Requirement for metalloendoprotease in exocytosis: evidence in mast cells and adrenal chromaffin cells

Exocytosis is initiated by the receptor-mediated influx of calcium that results in fusion of the secretory vesicle with the plasma membrane. We examined the possibility that calcium-dependent exocytosis in mast cells and adrenal chromaffin cells requires metalloendoprotease activity. Metalloendoprotease inhibitors and dipeptide substrates block exocytosis in these cells with the same specificity and dose dependency as that with which they interact with metalloendoproteases. Metalloendoprotease activity is identified in these cells with fluorogenic synthetic substrates, which also blocked exocytosis. Metalloendoprotease activity is highest in the plasma membrane of chromaffin cells. The metalloendoprotease appears to be required in exocytosis at a step dependent on or after calcium entry, since exocytosis initiated by direct calcium introduction in both mast cells and chromaffin cells is blocked by metalloendoprotease inhibitors.     

Metalloendoprotease inhibitors block protein synthesis, intracellular transport, and endocytosis in hepatoma cells

Fusion of membrane vesicles has been implicated in many intracellular processes including the transport of proteins destined for secretion or storage. Vesicular transport coupled with membrane fusion has been demonstrated for rough endoplasmic reticulum to Golgi and Golgi to plasma membrane transport as well as receptor mediated endocytosis and receptor recycling. Recent studies with inhibitors suggest that metalloendoproteases may mediate a wide variety of intracellular fusion events. Thus, in order to examine the potential role of metalloendoproteases in both transport/secretion and endocytosis/recycling we have used selected dipeptide substrates to probe these processes in human HepG2 cells. Using pulse-chase labeling, immunoprecipitation, and polyacrylamide gel electrophoresis we show that transport and secretion of newly synthesized proteins along the exocytotic route were completely inhibited by substrate dipeptides (e.g. Cbz-Gly-Phe-amide, where Cbz is benzyloxycarbonyl) but not by irrelevant dipeptides (e.g. Cbz-Gly-Gly-amide). The effect was rapid, reversible, and specific. The secretory pathway was blocked between the rough endoplasmic reticulum and Golgi as well as Golgi and plasma membrane as judged by the status of N-glycosylation intermediates. In addition, these inhibitors specifically inhibited protein synthesis without alterations in cellular ATP concentrations. However, cell-free amino acid incorporation was not inhibited. Receptor-mediated uptake of asialoglycoproteins was specifically and reversibly inhibited by dipeptide substrates. This effect appears to be secondary to inhibition of recycling as neither ligand binding nor internalization were affected. Thus the present observations suggest that metalloendoprotease activity may be involved in the regulation of multiple intracellular pathways perhaps at the level of vesicular fusion events.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Current status of the thiol redox model for the regulation of hexose transport by insulin.

Data obtained over the last two years pertinent to the thiol redox model for the modulation of hexose transport activity by insulin is summarized. The model proposes that activation of hexose transport in fat cells involves sulfhydryl oxidation to the disulfide form in a key protein component of the fat cell surface membrane. Theoretically, the rapid activation of transport by insulin may involve either the conversion of inactive membrane carriers to the active form as originally proposed, or the conversion of a low Vmax transport system to a high Vmax form. The present experiments showed that the percent inhibition of insulin-activated transport rates by submaximal levels of cytochalasin B was decreased compared to its effects on basal transport. Treatment of fat cells with N-ethylmaleimide inhibited cytochalasin B action but not transport activity. When insulin or the oxidant vitamin K5 was added to cells 5 minutes before the N-ethylmaleimide, the elevated transport activity was also resistant to the sulfhydryl reagent, but cytochalasin B retained its potent inhibitory effect on transport. The data demonstrate that unique properties characterize basal versus insulin-activated transport activity with respect to the sensitivity of cytochalasin B action to sulfhydryl blockade in isolated fat cells. The data are consistent with the concept that activation of transport activity reflects the conversion of a reduced (sulfhydryl) system characterized by a low Vmax to an oxidized (disulfide), high Vmax transport system.     

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

The role of small G-proteins in the regulation of glucose transport

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

Oligopeptide inhibitors of metalloendoprotease activity inhibit catecholamine secretion from bovine adrenal chromaffin cells by modulating intracellular calcium homeostasis

Synthetic oligopeptide inhibitors of metalloendoprotease activity were found to inhibit catecholamine release from intact bovine adrenal chromaffin cells. The efficiency of these compounds in blocking secretion was dependent on the type and dose of the secretagogues employed. By contrast, catecholamine release from digitonin-permeabilized cells stimulated with micromolar calcium was virtually not affected. Using a different model system mimicking protein-mediated membrane fusion during exocytosis (Bental, M., Lelkes, P.I., Scholma, J., Hoekstra, D., and Wilschut, J. (1984) Biochim. Biophys. Acta 774, 296-300) we found that exposure of chromaffin granules to a genuine metalloendoprotease, thermolysin, impaired their fusion competence with liposomes. The same oligopeptide inhibitors of metalloendoprotease activity that interfered with secretion from the intact cells were also found to cause an increase in 45Ca2+ efflux concomitant with a slight elevation of the free intracellular calcium concentration [( Ca2+]i) to levels not sufficient to elicit secretion. Subsequent stimulation of the cells in the presence of the potent inhibitors resulted in a reduced increase in the cytosolic calcium concentration, as compared to nontreated control cells. The reduction in the secretagogue-evoked rise in [Ca2+]i was also dependent on the time of pretreatment of the cells with the metalloendoprotease inhibitors. Consistently, none of these effects were seen with structurally similar oligopeptides that are not metalloendoprotease substrates/inhibitors. We conclude that potent inhibitors of metalloendoprotease activity and hence, presumably, the enzymes per se modulate stimulus-secretion coupling by interfering with calcium homeostasis rather than directly with membrane fusion.     

Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.     

Retention of insulin-stimulated D-glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins

Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D-glucose transport activity characteristic of insulin action on intact fat cells. Both control and insulin-stimulated D-glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000-dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin-activated D-glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vescles and D-glucose uptake into extracted vescles derived from untreated or insulin-treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.     

Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter

Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+-dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p-chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N-ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter.     

Dipeptide transport in isolated intestinal brush border membrane.

Transport of glycyl-L-leucine into isolated brush border membrane vesicles was studied. On the basis of the following observations it was postulated that glycyl-L-leucine was transported intact by a specific dipeptide mechanism. (1) The differing time course and Na-+ stimulation of glycine, L-leucine and glycyl-L-leucine. (2) The failure of glycine and L-leucine to inhibit glycyl-L-leucine transport. (3) Initial presence of dipeptide within the vesicle. (4) Inhibition of glycyl-L-leucine uptake by other dipeptides. (5) The occurrence of accelerated amino acid uptake in the presence of the dipeptide.     

Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli.

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca2+, Mg2+-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca2+, Mg2+-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome-containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under under anaerobic conditions is energized by ATP through the Ca2+, Mg2+-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.     

Rat myoblast fusion requires metalloendoprotease activity

The calcium-dependent fusion of cultured rat myoblasts to multinucleate myotubes appears to require the activity of a neutral metalloendoprotease at the time of fusion. Metalloendoprotease inhibitors and synthetic dipeptide substrates prevent myoblast fusion when added to fusion-competent myoblasts with the addition of calcium. Metalloendoprotease activity has been identified and partially characterized in myoblast membranes with a fluorogenic protease substrate, and is inhibited by the same compounds that prevent fusion.     

Transport properties of membrane vesicles fromAcholeplasma laidlawii

Membrane vesicles obtained from Acholeplasma laidlawii accumulate glucose as well as maltose and fructose against their concentration gradient in the absence of exogenous energy sources. Glucose uptake by membrane vesicles is inhibited by anaerobiosis and by electron transfer inhibitors, such as rotenone and amytal, but not by 2-heptyl-4-hydroxyquinoline N-oxide, antimycin A, cyanide and azide. Rotenone, cyanide and amytal also produce a rapid efflux of glucose from the membrane vesicles. Arsenate, oligomycin and N,N'-dicyclohexylcarbodimide do not inhibit glucose transport. Transport of glucose is markedly inhibited by proton conductors such as CCCP and pentachlorophenol. It is concluded that glucose transport can be driven by a high-energy state of the membrane or by the membrane potential.     

Rab蛋白在葡萄糖转运中的作用

在脂肪和骨骼肌细胞中,胰岛素可迅速刺激葡萄糖转运,即通常所说的GLUT4转运。 GLUT4转运是指Rabs与GTP结合时,促进囊泡与微管和微丝蛋白结合,并通过锚定和融合作用使GLUT4囊泡与目标膜结构融合。多数 Rab 家族成员广泛表达于各种组织细胞中,且在细胞内定位十分广泛,几乎存在于真核细胞所有的膜相关的细胞器的胞浆侧。 Rab 蛋白作为囊泡运输的分子开关,通过调节运输小泡的停泊和融合,在囊泡的形成、转运、粘附、锚定、融合等过程中起着重要的作用。 Rab蛋白受到多种上游调节蛋白的调节,同时调控着下游的多种效应蛋白,构成了复杂的调控网络：任何一个环节改变都可能会导致蛋白质转运的异常,进而引发疾病。本文系统阐述了Rab蛋白在葡萄糖转运过程中的作用及该领域的最新进展。     

Evidence that metalloendoproteases are involved in gamete fusion of Ciona intestinalis, ascidia.

The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.     

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by approximately 33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCzeta/lambda, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.     

Novel inhibitors of basal glucose transport as potential anticancer agents

Cancer cells commonly show increased levels of glucose uptake and dependence. A potential strategy for the treatment of cancer may be the inhibition of basal glucose transport. We report here the synthesis of a small library of polyphenolic esters that inhibit basal glucose transport in H1299 lung and other cancer cells. These basal glucose transport inhibitors also inhibit cancer cell growth in H1299 cells, and these two activities appear to be correlated.     

Cadmium and manganese transport in Staphylococcus aureus membrane vesicles.

The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.     

Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells

The rate-limiting step in the uptake and metabolism of Dglucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin-coated pits and vesicles, early endosomes, and tubulo-vesicular structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3-kinase, is discussed.