Changes in the digestive gland of the loliginid squid Sepioteuthis lessoniana (Lesson 1830) associated with feeding

Changes associated with feeding in the histological and cytological structure of the digestive gland of the loliginid squid Sepioteuthis lessoniana were examined, along with the nature of both the intracellular and extracellular enzymes produced by the gland. The timing of the release of the extracellular enzymes during the digestive cycle was also determined using a quantitative experimental program. Like that of all coleoid cephalopods, the digestive gland was characterised by one type of cell with several functional stages. As is the case for other loliginid squids, however, the digestive cells did not contain the large enzyme-carrying boules that characterise the digestive glands of most cephalopods. Instead, smaller secretory granules were found in the digestive cells and these may be the enzyme carriers. The prominent rough endoplasmic reticulum, large mitochondria and active Golgi complexes present in the digestive cells are characteristic of cephalopods and indicate a high metabolic activity. Like that of other cephalopods, endocytotic absorption of nutrients and intracellular digestion occurs in the digestive gland of this squid. From quantitative and qualitative examinations of structural changes in the digestive gland of S. lessoniana after feeding, a schedule of its function during the course of digestion was proposed. This indicated that digestion was very rapid, being completed in as little as 4 h in S. lessoniana. Extracellular digestive enzymes were only released after the first hour following feeding, which implies that they are stored in the stomach between meals to increase digestive efficiency.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

云南盘鳇消化系统解剖学、组织学及消化酶活性研究

采用形态学、组织学及酶学方法对云南盘逗(Discogobio yunnanensis)成体消化系统进行研究。结果表明, 云南盘逗消化系统有以下特征: 口下位, 口腔上皮分布有较多味蕾及杯状细胞, 食道粗大, 含有大量黏液细胞, 无胃, 肠道较长, 盘旋于体腔中, 成鱼盘旋10回, 肠道系数为5.06±0.61, 肠分为前中后三段, 肠腔中密布肠绒毛。消化腺为肝胰脏, 肝脏分为左右两叶, 胰脏弥散分布在肝脏中。消化系统不同部位消化酶活性大小不同, 脂肪酶活性: 肝胰脏>前肠>中肠>后肠, 胰蛋白酶、淀粉酶、碱性磷酸酶活性: 前肠>中肠>肝胰脏>后肠。云南盘逗口下位, 食道粗短, 肠道细长, 肠绒毛丰富, 肠道含较高的胰蛋白酶和淀粉酶活性, 消化系统所具有的这些特征与其以固着藻类为食有关。     

Biosynthesis of Astacus protease, a digestive enzyme from crayfish

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.     

大鵟消化系统蛋白水解酶种类和活性分析

采用蛋白水解酶复性电泳(G-PAGE)技术对大(Buteo hemilasius)消化系统5种器官腺胃、胰脏、十二指肠、空肠、大肠蛋白水解酶的种类和性质进行了研究,以期为研究野生鸟类的分类地位、系统演化提供基础资料,结果表明,①受pH值的影响和制约,大消化系统蛋白水解酶的活性在碱性、中性与酸性条件下递减;②在酸性条件下,45 ku蛋白水解酶存在于除腺胃外的各受检器官;③pH 7.0时,腺胃、胰脏酶谱相似,均含有683、5、342、0 ku的蛋白水解酶;④pH 8.0时,空肠和十二指肠的蛋白水解酶种类最多、活性最强,分别检出8种和7种蛋白水解酶。总之,pH值对蛋白水解酶的活性有明显的制约作用,46、41ku蛋白水解酶随着pH值的增高而失去活性,为酸性蛋白水解酶,250、2064、5 ku蛋白水解酶随着pH值的增高活性逐渐增强,为碱性蛋白水解酶。十二指肠和空肠的蛋白水解酶种类多、活性强,可能为蛋白质消化的主要场所。     

中华蟾蜍消化道6种酶的组织化学定位

目的研究中华蟾蜍消化道酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、腺苷三磷酸酶(ATPase)、非特异性酯酶(NSE)、过氧化物酶(POX)和琥珀酸脱氢酶(SDH)等6种酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术、石蜡切片技术、酶的组织化学方法和光密度定量分析。结果 ACP主要分布于胃贲门中贲门腺部,十二指肠和回肠中酶反应呈弱阳性。ALP主要分布于食管、十二指肠至回肠的粘膜上皮,十二指肠酶活性最高。ATPase在消化道各部位均有分布,胃中胃腺部和回肠粘膜上皮酶活性显著较高(P0.05)。NSE和POX在整个消化道粘膜上皮和粘膜固有层均有分布,胃各部位酶活性显著较低(P0.05)。SDH除在食管和直肠酶活性显著较低外,其它部位均有大量分布,十二指肠和回肠酶活性显著较高(P0.05)。结论中华蟾蜍消化道粘膜6种酶的分布同其它动物有相似之处,也有其自身特点。6种酶在消化道中的分布与消化道各部位的生理机能密切相关。     

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae)

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.     

Feeding and gut physiology in Acanthopleura spinigera (Mollusca)

The morphology and histology of the alimentary canal of the rock chiton Acanthopleura spinigera are described and the ability of regions of the gut to digest specific substrates investigated. The oesophagus is produced into a pair of thin-walled lateral pouches, the salivary glands or "sugar glands" which empty into the stomach. Folds of the capacious stomach are almost obscured by the large digestive gland over which is coiled the intestine. Histologically the gut consists of an outer layer of connective tissue, an inner muscular layer and a ciliated epithelium which varies in thickness from one region to the next. Proteases are most active in the stomach, digestive gland and anterior intestine at pH 6·5 and in the posterior intestine at pH 7·5-8·5. The digestion of lipoidal substance was greatest in the stomach and digestive gland and least in anterior intestine. There was little increase in the amount of digestion product obtained after 20 hours incubation. All regions of the alimentary canal and salivary gland were capable of digesting carbohydrates except that many low molecular weight carbohydrates were digested by salivary gland extracts only. The amylases were most active at pH 6–6·5. It is concluded that digestive enzymes are distributed throughout the intestinal tract but the amount of enzyme present varies from region to region, and is greatest just after feeding.     

黑眉锦蛇消化道6种酶的组织化学定位

目的研究黑眉锦蛇消化道酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、过氧化物酶(POX)、非特异性酯酶(NSE)、腺苷三磷酸酶(ATPase)、琥珀酸脱氢酶(SDH)等酶的分布。方法消化道分8个部位取材,应用冰冻切片、石蜡切片、酶组织化学技术及光密度定量分析。结果 ACP主要分布于十二指肠至回肠的黏膜上皮,十二指肠和空肠酶活性显著较高(P<0.05);ALP分布于食管、十二指肠至回肠的黏膜上皮,十二指肠酶活性最高(P<0.05);POX和NSE在整个消化道黏膜上皮和黏膜固有层中均有分布,胃幽门和直肠酶活性较低(P<0.05);ATPase在消化道除直肠未检测到酶活性外,其它部位均有分布,以十二指肠酶活性最高(P<0.05);SDH除食管未检测到酶活性外,其它部位均有分布,胃中胃腺部酶活性较高(P<0.05)。结论黑眉锦蛇消化道黏膜酶的分布同其它动物有相似之处,也有其自身特点。不同酶的分布和消化道各部位的生理机能密切相关。     

Elaboration of enzymes in the digestive gland of Viviparus bengalensis (Mollusca: Gastropoda) during the first hours of digestion

digestive gland in Viviparus bengalensis does not show prominent rhythmicity in enzyme secretion during the post-feeding period. Amylolytic, lipolytic and proteolytic enzymes are secreted at a slightly enhanced rate between the first and second hour after feeding, which is followed by gradual decline to become more or less constant. The secretion of different enzymes is synchronous. The absence of secretory rhythms during post-feeding period appears to be correlated with food-habits and feeding behaviour.     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。     

Biosynthesis of Astacus protease,a digestive enzyme from crayfish

Summary For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalfoenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to V.S./R.Z. (Sto 75/10) and to W.S./R.Z. (Sto 185/1-1)     

A field study on the physiology of digestion in the Antarctic krill, Euphausia superba, with special regard to chitinolytic enzymes

Endo- and exochitinase activities were determined in the stomachand midgut gland of the Antarctic krill, Euphausia superba.along a transect west of the Antarctic Peninsula. Activitieswere compared with the digestive enzymes protease, cellulase(1,4-ß-D-glucanase) and laminarinase (1,3-ß-D-glucanase)The chlorophyll and protein contents in the surface water ofthe corresponding stations were determined. Enzyme activitieswere characterized by high individual and spatial variations.Chitinolytic activity in the stomach correlated well with alldigestive enzymes investigated. In the midgut gland, a correlationwith cellulase and laminannase was evident. The amount of chlorophylla and phytoplankton protein in the surface water was not correlatedwith enzyme activity. Specific enzyme activity was higher inthe stomach than in the midgut gland. showing individual ratiosfor each enzyme. Elevated endochitinase activity in the stomachsuggests that chitinous food is digested to oligomers in thestomach, while the subsequent degradation to amino sugars occurspredominantly in the midgut gland.     

Transpancreatic transport of digestive enzyme

When porcine α-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [³H]- leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase of about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrpsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.     

东北虎幼体消化系统蛋白水解酶的初步研究

蛋白水解酶在许多生命活动中是必需的物质(Vassalli and Pepper,1994)。蛋白质的酶解修饰(Xuet al.,1999)、细胞迁移、组织再生与修复、消化系统对蛋白质的消化等均与蛋白水解酶有关(Baimbridgeet al.,1992),且蛋白水解酶功能失调会导致许多疾病(Teichertet al.,1989)。东北虎(     

凹耳蛙消化道组织学和嗜银细胞形态观察

为了揭示凹耳蛙(Odorrana tormota)消化道的基本特征,运用石蜡切片法和龙桂开银浸法对凹耳蛙消化道组织学结构及嗜银细胞的形态与分布密度进行了观察。结果显示:①凹耳蛙的胃壁具明显的纵行皱襞和胃小凹,胃腺发达,小肠可分为十二指肠和回肠,杯状细胞分散在十二指肠上皮细胞之间,十二指肠中未见十二指肠腺分布。②凹耳蛙嗜银细胞见于消化道全长,呈毛笔头样、锥体形、梭形、椭圆形和长条形等;幽门腺上皮和十二指肠绒毛上皮中的嗜银细胞具指向腺泡腔或肠腔的突起,提示其可能具有腔分泌的功能。嗜银细胞的分布密度胃幽门部最高,十二指肠和胃体其次,食道最低。据此认为胃既是凹耳蛙的主要消化器官,也是消化道中主要的内分泌器官;十二指肠是凹耳蛙消化道中的主要吸收部位,同时也具有内分泌功能;消化道嗜银细胞具有内分泌的功能,还可能具有腔分泌的功能。     

Microstructural and histochemical advances on the digestive gland of the common cuttlefish,Sepia officinalis L.

The cephalopod digestive gland is a complex organ that, although analogous to the vertebrate liver, has additional functions, with special (albeit not exclusive) note on its active role in digestion. Although the structure of the digestive cell and its main constituents are well known (among which “boules” and brown bodies are distinctive features), histological details of other cell types and the general structure of the digestive gland need still further research. By a thorough combination of histological and histochemical techniques, it is shown that the digestive gland diverticula of the common cuttlefish (Sepia officinalis L.) are comprised of three essential cell types: digestive, basal and excretory. Basal (“pyramidal”) cells are multi-functional, being responsible for cell replacement and detoxification, mainly through the production of calcic spherulae containing metals like copper and lead in a complex organic matrix of proteins and ribonucleins. Since copper- and lead-positive spherulae were almost absent from other cell types and lumen of the tubules, it appears that controlled bioaccumulation of these metals, rather than excretion, is the main detoxification mechanism. The results show that the organ is crossed by an intricate network of blood vessels, especially arteries and arterioles, whose contents share histochemical properties with a particular set of “boules” that are shed into the lumen of diverticula for elimination, suggesting that the organ actively removes unwanted metabolites from the haemolymph. Conversely, the rarer excretory cells appear to be specialized in the elimination of salts. Although the exact nature of many excretory and secretory products, as the metabolic pathways that originate them, remain elusive, the findings suggest an intricate interaction between the different cell types and between these and the surrounding media: haemolymph and digestive tract.     

Development of a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis L. (Mollusca Cephalopoda): effect of Cu, Zn and Ag on enzyme activities and cell viability

The purpose of this study was to establish a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis in order to observe the effect of heavy metals on digestive enzyme activities. Digestive cells were isolated using a pronase enzyme that was removed by several washings of the cell suspension. Cell viability was tested by the MTT assay (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium) and microscopic analysis. The results showed that isolated digestive cells could be maintained 24 h with preservation of whole digestive functionality, measured in terms of MTT test. In fact, the viability was maintained at a high level during 24 h and the intra- and extracellular digestive enzyme activities became stabilised rapidly. Furthermore, suspension cells responded to calcium ionophore and 8-Bromo-cAMP by an unspecific secretion of extracellular digestive enzyme, trypsin, which demonstrated that isolated digestive cells were functional. Using the bioassay, ecotoxicological studies showed that heavy metals could have effects on digestive enzyme activities after 24 h of an incubation time of the metal with the cells. In fact, zinc and silver affected trypsin and/or cathepsins specific activity of the cells. On the contrary, copper had no effect on digestive enzyme activities. Zinc, which is a trace element in all living animals, generated two different responses of cathepsins and cell viability. At a low concentration (0.02 μM), it increased viability and cathepsins specific activity, whereas at a high concentration (0.02 mM), zinc inhibited the cathepsins specific activity with an inhibition of cathepsins. For silver, whatever the tested concentration (0.02 mM or 0.02 μM), it has no impact on digestive gland isolated cell viability. Nevertheless, heavy metal induced high disturbance of enzymatic systems.     

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.