Platelet-activating factor (PAF) mediation of rat anaphylactic responses to soluble immune complexes. Studies with PAF receptor antagonist L-652,731

A new synthetic compound, L-652,731 (trans-2,5-(3,4,5-trimethoxyphenyl) tetrahydrofuran), which has been demonstrated by Hwang et al. to be a potent and specific platelet-activating factor (PAF) receptor antagonist causes 100% inhibition of 1 microM PAF-induced neutrophil degranulation at 50 microM, but has no effect on neutrophil degranulation induced by precipitating immune complexes (323 micrograms/ml), fMet-Leu-Phe (10(-7) M), or the calcium ionophore A23187 (10(-5) M). Intravenous infusion of 1 mumol L-652,731 results in almost 100% inhibition of hypotension induced by PAF but not that induced by isoproterenol, histamine, bradykinin, or acetylcholine. With the use of this novel PAF receptor antagonist, the in vivo mediator role of PAF in the soluble immune complex-induced hypotension, extravasation, vascular lysosomal hydrolase secretion, and neutropenia in rats was determined. The hypotension, extravasation, and lysosomal hydrolase release induced by immune complex infusion take 2 to 10 min longer to occur than the same responses elicited by PAF infusion. The neutropenia response is immediate with both stimuli. L-652,731 when orally administered to rats (20 mg/kg, 1.5 hr before PAF infusion) inhibited PAF-induced hypotension (69%), extravasation (76%), vascular lysosomal hydrolase release (79%), and neutropenia (73%). The same L-652,731-dosing regimen inhibited immune complex-stimulated hypotension (87%), extravasation (77%), and vascular lysosomal hydrolase release (31%). The initial and complete neutropenia induced by immune complex infusion was not inhibited in L-652,731-pretreated rats, but the rate of return of neutrophils to the blood was faster in the latter rats. Rats with blocked circulation to the liver still exhibited extensive extravasation and vascular lysosomal hydrolase release in response to PAF, but there was no extravasation and greatly reduced hydrolase release in response to immune complexes. Thus PAF is indicated to be a major mediator of soluble immune complex-induced hypotension and vascular permeability and a minor mediator of immune complex-induced lysosomal hydrolase release in rats. PAF probably does not mediate the initial and complete neutropenia stimulated by immune complexes. The liver is probably the major site for PAF production in response to circulating immune complexes.     

An established light ear mutant (C57BL/6J-Pdeb(rd1) le) mouse cell line exhibits a block to secretion of lysosomal enzymes

The hypopigment mutant mice, light ear, pallid, and beige, possess defects in melanosomes, lysosomes, and platelet dense granules, suggesting that these organelles share a common biogenesis and processing. Light ear and pallid mutants are animal models for Hermansky Pudlak syndrome, whereas the beige mouse is an animal model for Chediak Higashi syndrome. An established skin cell line from the light ear mouse was tested along with pallid and beige cell lines for mutant effects on secretion of lysosomal hydrolase activities of six different lysosomal glycosidases and the trafficking of N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythrosphingosine (C(5)-DMB-ceramide). There were no consistently significant differences between the pallid and the beige mutant cell lines or between these two mutant lines and the control cell line in the percentage secretion of lysosomal hydrolase activities. The light ear mutant cell line, however, displayed a significantly lower percentage secretion of lysosomal hydrolase activities than all other cell lines tested. The light ear mutant cells processed C(5)-DMB-ceramide completely, as seen in the control cell line, whereas pallid and beige cell lines retained fluorescent material and exhibited a block in the complete processing of C(5)-DMB-ceramide 20 h after labeling. The block to secretion of lyososomal hydrolase activities in the light ear mutant cell line will be useful for further studies on this mutant's lysosomal defect.     

Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel,Mytilus edulis,and changes induced by thermal stress

Latent beta-glucuronidase and glucosaminidase activities have been demonstrated in small cytoplasmic particles, which may possibly be primary lysosomes, as well as some larger granules of the digestive cells of the common mussel. Latency was indicated by increased staining of these structures following incubation in buffer at pH 4.5 at 37 degrees C. The exposure of mussels to temperatures of 25-28 degrees C over a period of four days induced a significant decrease in the latency of lysosomal glucosaminidase. Thermal death produced labilization of lysosomes although selective release of hydrolase activity was indicated by the differential latency of glucosaminidase and glucuronidase. The injection of hydrocortisone induced a significant increase in latency in stressed animals, indicating that the stress response involved changes in structure and function of membranes.     

Hydrolase secretion is a consequence of membrane recycling

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.     

Unique inhibitory profile of platelet activating factor induced calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophils towards pertussis toxin and phorbol ester

Platelet activating factor has been found to increase the intracellular level of free calcium (as monitored by the fluorescent calcium indicator quin-2) and to stimulate the turnover of the polyphosphoinositides in rabbit neutrophils. Calcium mobilization induced by platelet activating factor, in contrast to previous reports with chemotactic factors, is unaffected by pertussis toxin; on the other hand, stimulated polyphosphoinositol hydrolysis and granule enzyme secretion are potently antagonized under the same conditions. The calcium, as well as the secretory responses to the lipid mediator are largely dependent on the presence of extracellular calcium. Internal contributions to the quin-2 signal are only detectable at relatively high concentrations of platelet activating factor. Calcium mobilization and secretion stimulated by platelet activating factor are inhibited following a short incubation with phorbol 12-myristate 13-acetate. These results are discussed in terms of the possibility that platelet activating factor activates neutrophils via dual pathways, the first involving direct interaction with phorbol ester inhibitable calcium channels and the other the stimulation in a manner dependent on a guanine nucleotide binding protein of the phospholipase C specific for polyphosphoinositides.     

Protective effects of CG-4203, a novel stable prostacyclin analog, in traumatic shock

We studied the effects of CG-4203, a novel stable prostacyclin analog, in a severe model of traumatic shock in rats. Traumatic shock was produced by Noble Collip drum trauma and was characterized by marked hypotension, a 4- to 5-fold increase in plasma cathepsin D and myocardial depressant factor activities, and survival time of 95 +/- 15 minutes. Treatment with CG-4203 (100 ng/kg/min) significantly prolonged survival time to 194 +/- 20 min (p less than 0.002). Traumatized rats treated with CG-4203 exhibited significantly lower plasma activities of the lysosomal hydrolase cathepsin D (p less than 0.05). Furthermore, the plasma accumulation of myocardial depressant factor (MDF) activity was also significantly blunted in traumatized CG-4203 treated rats when compared with traumatized rats receiving only the vehicle (p less than 0.01). Our results suggest that a combination of membrane stabilizing and anti-proteolytic effects and inhibition of platelet aggregation may mediate the protective effects of CG-4203 in traumatic shock.     

Lysosomal enzyme secretion by macrophages during intracellular storage of particles.

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase beta-N-acetylglucosaminidase. Thus macrophage lysosomal enzyem secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-beta-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.     

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.     

Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line.

Lysosomal enzyme secretion by the murine macrophage-like cell line, P388D1, was compared with that of normal peritoneal macrophages. Unlike macrophages, lysosomal hydrolase secretion by P388D1 cells occurred spontaneously in vitro and was not further stimulated by the presentation of inflammatory agents such as zymosan and asbestos.     

Hormonal regulation of acid cholesterol ester hydrolase activity: effects of triiodothyronine and 17 alpha-ethynylestradiol

Cholesterol ester hydrolase activity was measured in isolated rat hepatocytes and adipocytes. Administration of triiodothyronine to rats resulted in a specific and selective increase in lysosomal acid (pH 4.5) cholesterol ester hydrolase activity in hepatocytes. Since the majority of lipoprotein degradation occurs in liver parenchymal cells (hepatocytes), the stimulation of liver (hepatocyte) acid cholesterol ester hydrolase activity by triiodothyronine could contribute to the hypocholesterolemic action of thyroid hormones. Treatment of rats with 17 alpha-ethynylestradiol to increase the hepatic degradation of lipoprotein did not change acid cholesterol ester hydrolase activity in liver, indicating that the thyroid hormone induced stimulation of acid cholesterol ester hydrolase activity in hepatocytes is not a secondary effect owing to the increased hepatic catabolism of low density lipoproteins (LDL). In contrast to the results with hepatocytes, hyperthyroidism did not increase acid cholesterol ester hydrolase activity in rat adipocytes.     

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.     

The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles

Interleukin 1beta (IL-1beta), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a "leaderless" secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1beta out of the cell. Indeed, although most of the IL-1beta precursor (proIL-1beta) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1beta and the endolysosomal hydrolase cathepsin D or for both IL-1beta and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1beta is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1beta secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1beta from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1beta but deplete the vesicular proIL-1beta content, indicating that exocytosis of proIL-1beta-containing vesicles is regulated by ATP and osmotic conditions.     

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Neomycin (0.1-1 mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID50 0.2 mM) and formation of [3H]inositol mono-, bis- and trisphosphate (ID50 0.6-0.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0.1-1 mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (greater than 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.     

Alteration of platelet activating factor (PAF) metabolism in rat pulmonary alveolar macrophages and plasma by cigarette smoking.

The pathophysiological role of platelet activating factor (PAF) in smoking-induced disorders was examined in rats exposed daily to smoke for 10, 18 and 26 weeks. The concentration of PAF in bronchoalveolar lavage fluid and the activities of PAF biosynthetic and catabolic enzymes in alveolar macrophages and in plasma were determined. The concentration of PAF in lavage fluid of the smoke-exposed group was significantly lower than that in the sham group for each duration of smoke exposure. The PAF biosynthetic enzyme, acetyl transferase, activity in alveolar macrophages of smoked group was less than that in the sham group although the difference was not statistically significant. PAF catabolic enzyme, acetyl hydrolase, activities in alveolar macrophages and in plasma were all significantly higher in every smoked group than in the sham group. These data indicate that cigarette smoking alters PAF metabolism in the respiratory tract and in plasma and such an alteration may contribute, at least in part, to smoking induced cardiopulmonary disorders.     

Effect of hyperinsulinemia on acid cholesterol ester hydrolase activity in liver, heart and epididymal fat pad preparations from rats and mice

The effect of insulin on lysosomal acid cholesterol ester hydrolase activity was studied in liver, heart and fat pad preparations from rats and mice. Hyperinsulinemia was induced for a period of 6 days in rats by the subcutaneous administration of exogenous insulin by an osmotic minipump. The effect of more chronic endogenous hyperinsulinemia was studied using genetic strains of diabetic (db/db) mice at 12 weeks of age. Mouse liver and heart preparations were characterized as having an acid pH optimum of 4.5-5 for cholesterol ester hydrolase activity; a smaller but distinct pH optimum could also be observed at pH 7. In contrast, hydrolase activity in mouse fat pad preparations had only one distinct pH optimum of 6.5. Hyperinsulinemia in rats and mice resulted in a significant decrease in acid cholesterol ester hydrolase activity in heart preparations, but had no consistent effect on acid hydrolase activity observed in liver and fat pad preparations. This decrease in lysosomal acid cholesterol ester hydrolase activity in cardiac tissue due to hyperinsulinemia cannot be related to any changes in lipoprotein turnover caused by insulin or diabetes.     

Effect of thyroid hormones on acid cholesterol ester hydrolase activity in rat liver,heart and epididymal fat pads

The regulation of acid cholesterol ester hydrolase activity by thyroid hormones was studied in subcellular fractions from rat liver, heart, and epididymal fat pads; hydrolase activity was determined at pH 5 with a glycerol-dispersed cholesterol oleate substrate preparation. Acid cholesterol ester hydrolase activity was decreased in liver preparations from thyroidectomized rats relative to activity in livers from euthyroid control rats. Administration of triidothyronine to either euthyroid or hypothyroid (thyroidectomized) rats resulted in an increase in acid cholesterol ester hydrolase activity in liver preparations. Similar effects of thyroidectomy and the administration of triiodothyronine on acid cholesterol ester hydrolase activity were observed with fat pad preparations. In contrast, no effect of thyroid hormones was observed on acid cholesterol ester hydrolase activity in heart. These results suggest that thyroid hormones may regulate the catabolism of serum lipoproteins, in part, by alterations in lysosomal acid cholesterol ester hydrolase activity in liver and epididymal fat pads.     

Studies on Cholesterol Ester Formation and Hydrolysis in Liver Disease: A Selective Review

Plasma cholesterol esters are formed within the circulation by lecithin-cholesterol acyltransferase (LCAT), an enzyme produced by the liver. Patients with hepatocellular disease have low plasma LCAT activity. This largely accounts for the decreased levels of cholesterol esters observed in such patients and appears due to impaired hepatic production of the enzyme. In contrast, activity of the LCAT reaction in patients with cholestasis seems variable and is the subject of controversy, largely because the influence of abnormal cholestatic lipoproteins on the reaction requires further clarification.Human liver contains a lysosomal cholesterol ester hydrolase (CEH) which may play an important role in hepatic cholesterol homeostasis. In patients with liver damage there is no concrete evidence of circulating CEH activity, but recent studies show elevated activity of hydrolase within the liver itself in acute hepatitis. Hepatic activity of another lysosomal enzyme, acid phosphatase, is not increased, suggesting that high CEH in hepatitic liver does not simply reflect a general increase in lysosomal enzymes. The pathogenesis and significance of altered CEH activity in liver disease require further study.     

Effect of arginine and its analogs on the activity of lysosomal and nonlysosomal peptide hydrolases in rat tissues

The autolysis intensity and proteolysis activity at pH 4,5, 7,4, 8,5 and lysosomal and nonlysosomal peptide hydrolase activity have been studied in brain and liver tissues of rats. L-arginine has been found to increase the peptide hydrolase activity in neutral and alkaline media in case of autolysis and proteolysis estimation according to the amino nitrogen increase. When the peptide hydrolase activity is estimated according to the increase of folin-positive components its decrease under the action of arginine in neutral and alkaline media has been revealed. Arginine doesn't change the lysosomal peptide hydrolase activity. In both tissues under the influence of arginine the nonlysosomal peptide hydrolase activity defined by amino nitrogen increases, estimated by the folin-positive components--decreases. Arginine shows the specific influence on the nonlysosomal peptide hydrolase activity. The L-arginine analogues (D-arginine, guanidine) and products of the arginase reaction (ornithine and urea) don't exert such an effect on the nonlysosomal proteolysis.     

Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, β-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that α-mannosidase, β-fucosidase, and β-galactosidase are secreted into the salt solution and that secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for α-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.