Stimulated Enzyme Release Research Articles

Release of digestive enzymes from the crustacean hepatopancreas: effect of vertebrate gastrointestinal hormones

Vertebrate gastrointestinal hormones were tested on their ability to liberate digestive enzymes from the crustacean midgut gland. CCK-8 (desulfated form), gastrin, bombesin, secretin, and substance P were detected to release enzymes. Maximal concentrations observed were 5 nM CCK for protease release, 1 nM gastrin for protease and 100 nM for amylase release, 100 nM bombesin for protease release, 10 nM secretin for amylase and protease release, and 100 nM substance P for protease release. Unlike in vertebrates, glucagon was unable to stimulate enzyme release in crustaceans, this also applies to the counterpart insulin. These results may support the assumption that Crustacea possess endogenous factors resembling the above mentioned vertebrate hormones, at least in such a way that the appropriate receptors have the capacity to accept these hormones.

The C-terminal part of human melanoma growth stimulating activity (MGSA/GRO alpha) binds to neutrophils but does not stimulate enzyme release

CXC chemokines are known to bind to their receptors on neutrophils and activate them through the N-terminal ELR box. In this study, we show that the C-terminal alpha helix of human MGSA does bind to neutrophils, but contrary to complete MGSA, is unable to promote enzyme release by neutrophils. These results raise the possibility of MGSA being a multi-functional molecule with various domains of activation.

Leukocyte proteinase release during storage of red cell concentrates.

The release of polymorphonuclear leukocyte proteinases in buffy-coat-depleted red cell concentrates was examined during a storage period of 35 days. Collagenase, gelatinase and elastase predominantly induce breakdown of connective tissue. However, when released by cell disintegration during red cell concentrate storage, the considerable proteolytic activities of these enzymes might influence the quality of the stored blood. During the observation period a considerable decrease in the polymorphonuclear leukocyte count was observed, accompanied by increases in the levels of collagenase, gelatinase and elastase. Compared with the enzyme levels on the day of red cell concentrate preparation, collagenase increased 20-fold, gelatinase 6-fold and elastase 100-fold during the storage period. When cells were treated with the chemoattractant hexapeptide, N-formyl-nle-leu-phe-nle-tyr-leu, and the degranulation promoting cytochalasin B, gelatinase exhibited the highest secreted concentration in the freshly prepared red cell concentrate, exceeding the maximum of spontaneously released elastase by 4- to 6-fold. However, these compounds stimulated enzyme release only during the first day after red cell concentrate preparation. Thereafter, no differences between stimulated and non-stimulated samples were observed. The data indicate that polymorphonuclear leukocytes contain a large storage pool of proteolytic enzymes. These enzymes together with other polymorphonuclear leukocyte enzymes, e.g. hydrolases and oxidoreductases, might alter the erythrocyte membrane surface and thus influence the storage quality of the prepared red cell concentrate.

Selective induction of release of hydrolases from Crassostrea virginica hemocytes by certain bacteria

Serum-free Crassostrea virginica hemocytes were maintained in a medium to which was added one of four species (or strains) of bacteria: Escherichia coli, Enteric Group 17 bacteria, Micrococcus roseus, and Klebsiella oxytoca. The hemocytes were exposed to each type of bacteria for 1, 2, 3, and 4 hr after which they were removed and the activities of two hydrolases, acid phosphatase and α-mannosidase, in the maintenance medium were ascertained. It was determined that at 25°C both E. coli and Enteric Group 17 bacteria induced a linear release of both enzymes from hemocytes into the maintenance medium while neither M. roseus nor K. oxytoca stimulated enzyme release. It is concluded that there is selective induction of release of hydrolases from oyster hemocytes by only certain bacteria.

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BxPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca 2+] i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca 2+] i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK ( 4 · 10 −13 M to 4 · 10 −10 M ) in 77% of cells tested. In contrast [Ca 2+] i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP 3) resulted in a marked increase in medium calcium concentration indicating IP 3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP 3-sensitive intracellular Ca 2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.

The effect of leukotrienes on porcine alveolar macrophage function

The ability of the synthetic leukotrienes LTB 4, LTC 4, LTD 4 and LTE 4 to stimulate porcine AM was compared with that of two known AM stimuli: zymosan, a particulate stimulus and phorbol myristate acetate (PMA), a soluble stimulus. The criteria for AM stimulation were: (1) increased generation of superoxide anion (O 2 −), (2), the release of the lysosomal enzymes N-acetyl-β-D-glucosaminidase (NABG) and arylsulfatase and (3) an increase in surface free energy. Whereas zymosan and PMA both stimulated AM according to each of the three criteria, the effect of leukotrienes was relatively minor. LTB 4 (10 −6 M) and LTD (10 −6 M) caused a modest reduction in spontaneous O 2 release by AM.LTD 4 (10 −6 M) also caused a small (18%), but significant (p < 0.05), reduction in the additional O 2 release induced by PMA. LTC 4 and LTE 4 did not alter spontaneous O 2 release. Neither spontaneous nor stimulated enzyme release was systematically altered by any of the leukotrienes. LTD 4 caused a cysteine-inhibitable, dose-dependent increase in surface free energy with a plateau at concentrations above 10 −8M. The increased surface free energy induced by LTD 4 may be a consequence of binding to a surface dipeptidase.

Ca2+-Stimulated Secretion of α-Amylase During Development in Barley Aleurone Protoplasts

The effects of gibberellic acid (GA(3)) and Ca(2+) on the synthesis and secretion of alpha-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA(3), CaCl(2), or both. Incubation of protoplasts in medium containing both GA(3) and Ca(2+), however, causes an increase in the alpha-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA(3) or Ca(2+) alone. Isoelectric focusing shows that adding Ca(2+) to incubation media containing GA(3) increases the levels of alpha-amylase isozymes having high isoelectric points (pI). In the presence of GA(3) alone, only isozymes with low pIs accumulate. The increase in alpha-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca(2+) to maintain elevated levels of alpha-amylase release. Immunoelectrophoresis shows that Ca(2+) stimulates the release of low-pI alpha-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA(3) alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl(2). The response is not specific for Ca(2+) since Sr(2+) can substitute, although less effectively than Ca(2+). Pulse-labeling experiments show that alpha-amylase isozymes produced by aleurone protoplasts in response to GA(3) and Ca(2+) are newly synthesized. The effects of Ca(2+) on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on alpha-amylase stability or on protoplast viability. We conclude that Ca(2+) directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca(2+)-stimulated enzyme release.

Basic polyaminoacids and protamine stimulate enzyme release from frog muscles.

Polylysines, polyornithine, and protamine increased the release of creatine kinase (CK) and lactate dehydrogenase (LDH) from frog muscles. With 5 micrograms/ml (50 nM) of a polylysine of molecular weight 100,000, enzyme release reached peak values (ten-fold above control) within 30 minutes; subsequent removal of the polylysine did not reduce significantly the release rates. The effectiveness of the polycations increased with the degree of polymerization. Their effects were prevented by pretreatment of the muscles with polyanions (heparin or polyglutamic acids) and were reversed when heparin was applied within 30 minutes of exposure to polylysine. The initial stimulation of enzyme release by polylysine was attenuated by Ca-free solutions and by low temperature (4 degrees C). It is suggested that, while fiber damage can explain the increased loss of sarcoplasmic enzymes after prolonged treatment of frog muscles with polycations, other mechanisms (perhaps exocytosis) are involved in the stimulation of CK and LDH release during brief exposures to nanomolar concentrations of polylysines or polyornithine.

Granulocyte functions during maturation of human promyelocytic leukemia cells

Human promyelocytic leukemia cells (HL-60) mature into functional granulocytes in response to in vitro treatment with several classes of chemical agents. Compounds that increase intracellular adenosine 3′:5′-cyclic monophosphate induce a modified program of maturation in which the cells demonstrate functional properties characteristic of mature phagocytic cells while remaining morphologically immature. We compared the developmental programs initiated by two well-studied inducers, retinoic acid and dimethyl sulfoxide, with the programs initiated by two inducers known to raise intracellular adenosine 3′:5′-cyclic monophosphate; N 6, O 2-dibutyryl adenosine 3′:5′-cyclic monophosphate and the combination of prostaglandin E 2 and theophylline. In response to the increase in intracellular adenosine 3′:5′-cyclic monophosphate, the cells ceased proliferation, expressed chemotactic receptors and demonstrated stimulated enzyme release within 24 h. Chemotaxis, adherence, NBT reduction and superoxide production appeared by 72 h, although the cells remained unchanged morphologically. A similar developmental program was induced by dimethylsulfoxide, but appearance of the markers was delayed by 48 h. Expression of these markers was delayed and incomplete in response to retinoic acid.

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium.

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.

Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis.

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.

The Induction of Lysosomal Enzyme Release from Leucocytes of Normal and Emphysematous Subjects and the Effects of Tobacco Smoke upon Phagocytosis

1. The lysosomal enzymes of circulating polymorphonuclear leucocytes contain a potent elastase; release of this enzyme within the lung is thought to be responsible for the destruction of elastic tissue in pulmonary emphysema. 2. The release of lysosomal enzymes from blood leucocytes of normal and emphysematous subjects during phagocytosis of particulate material was studied In vitro. Acid phosphatase and acid ribonuclease were used as markers of lysosomal enzyme release, no sufficiently sensitive assay for elastase being available. Cigarette smoke was separated into ‘particulate’ and ‘soluble’ fractions. In a preliminary study, the particulate fraction stimulated enzyme release; in the experiments reported here, latex particles were used to produce this effect. 3. Approximately one-third of the total lysosomal enzyme content was released to the exterior of the cell during phagocytosis of latex particles. In this respect there was no difference between normal and emphysematous subjects. 4. The effects of the non-particulate soluble fraction of cigarette smoke on phagocytosis-induced enzyme release were studied. This fraction inhibited enzyme release from polymorphonuclear leucocytes of normal subjects but not from those of emphysematous patients. When the ‘cigarette-smoke solution’ was replaced by the respiratory inhibitor, antimycin A, a similar inhibition of enzyme release occurred. The inhibition of phagocytosis in cells of normal subjects is presumed to be due to a respiratory inhibitor such as carbon monoxide in the soluble fraction of the smoke. We postulate that the polymorphonuclear leucocytes of emphysematous patients are adapted to hypoxic conditions so that inhibition of enzyme release does not occur.

Prostaglandins and enzyme secretion from dispersed rat pancreatic acinar cells

The role of prostaglandins in exocrine pancreatic enzyme secretion was studied. The effects of three inhibitors of prostaglandin and thromboxane syntheses, were evaluated on release of amylase from dispersed rat pancreatic acinar cells. Mepacrine inhibited, while indomethacin and imidazole had no effect on basal or carbachol or cholecystokinin stimulated enzyme release. Exogenous arachidonic acid or various prostaglandins (E 1, E 2, F 2α, I 2), also did not affect the secretory process. Acinar cells actively incorporated radioactive arachidonic acid, principally into phospholipids (especially phosphatidylcholine), however release of the free fatty acid and subsequent synthesis of radioactive endogenous prostaglandins was not stimulated by the presence of different pancreatic stimulants. Pancreatic microsomes were found to be lacking in cyclo-oxygenase, an enzyme involved in endegenous synthesis of prostaglandins. The data suggest that prostaglandins are not involved directly in excitation-secretion coupling in the exocrine pancreas.

Fluorescent probe detects redistribution of cell calcium during stimulus–secretion coupling

STIMULUS-secretion coupling in pancreatic acinar cells is believed to be mediated by a rise in intracellular Ca2+ activity1–5. Based on the observation that physiological secretagogues trigger a rapid increase of Ca2+ efflux3,4 and stimulate enzyme release even in the absence of extracellular calcium2,4,5, it has been proposed that stimulus–secretion coupling involves a redistribution of intracellular calcium3,5,6. Since most calcium in pancreatic acinar cells is bound to membranes or is sequestered in mitochondria7, we have attempted to monitor these calcium stores during secretion by using the fluorescent probe chlorotetracycline (CTC). This probe forms highly fluorescent complexes with Ca2+ and Mg2+ in the presence of biological membranes or detergent miceles8,9 and has been successfully used in isolated mitochondria10,11 and sarcoplasmic reticulum vesicles12 to monitor calcium movements induced by substrates and inhibitors. In this report, we show that fluorescence of CTC associated with pancreatic acinar cell membranes decreases rapidly on stimulation of enzyme secretion and that this intensity change is preferential for CTC complexed to calcium.

Changes in plasma hydrolase activities in hereditary and streptozotocin-induced diabetes

Reports of elevated plasma levels of acid hydrolases in diabetic patients prompted us to investigate these enzymes in genetically diabetic (db/db) mice and in streptozotocintreated rats and mice. The homozygous (db/db) mice showed decreased, rather than increased, levels of plasma hydrolases as compared to their heterozygous (db/+) nondiabetic controls. Similarly, mice (Swiss and db/+) with streptozotocin-induced diabetes showed lowered plasma hydrolase activities. On the other hand, drug-induced diabetes in rats was accompanied by increased hydrolase levels, the increase being reversed by insulin treatment. In investigating the underlying mechanism of the changes observed in rats, leukocytes and blood platelets were ruled out as sources of the additional plasma lysosomal enzymes, and evidence was obtained suggesting that the diabetic animal was normal in regard to the ability to remove lysosomal glycosidase from blood. However, perfusion of diabetic liver resulted in release of more acid hydrolase activity than did perfusion of normal liver, and perfusion with glucagon stimulated enzyme release. These results suggest that liver is a possible source of the added enzyme found in streptozotocin-treated rat plasma.

Pancreatic acinar cells: Use of a Ca ++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling

The Ca ++ ionophore A23187 had no effect on the release of amylase by mouse pancreas fragments in the absence of Ca ++ but when Ca ++ was re-added to the medium amylase release was observed in a pattern which mimicked that produced by normal stimulants. Uptake of 45Ca ++ by pancreatic fragments was increased by A23187. Tetracaine and dinitrophenol at concentrations which block cholinergic stimulated enzyme release blocked ionophore induced release whereas atropine did not. None of the inhibitors studied affected the ionophore induced Ca ++ uptake.