec) in the presence of cysteine or reducing agents. Substrate specificity, pH profile, and inhibitor sensitivity indicated that the proteolytic enzyme(s) belonged to the cysteine class of endopeptidases and were most similar to mammalian cathepsins L, B, and H, respectively. Intense protease activity was also detected with carbobenzoxy-glycyl-L-prolyl-L-arginine-7-amido-4-methylcoumarin (CBZ-gly-pro-arg-NHMec), a substrate for the serine proteases, plasmin and thrombin. Substrate gel electrophoresis revealed intense gelatinolytic activity in all body regions; however, the ES extract presented a pattern different from that of the adult body, suggesting that distinct proteolytic enzymes are expressed during development. Gelatinolytic activity was inhibited at low pH and in the presence of serine protease inhibitors but not cysteine protease inhibitors. Collectively, the results indicate the presence of 2 major classes of proteolytic enzymes, cysteine and serine proteases. Differential expression of these proteases may be important for the successful completion of the parasite's life cycle, as well as survival of the adult.

Effect of chronic ethanol feeding on digestive enzyme synthesis and mRNA content in rat pancreas.

Chronic ethanol ingestion is a primary factor in the development of pancreatitis in humans. Alterations in both enzyme secretion and protein synthesis have been implicated as causative factors. We determined the effect of chronic ethanol feeding on the content and synthesis rates of digestive enzymes in dispersed acini from rats that were pair-fed isocaloric diets with or without ethanol for 3-6 months. Total protein content and synthesis were unchanged. The relative synthetic rates of individual digestive enzymes were analyzed using scanning laser densitometry of 1-D sodium dodecylsulfate (SDS) gels. The content of all measurable digestive enzymes except amylase increased in acini from ethanol-fed rats. Relative synthetic rates were examined in pancreatic acini labeled in vitro with [35S]methionine. Liquid scintillation counting of radiolabeled digestive enzymes extracted from gel slices revealed that amylase synthesis in ethanol-fed rats decreased 2.8-fold compared with controls whereas the synthetic rates of proelastase 1 and 2, chymotrypsinogen, and trypsinogen increased by 1.5-, 1.4-, 1.8-, and 1.6-fold, respectively. Total cellular RNA was extracted from control and ethanol-fed rats and subjected to Northern and dot blot analysis. Amylase mRNA decreased in ethanol-fed rats whereas chymotrypsinogen and trypsinogen mRNA content increased, indicating that the effect of ethanol on expression of these genes was regulated at a step prior to translation. Elastase mRNA content was not altered, suggesting that the increased synthesis of proelastase may be regulated posttranscriptionally.

Ultrastructure of the midgut of Phrixocephalus cincinnatus (Wilson), a blood-feeding parasitic copepod of flatfishes.

Phrixocephalus cincinnatus is a blood-feeding copepod parasite inhabiting the choroid of the eye of the Pacific sanddab Citharichthys sordidus off the coast of southern California. The present study examined the fine structure of the midgut using both light and electron microscopy. The anterior midgut or stomach was lined with a tall columnar epithelium. The apical surfaces of the cells formed vesicles that pinched off and entered the gut lumen. Posteriorly, the midgut consisted of 2 morphologically distinct cell types; vacuolar and nonvacuolar. These cells probably were different developmental and functional stages of a single kind of columnar epithelial cell. The nonvacuolar cells possessed basally located nuclei, rough endoplasmic reticulum, and long microvilli indicative of an absorptive function. The vacuolar cells were characterized by short microvilli, endocytic vesicles, primary and secondary lysosomes, well-developed rough endoplasmic reticulum, and Golgi apparatus, suggesting that they were active in endocytosis as well as the synthesis of digestive and lysosomal enzymes. Distension of the apical surface of vacuolar cells with enzyme-containing vesicles resulted in the liberation of the distal part of the cell into the gut lumen. Subsequent lysis of the cell membrane released enzyme-containing vesicles, suggesting that digestive enzymes are discharged by apocrine secretion of the vacuolar cells. Both vacuolar and nonvacuolar cells absorb nutrients resulting from the extracellular digestion of host blood.

Ultrastructure of the holdfast of Phrixocephalus cincinnatus (Wilson), a blood-feeding parasitic copepod of flatfishes.

Phrixocephalus cincinnatus is a parasitic copepod inhabiting the eye of flatfishes. Specimens of infected Pacific sanddabs Citharichthyes sordidus were collected from Santa Monica Bay, California, and the copepods were fixed and examined using standard light and transmission electron microscopy. The holdfast consisted of numerous, intertwined rootlets originating from the cephalothorax of the parasite and embedded in the choroid of the host's eye. A large hematoma, extravasated blood, and necrotic host cells surrounded the holdfast. Electron microscopy revealed that the rootlets were encased with a trilaminar cuticle consisting of an outer epicuticle and an external and internal procuticle. The outer surface of the epicuticle was covered with microvillosities that contacted host tissue. In regions where host macrophages contacted the cuticle, the external layer of procuticle was expanded by large, cholesterol-like crystal deposits. Internally, the rootlets were comprised of at least 3 types of large cells. Epithelial cells contacted the internal layer of procuticle and contained numerous mitochondria. The cytoplasm of stellate cells was dominated by rough endoplasmic reticulum or large vacuoles. Ovoid cells contained distinct proteinaceous granules of unknown chemical composition. These observations revealed that the rootlets are structurally complex and suggest that the holdfast may be actively involved with molecular exchange between parasite and host.

Cholecystokinin-induced changes in polysome structure regulate protein synthesis in pancreas.

Translational regulation of digestive enzyme synthesis during short-term stimulation by cholecystokinin-octapeptide (CCK-OP), was examined in minced rabbit pancreas by measuring protein synthesis and monitoring alterations in the size of polysomes attached to the rough endoplasmic reticulum (RER). The effect of CCK-OP on protein synthesis was determined by measuring [3H]leucine incorporation into trichloroacetic-acid-precipitable proteins. Concentrations of CCK-OP that caused maximal enzyme secretion (10 and 30 nM) decreased protein synthesis by approx. 50% compared to control. Protein synthesis returned to the control level 60 min after terminating the action of CCK-OP. Autoradiography of [35S]methionine-labeled proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that CCK-OP reversibly inhibited the synthesis of all of the major groups of digestive enzymes. Northern blot analysis revealed that CCK-OP did not alter the cellular content of amylase and elastase mRNA. Incubation with CCK-OP caused a decrease in the size distribution of RER-bound polysomes. Polysome profiles returned to the control pattern 60 min following termination of the stimulus. These results suggest that the inhibitory effects of CCK-OP on the synthesis of digestive enzymes is regulated at translation by decreasing the number of RER-bound ribosomes that are actively translating digestive enzyme mRNA.

Intracellular mechanisms involved in short-term regulation of net protein synthesis in pancreatic acini.

The mechanisms regulating the net synthesis of digestive enzymes during short-term stimulation by agonists were examined in pancreatic acini isolated from the rat. Dispersed pancreatic acini were stimulated for up to 60 min with various concentrations of cholecystokinin octapeptide (CCK-OP), carbachol, A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA). The effects of these agonists on net protein synthesis was determined by measuring the incorporation of [3H]leucine or [35S]methionine into protein. Carbachol, PMA, A23187 and concentrations of CCK-OP of 100 pM and greater caused inhibition of protein synthesis. Fluorography of [35S]methionine labeled acinar cell proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that the agonists inhibited the synthesis of the digestive enzymes. Northern blot analysis using cDNA probes revealed that CCK-OP, carbachol and PMA did not alter the cellular content of amylase, lipase and elastase mRNA. The protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine failed to reverse the inhibitory effects of CCK-OP, carbachol and PMA on protein synthesis. CCK-OP and PMA activated phospholipase A (PLA) which liberated lysophosphatidylcholine (LPC) and free fatty acids from membrane phosphatidylcholine. Exogenously added PLA2 (Naja naja venom) inhibited protein synthesis and increased LPC to a similar extent as CCK and PMA. The results suggest that the inhibitory effects of CCK and carbachol on net protein synthesis are due to their effects on intracellular calcium and PLA-mediated breakdown of phosphatidylcholine rather than protein kinase C activation.

Comparisons of histones in retinal and brain nuclei from newborn and adult mice.

Histone proteins from purified nuclei of neonatal and adult mouse retinas were analyzed and compared utilizing SDS-polyacrylamide gel electrophoresis. Identical procedures were applied to examine the histones extracted from the brains of the same animals. In the newborn and mature retina and brain, 8 histone fractions have been separated, identified and quantified by scanning densitometry. These are the linker histone (H1), consisting of 3 subfractions (H1a, H1b, H1(0); the semi-histone uH2A (A24); and the 4 nucleosome core histones (H2A, H2B, H3, H4). Developmental differences are exhibited by the linker histone in both brain and retinal cells. The greatest differences between the histone patterns of retina and brain are also in the H1 group. Because the linker histone is subject to the greatest variability. H1 was selectively extracted with 5% perchloric acid from both neonatal and adult brain. This procedure established that the observed differences are a developmental phenomenon and are not due to interactions of the linker histones with other nuclear proteins. The ratio of the non-histone chromosomal proteins to total histone was found to be significantly greater in both adult and neonatal brain compared to retina at either age.

Histone proteins in fetal and adult human retinas.

The DNA-binding histone proteins from the human retina are described for the first time. Retinas were obtained from male and female donors ranging in age from 11 to 72 years old. Retinas from two human fetuses at approximately four months of gestation were also examined for their histone content. Histones extracted from purified nuclei were separated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Eight histone fractions were identified in all retinal samples, and were quantified by scanning transmittance densitometry. These fractions included three subfractions of linker histones (H1a, H1b, H1(0)), four nucleosomal core histones (H2A, H2B, H3, H4), and the modified core histone, A24 (uH2A), comprised of H2A covalently bound to the non-histone protein, ubiquitin. When fetal and adult retinal histones were compared, the relative amounts of the linker histone subfractions proved to be different. In the adult retinas, H1a diminished in amount whereas H1b and H1(0) were increased. No alterations were detected in the core histones. The developmental changes in linker histones may be related to modifications of chromatin compaction which accompany cell differentiation. Species differences in the pattern of linker histones were detected when the nuclear proteins of human and mouse retinas were compared. Non-histone nuclear proteins are more abundant in the adult human retina than in the mouse retina or the fetal human retina.

A new genus of cyclopoid copepod (Chondracanthidae) parasitic on halibut from California inshore waters.

Auchenochondria lobosa gen. et sp. n. was recovered from the oral cavity of the California halibut, Paralichthys californicus (Ayers), collected in Agua Hedionda Lagoon, Carlsbad, California. The new genus is closely related to Prochondracanthus Yamaguti 1939, and Rhynchochondria Ho 1967, but can be distinguished from them by the possession of cephalothoracic processes, a long neck, a 2-segmented abdomen, and a distinctly 6-segmented first antenna. Auchenochondria lobosa gen. et sp. n. is the only known species of chondracanthid in which the mandible of the male is equipped with more teeth on both the convex and concave surfaces than that of the female.