Increased lung uptake of liposomes coated with polysaccharides

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Morphological changes in rat pancreatic slices associated with inhibition of enzyme secretion by high concentrations of secretagogues

Stimulation of enzyme secretion in rat pancreatic slices by cholinergic agonists or by cholecystokinin-pancreozymin (CCK-PZ) and its peptide analogs showed a biphasic dose response curve. The optimal concentrations eliciting an efficient rate of enzyme secretion were 1 microM for carbamylcholine or acetylcholine, and 5 nM and 20 nM for CCK-PZ octapeptide and CCK-PZ, respectively. At higher concentrations of secretagogues, however, the rate of secretion progressively declined, and almost complete inhibition was achieved at 1 mM of carbamylcholine or acetylcholine and at 0.1 microM of CCK-PZ or its octapeptide analog. Atropine displaced the dose-response curve for carbamylcholine to the right so that in the presence of 7 microM atropine a concentration of 1 mM carbamylcholine now gave an optimal rate of enzyme secretion. The ionophore A-23187 which bypasses the receptor and elicits enzyme secretion did not relieve the inhibition caused by supraoptimal concentrations of secretagogues, indicating that the inhibition occurs at the cellular rather than at the receptor level. Secretin had no effect on the inhibition of enzyme secretion by a high concentration of carbamylcholine, indicating that the inhibition was not caused by lack of water and electrolyte secretion. The energy-producing metabolism was not affected since the ATP level in the pancreatic slices was the same in the presence of either inhibitory or optimal concentrations of secretagogues. The inhibition of enzyme secretion was reversible since restoration of efficient enzyme secretion occurred after removal of carbamylcholine (1 mM) by washing, followed by addition of an optimal concentration of CCK-PZ octapeptide. Morphological studies revealed that the presence of inhibitory concentrations of secretagogues caused severe distortion of the lumen structure: disruption of the filamentous system surrounding the lumen, disappearance of microvilli, and production of distended evaginations of the luminal membrane containing cellular material. These changes eventually caused a reduction in the size of the lumen which becomes plugged with secretory material. It is suggested that these changes in the microtubular microfilamentous system could account for the inhibition of enzyme secretion.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.     

Calcium requirements in the action of cholecystokinin-pancreozymin on pancreatic acinar cell metabolism.

The present study utilized ionophore A23187 to determine the role of Ca²⁺ in pancreatic acinar cell metabolism. The ionophore A23187 in the presence of EGTA increased efflux of Ca²⁺ from the rat pancreatic fragments. Ionophore and CCK-PZ were equally effective in the presence of extracellular Ca²⁺ in stimulating ¹⁴C-labeled protein secretion. The ionophore decreased synthesis of new protein more effectively than CCK-PZ in the presence of extracellular Ca²⁺. The effect of ionophore and CCK-PZ in combination was greater than either agent alone. Phospholipid labeling was not stimulated by A23187 in the presence of extracellular Ca²⁺ in contrast to CCK-PZ. With CCK-PZ, the effect was dependent on the concentration of extracellular Ca²⁺. Protein phosphorylation was stimulated ～ 109% by CCK-PZ and ～ 39% by ionophore. CCK-PZ stimulated protein phosphorylation in the 100,000 g supernatant whereas A23187 was ineffective. Ionophore A23187 inhibited glucose oxidation whereas CCK-PZ stimulated glucose oxidation. These data suggest that more than one kinase system might be involved in metabolic responses to hormonal stimulation of the pancreas viz. a phosphorylase kinase may be directly activated by Ca²⁺ causing protein discharge whereas other kinase system may require binding of the hormone to receptor leading to other events besides protein discharge.     

Intracellular control of human neutrophil secretion. I. C5a-induced stimulus-specific desensitization and the effects of cytochalasin B.

Human neutrophils released the granule constituents myeloperoxidase and lysozyme, but not the cytoplasmic enzyme lactic dehydrogenase, when pretreated with cytochalasin B and stimulated with purified human C5a. Prior exposure to C5a before the cytochalasin B, however, abrogated the subsequent secretory process. Interaction of neutrophils with C5a was shown to result in a concentration-dependent rapid desensitization that could not be overcome by later addition of cytochalasin B or of cytochalasin B and C5a. The effect was relatively stimulus specific in that neutrophils desensitized in this manner could be induced to release granule enzymes by casein or by complement-coated zymosan particles. Cytochalasin B effects on neutrophils appear to mimic those of surface binding of soluble stimuli such as C5a and immune complexes. It is suggested that desensitization in concert with surface stimulation may represent an important intracellular mechanism for limiting neutrophil secretion.     

Effects of some gastrointestinal peptides on pancreatic growth in rats (preliminary results)

The purpose of this study was to estimate the effects of cholecystokinin (CCK), somatostatin (SS) pancreatic polypeptide (PP) and their interaction with each other, given them in single doses, on pancreatic secretion and pancreatic growth after long-term treatment in rats. The acute secretory effects of the above mentioned peptides were studied on conscious rats supplied with pancreatic, gastric and jugular vein cannulae. The pancreatic growth was characterized by measurements of pancreatic weight, desoxyribonucleic acid (DNA), protein, trypsin and amylase content after 5 days treatment. Amylase output was increased by caerulein alone, and given it in combination with somatostatin (SS), while its value decreased by SS alone. After 5 days treatment, the pancreatic weight, trypsin and amylase activity (hypertrophy) was increased by caerulein, and these values were not altered by S alone. In combinative administration of caerulein with somatostatin, the stimulatory effect by caerulein was decreased. PP given alone or in combination with caerulein decreased both the basal and stimulated amylase output. PP given for 5 days decreased pancreatic trypsin and amylase contents and counteracted the stimulatory effect by caerulein to these enzymes' contents. It has been concluded that: 1. caerulein stimulates both pancreatic enzyme secretion and pancreatic growth; 2. somatostatin inhibits the pancreatic secretion and caerulein induced pancreatic growth, but it does not affect the spontaneous growth of pancreas; 3. pancreatic polypeptide inhibits the pancreatic secretion and decreases pancreatic trypsin and amylase contents.     

Role of the cytoskeleton in laminin induced mammary gene expression

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Effects of cytochalasin B, colchicine and vincristine on the metabolism of isolated fat-cells

1. Colchicine and vincristine only slightly inhibit the metabolism of glucose to CO(2) and lipids by isolated fat-cells. 2. Prolonged incubation with these agents causes no further inhibition. 3. Cytochalasin B, however, inhibits glucose metabolism to both CO(2) and lipids in fat-cells. 4. However, at a concentration that causes a strong inhibition of glucose metabolism cytochalasin B is without effect on the metabolism of pyruvate, lactate or arginine to these end products. The uptake of labelled alpha-aminoisobutyrate is likewise not modified. Similarly it does not affect release of glycerol or free fatty acid, or the actions of adrenaline, insulin or caffeine on these parameters. At 10mug/ml it slightly lowers ATP concentrations, an effect that does not occur at 2mug/ml. 5. The transport of fructose into adipocytes by a specific fructose-transport system is also not affected by the agent, but the uptake of 2-deoxyglucose is strongly inhibited. It is concluded that cytochalasin B may specifically inhibit the glucose-transport system of isolated fat-cells. 6. Cytochalasin A has a much weaker action than cytochalasin B on glucose metabolism.     

Process of re-establishment of beta-adrenergic induced protein secretion after cytochalasin D inhibition in rat parotid gland. Effect of cholinergic agonist, phorbol ester and calcium.

In rat parotid gland, 3H-protein secretion is stimulated by beta-adrenergic receptor activation (via cAMP) and also by cholinergic receptor activation (via IP3, calcium and diacylglycerol). The disorganization of microfilament system by cytochalasin D induced an inhibition of beta-adrenergic induced 3H-protein secretion whereas it did not modify the cholinergic muscarinic one. Cytochalasin D induced the formation of vacuoles in the parotid cell. In this work we show that the activation of muscarinic receptors (with carbachol) partially abolished the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. Since carbachol induced both intracellular calcium increase and protein kinase C activation, we decided to test separately the effect of calcium (using the calcium ionophore A23187) and protein kinase C activation (using phorbol ester) on the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. A23187, in the presence of calcium in the external medium was able to partially abolish cytochalasin D effect (ie re-establishing protein secretion) whereas activation of protein kinase C by phorbol 12-13 di-butyrate had no effect. These results suggest that protein kinase C is not involved in re-establishing a 'normal' secretion phenomenon whereas calcium does interfere. Furthermore, our fluorescence study shows that, when cytochalasin D is present in the incubation medium, the actin network is disturbed even in the presence of carbachol. This indicates that a calcium entry in the cell is not sufficient to restore a 'normal' actin network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulus-secretion coupling in platelets. Effects of drugs on secretion on adenosine 5'-triphosphate.

The mechanism of stimulus-secretion coupling in platelets was investigated by observing the effects of drugs on the kinetics on ATP secretion induced by either thrombin or the divalent cation ionophore A23187. The actual secretion is the same with either of these agents, since the rate constants and activation energies of secretion are the same and since drugs that affect the final, enzyme-independent steps of thrombin-induced secretion have the same effect on ionophore-induced secretion. Drugs that affect early steps of thrombin-induced secretion have no effect on ionophore-induced secretion. Drugs that act through cAMP (PGE1, theophylline, dibutyryl-cAMP) slow an early step in the mechanism of thrombin-induced secretion and completely block at higher levels, with the required concentration of inhibitor dependent on thrombin concentration. The inhibition of rate appears to be all-or-none, with no intermediate rates observed. By replacing thrombin with trypsin, which makes it possible to observe a complete change in rate-determining step from an enzyme-dependent to an enzyme-independent platelet step, it was found that these drugs slow the rate only when the enzyme-independent step is rate determining. These drugs have no effect on A23187-induced secretion. It was concluded that cAMP inhibits at a step after the enzyme step but before the final step by interfering with transmission of the stimulus-secretion coupling signal. Disruption of microfilament function by cytochalasin B (10 muM) accelerates the rate of secretion induced by either thrombin or ionophore. The microtubule agents colchicine, vinblastine, and vincristine had effects only at concentrations above those usually considered necessary for the specific inhibition of microtubule function. Drugs that inhibit prostaglandin synthesis (aspirin, indomethacin, eicosatetraynoic acid), drugs that block ATP production (antimycin A, deoxyglucose), or several other drugs previously reported to inhibit platelet function had no effect on secretion.     

Pancreatic response of anaesthetized and conscious rats to bolus injection of cholecystokinin-pancreozymin

Pancreatic secretion was studied in anaesthetized rats tested immediately after surgery or in conscious rats tested 48 hr after the cannulation of the pancreatic duct. Pancreatic flow, protein output and enzyme output were measured over a 30-min period in the unstimulated state and after the intravenous injection of bolus doses of cholecystokinin-pancreozymin (CCK-PZ) ranging from 1.25 to 20 Crick-Harper-Raper units (CHRU). Each animal received three doses of CCK-PZ, as either ascending or descending doses. In anaesthetized rats there was a linear relationship between the log-dose of CCK-PZ and the flow, protein and enzyme output with both the ascending and descending doses. In contrast, in conscious rats flow was unaffected by CCK-PZ, and protein output was greatest after the first dose, whether this was given in the ascending or descending doses. At all CCK-PZ levels flow in anaesthetized rats was less than that seen in conscious animals, but at doses of CCK-PZ above 5.00 CHRU protein output was greater in anaesthetized rats than in conscious rats. Ultrastructural studies of the pancreas showed areas of focal cytoplasmic degeneration and possible blockage of the duct with cellular debris after administration of high doses of CCK-PZ to conscious rats. These changes may be responsible for the reduced protein output with the second and third dose of CCK-PZ in these animals. No such changes were seen in anaesthetized rats after similar doses of CCK-PZ. These studies show fundamental differences in the response of the pancreas to CCK-PZ in anaesthetized and conscious rats. The mechanism for this difference is not clear, but it may represent a change in the normal response to CCK-PZ in the anaesthetized rats as a result of the effects of acute operative trauma, possibly acting through changes in pancreatic blood flow.     

Vanadate stimulates rat pancreatic enzyme secretion through the release of calcium from an intracellular store

Orthovanadate accelerates 45Ca efflux and enzyme secretion from the rat pancreas incubated in either control (2.5 mM Ca) or nominally Ca-free buffers. Secretion induced by vanadate does not appear to be mediated by changes in either adenylate cyclase or sodium pump activity. Instead, vanadate appears to act at an intracellular site to cause the release of calcium from the same pool mobilised by acetylcholine. Vanadate action is not inhibited by DIDS. The effect of pH on vanadate action may be accounted for by changes in the distribution of the vanadates. Vanadyl sulphate inhibits secretion evoked by acetylcholine. This suggests that intracellular reduction of vanadate (+5 oxidation state) to the +4 oxidation state may account for an inhibitory component observed during stimulation with vanadate.     

Colchicine inhibition of plasma protein release from rat hepatocytes

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.     

Microfilaments regulate the rate of exocytosis in rat basophilic leukemia cells

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.