Secretion In Permeabilized Cells Research Articles

Role of ADP-ribosylation Factor 6 (ARF6) in Gastric Acid Secretion

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.

Synaptotagmin Regulation of Secretory Mode

Vesicular secretion of neurotransmitter can take place through two distinct mechanisms: full fusion, in which a fusion pore opens and dilates and the vesicle completely releases its contents; and kiss-and-run, in which the fusion pore opens but does not dilate. Wang et al. performed amperometric measurements of catecholamine release in cultured pheochromocytoma 12 (PC12) cells to distinguish between these two forms of secretion and to investigate the role of synaptotagmins (Syts) in regulating the choice of secretory mode. Syts are a family of synaptic vesicle proteins that contain two calcium-binding C 2 domains (C 2 A and C 2 B). Full fusion events led to brief "spikes" of high concentrations of released catecholamine, whereas kiss-and-run events led to more prolonged release of low concentrations of transmitter. Overexpression or pharmacological manipulations that increased Syt IV--the expression of which is increased by seizures--led to increased frequency and duration of kiss-and-run episodes. Mutational analysis of transfected Syt I and Syt IV, in combination with analysis of the calcium dependence of secretion in permeabilized cells, indicated that Syt I promoted full fusion (through its C 2 A domain) whereas Syt IV promoted kiss-and-run secretion (through its C 2 B domain). The authors speculate that prolonged release of low levels of neurotransmitter in response to increased synaptic Syt IV concentrations could desensitize postsynaptic receptors, thereby silencing the synapse and serving as a homeostatic mechanism to counteract seizure activity. C.-T. Wang, J.-C. Lu, J. Bai, P. Y. Chang, T. F. J. Martin, E. R. Chapman, M. B. Jackson, Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424 , 943-947 (2003). [Online Journal]

Localization of phosphatidylinositol 4,5-P(2) important in exocytosis and a quantitative analysis of chromaffin granule motion adjacent to the plasma membrane.

A slow ATP-dependent priming step precedes a rapid, Ca(2+)-dependent triggering step in exocytosis in chromaffin cells and in most, if not all, differentiated secretory cells. A major component of ATP-dependent secretion in permeabilized cells reflects the maintenance of the polyphosphoinositides, especially PtdIns-4,5-P2. Here we summarize recent experiments with PH-GFP (binds to PtdIns-4,5-P2) that indicate that PtdIns-4,5-P2 is localized primarily on the plasma membrane in chromaffin cells, and that it is this pool that plays a role in exocytosis. It is demonstrated that transiently expressed PH-GFP inhibits secretion in subsequently permeabilized cells. Recent studies using total internal reflection fluorescent microscopy (TIRFM) to measure chromaffin granule motion adjacent to the plasma membrane are also summarized. The quantitative analysis indicates that chromaffin granule motion is highly restricted and suggests that chromaffin granules are caged or tethered immediately adjacent to the plasma membrane.

Synaptotagmin III/VII Isoforms Mediate Ca2+-induced Insulin Secretion in Pancreatic Islet β-Cells

Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.

Latrotoxin Stimulates Secretion in Permeabilized Cells by Regulating an Intracellular Ca2+- and ATP-dependent Event: A ROLE FOR PROTEIN KINASE C

alpha-Latrotoxin, a component of black widow spider venom, stimulates transmitter release from nerve terminals and intact chromaffin cells and enhances secretion from permeabilized chromaffin cells already maximally stimulated by Ca(2+). In this study we demonstrate that chromaffin cells contain a protein antigenically similar to the cloned Ca(2+)-independent receptor for alpha-latrotoxin. Although this receptor has homology to the secretin family of G-protein-linked receptors, pertussis toxin has no effect on the ability of alpha-latrotoxin to enhance secretion, suggesting that neither G(i) nor G(o) is involved in the response. Furthermore, in the absence of Ca(2+), alpha-latrotoxin does not stimulate polyphosphoinositide-specific phospholipase C. alpha-Latrotoxin specifically enhances ATP-dependent secretion in permeabilized cells. An in situ assay for protein kinase C reveals that alpha-latrotoxin augments the activation of protein kinase C by Ca(2+), and use of protein kinase inhibitors demonstrates that this activation is important for the toxin's enhancing effect. This enhancement of secretion requires Ca(2+) concentrations above 3 microm and is not supported by Ba(2+) or nonhydrolyzable guanine nucleotides, which do not stimulate protein kinase C. We conclude that alpha-latrotoxin stimulates secretion in permeabilized cells by regulating a Ca(2+)- and ATP-dependent event involving protein kinase C.

α-Latrotoxin and its receptors CIRL (latrophilin) and neurexin 1α mediate effects on secretion through multiple mechanisms

α-Latrotoxin and its plasma membrane receptors cause a number of distinct effects in secretory cells. First, by tethering α-latrotoxin to the plasma membrane, CIRL/latrophilin and neurexin 1α facilitate α-latrotoxin-induced channel formation. The stimulation of secretion by α-latrotoxin in neuroendocrine cells is a consequence of Ca 2+ influx through these α-latrotoxin-induced channels. In addition to channel formation, α-latrotoxin enhances secretion in permeabilized cells through interaction with the plasma membrane receptor CIRL/latrophilin. Finally, overexpression of CIRL/latrophilin inhibits Ca 2+-dependent secretion in permeabilized chromaffin cells in the absence of α-latrotoxin. This effect represents a ‘constitutive’ action of the G-protein coupled receptor to specifically inhibit an ATP-dependent priming step in the secretory pathway. The effect suggests that the receptor may have an important modulatory role in synaptic transmission.

Identification of a Plasma Membrane-associated Guanine Nucleotide Exchange Factor for ARF6 in Chromaffin Cells: POSSIBLE ROLE IN THE REGULATED EXOCYTOTIC PATHWAY

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.

A Critical Role for N-ethylmaleimide–Sensitive Fusion Protein (NSF) in Platelet Granule Secretion

The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood.N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both -granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant -SNAP–stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant -SNAP–stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion.

A Critical Role for N-ethylmaleimide–Sensitive Fusion Protein (NSF) in Platelet Granule Secretion

AbstractThe molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood.N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both α-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant α-SNAP–stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant α-SNAP–stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion.

A Ca2+-Independent Receptor for α-Latrotoxin, CIRL, Mediates Effects on Secretion via Multiple Mechanisms

alpha-Latrotoxin (alpha-Ltx), a component of black widow spider venom, stimulates secretion from nerve terminals and from PC12 cells. In this study we examine the effects of expression of a newly cloned Ca2+-independent receptor for alpha-Ltx (CIRL) on secretion from bovine chromaffin cells. We first characterized the effect of alpha-Ltx on secretion from untransfected cells. alpha-Ltx, by binding in a Ca2+-independent manner to an endogenous receptor, causes subsequent Ca2+-dependent secretion from intact cells. The stimulation of secretion is correlated with Ca2+ influx caused by the toxin. In permeabilized cells in which the Ca2+ concentration is regulated by buffer, alpha-Ltx also enhances Ca2+-dependent secretion, indicating a direct role of the endogenous receptor in the secretory pathway. Expression of CIRL increased the sensitivity of intact and permeabilized cells to the effects of alpha-Ltx, demonstrating that this protein is functional in coupling to secretion. Importantly, in the absence of alpha-Ltx, the expression of CIRL specifically inhibited the ATP-dependent component of secretion in permeabilized cells without affecting the ATP-independent secretion. This suggests that this receptor modulates the normal function of the regulated secretory pathway and that alpha-Ltx may act by reversing the inhibitory effects of the receptor.

The Regulatory Site of Functional GTP Binding Protein Coupled to the High-Affinity Cholecystokinin Receptor and Phospholipase A 2 Pathway Is on the G β Subunit of G q Protein in Pancreatic Acini

A non-hydrolysable guanosine nucleotide analog, GTP[S] at 200 μM, stimicrolated amylase secretion which was inhibited by an anti-phospholipase A 2 (PLA 2) antibody in permeabilized pancreatic acini, indicating that the PLA 2 pathway is linked to the GTP binding protein. A high affinity cholecystokinin (CCK) receptor agonist, CCK-OPE (10 μM), and a low affinity receptor agonist,CCK-8 (0.1 μM), both caused amylase secretion in permeabilized cells. The action of CCK-OPE was abolished by the G β antibody but not by the G alpha-q,11 antibody, whereas the opposite was true of the CCK-8 response. Biscoclaurine alkaloid isotetrandrine (10 μM), a specific inhibitor of PLA 2-coupled G proteins, abolished Ca 2+ oscillations and amylase secretion induced by CCK-OPE (0.1-100 nM), but not by CCK-8 (10 pM) in intact acini. Gp antagonist-2A (10 μM), which inhibits the activation of Gq, also inhibited the actions of CCK-OPE (10 pM-1 μM) in intact acini. These observations indicate that the functional unit of the heterotrimeric G protein coupled to the high affinity CCK receptor appears to be different from that linked to the low affinity CCK receptor/G q-alpha pathway. The regulatory site of this G protein coupled to the high affinity CCK receptor is on the β subunit of G q protein which elicits Ca 2+ oscillations and monophasic amylase secretion via the PLA 2 pathway.

Anti-syntaxin Antibodies Inhibit Calcium-Dependent Catecholamine Secretion from Permeabilized Chromaffin Cells

Adrenomedullary chromaffin cells release catecholamines in response to the intracellular calcium rise upon stimulation by different secretagogues. The presence of syntaxin 1, a protein presumably involved in docking of synaptic vesicles to presynaptic membranes, has been investigated in chromaffin cells. The study using two different monoclonal antibodies shows that syntaxin 1 is present in the chromaffin cell membrane fraction. Functional experiments demonstrate that anti-syntaxin antibodies inhibit calcium-dependent secretion in permeabilized cells. These results suggest that syntaxin 1 is an important component of the secretory machinery in chromaffin cells.

Correlation between secretion and phospholipase D activation in differentiated HL60 cells

Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself.

Regulation of norepinephrine secretion in permeabilized PC12 cells by Ca2(+)-stimulated phosphorylation. Effects of protein phosphatases and phosphatase inhibitors.

Protein phosphatases and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized PC12 cells. The addition of an exogenous type 2A protein phosphatase caused as much as a 70% decrease in Ca2(+)-dependent norepinephrine secretion. In the presence of okadaic acid, a potent inhibitor of type 2A protein phosphatases, phosphatase 2A had no effect on secretion. The addition of exogenous calcineurin, a Ca2(+)-calmodulin-stimulated phosphatase, also caused decrease in Ca2(+)-dependent secretion, but on a molar basis it was less effective than phosphatase 2A. Two phosphatase inhibitors, 1-naphthylphosphate and sodium pyrophosphate, caused 75-100% increases in the amount of norepinephrine secreted in the absence of Ca2+ without affecting the amount of norepinephrine secreted in the presence of Ca2+. This stimulation of Ca2(+)-independent secretion by 1-naphthylphosphate and pyrophosphate suggests that there is a slow rate of Ca2(+)-independent phosphorylation and that phosphorylation triggers secretion. Unlike the results obtained in the presence of ATP, secretion in the presence of adenosine-5'-O-(3-thiotriphosphate), ATP gamma S, was not affected by the addition of type 2A protein phosphatase or by the addition of phosphatase inhibitors. These results are consistent with secretion in these permeabilized cells being regulated by a Ca2(+)-stimulated phosphorylation.

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: Possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2- 3H] inositol were permeabilized by ATP 4−, exposed to guanine nucleotides and resealed by Mg 2+. Addition of guanosine 5′-0-(3-thio triphosphate) (GTPγS) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [ 3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5′-0-(2-thiodiphospate) (GDPβS) or by neomycin. Surprisingly, addition of GDPβS (100–600 uM) on its own resulted in a dose-related increase in [ 3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP>GTPγ S>ATP>CTP. The stimulatory effect of UTP, ATP and CTP, but not GTPγS or GDPβS, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTPγS decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.