Plasma Membrane Of Chromaffin Cells Research Articles

Annexin A2, an essential partner of the exocytotic process in chromaffin cells

Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, such as neuroendocrine cells. In chromaffin cells, cytosolic annexin A2 is recruited to the plasma membrane upon cell stimulation. Here, we review the latest evidence detailing the functional importance of annexin A2 in different stages of exocytosis. These include the recruitment of annexin A2 to the plasma membrane near soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, the role of annexin A2 in the formation of lipid domains at exocytotic sites, and finally the annexin A2 bundling of actin microfilaments associated with chromaffin granules. These structures induce first the coalescence of lipid domains required for the formation of the exocytotic site, and in the second time, exert mechanical force on the granule to favor fusion pore expansion and squeeze the granule to facilitate catecholamine release. Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, including neuroendocrine cells. Upon cell stimulation, annexin A2 translocates from the cytosol to the plasma membrane of chromaffin cells and bundles actin filaments associated with chromaffin granules. This promotes the formation of lipid domains required for granule docking, and facilitates catecholamine release by compressing the granule. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Monoamines suppressed phosphorylation of SNAP-25 in mouse brain

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localizationin vivoare lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intracellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42

Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin-1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin-1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin-1L is an essential component of the exocytotic machinery. Silencing of intersectin-1 prevents secretagogue-induced activation of Cdc42 revealing intersectin-1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin-1L in endocytosis to a function in exocytosis and support the idea that intersectin-1L is an adaptor that coordinates exo-endocytotic membrane trafficking in secretory cells.

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E.

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.

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.

Regulated secretion in chromaffin cells: an essential role for ARF6-regulated phospholipase D in the late stages of exocytosis.

ARFs constitute a family of structurally related proteins that forms a subset of the ras GTPases. In chromaffin cells, secretagogue-evoked stimulation triggers the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. Both PLD activation and catecholamine secretion are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6. ARNO, a potential guanine nucleotide exchange factor for ARF6, is expressed and localized in the plasma membrane of chromaffin cells. Using permeabilized cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits both PLD activation and catecholamine secretion. Chromaffin cells express PLD1 at the plasma membrane. We found that microinjection of the catalytically inactive PLD1(K898R) dramatically reduces catecholamine secretion monitored by amperometry, most likely by interfering with a late postdocking step of calcium-regulated exocytosis. We propose that ARNO-ARF6 participate in the exocytotic reaction by controlling the plasma membrane-bound PLD1. By generating fusogenic lipids at the exocytotic sites, PLD1 may represent an essential component of the fusion machinery in neuroendocrine cells.

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells.

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.

Restriction of secretory granule motion near the plasma membrane of chromaffin cells.

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

A Pleckstrin Homology Domain Specific for Phosphatidylinositol 4,5-Bisphosphate (PtdIns-4,5-P2) and Fused to Green Fluorescent Protein Identifies Plasma Membrane PtdIns-4,5-P2 as Being Important in Exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.

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.

Tracking Single Secretory Granules in Live Chromaffin Cells by Evanescent-Field Fluorescence Microscopy

We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epiillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 600 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of ∼30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves ∼70 nm space around a granule. The “cage” itself moves only slowly ( D = 2 × 10 −12 cm 2/s). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

Polarized Expression of the Antidepressant-Sensitive Serotonin Transporter in Epinephrine-Synthesizing Chromaffin Cells of the Rat Adrenal Gland

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localizationin vivoare lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intracellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.

A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca 2+-dependent exocytosis in chromaffin cells

SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca 2+-evoked catecholamine release from digitonin-permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca 2+-dependent catecholamine release with an IC 50 = 20 μM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca 2+-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.

Adenosine 5′-triphosphate modulation of nitrobenzylthioinosine binding sites in plasma membranes of bovine chromaffin cells

Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of [ 3H]NBTI to plasma membranes of chromaffin cells was conducted in the presence or absence of adenosine 5′-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing B max values of 1.62 ± 0.20 pmol/mg protein for controls and 3.22 ± 0.31 pmol/mg protein in the presence of ATP. However, the affinity constant ( K D) was not significantly modified. The non-metabolizable ATP analogue, 5′-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap 4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role for ATP, non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggested.

Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.

1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of annexins in the human neuroblastoma SH-SY5Y

The annexins are a family of structurally related calcium dependent membrane-binding proteins [ I ] which can be shown to bind to phospholipid membranes in the presence of calcium [2]. The annexins have been identified in a wide range of tissues from many species yet their physiological roles have not been clearly elucidated. They have been implicated in a variety of important cellular processes [3]. In particular annexin I 1 seems to be a regulatory factor in the control of exocytosis [4,5]. Annexin 11 is one of the best characterised components of the annexin family, occurring as a 36kD monomer and as a 90kD heterotetramer complex, containing two molecules of the 36kD heavy chain and two molecules of an 1 IkD light chain [6]. Evidence for the involvement of annexin 11 in the regulation of exocytosis is provided by the observations that the 36kD molecule can restore secretion in digitonin permeablised chromaffin cells [4] and that the annexin heterotetramer can restore secretion in streptolysin-0 permeabilised chromaffin cells, but must be phosphorylated by protein kinase C (PKC) to be effective [ 5 ] . The annexin 11 heavy chain is a major substrate for PKC and is phosphorylated at Ser-25 in the NH2 terminal region [7]. Annexin 11 has also been shown to promote the aggregation of chromaffin cell secretory granule membranes in a calcium dependent manner [8], and electron microscopic studies have shown that annexin 11 is concentrated between chromaffin granules and the plasma membrane in chromaffin cells [9]. Previous studies by our group have shown that the human neuroblastoma SH-SY5Y expresses depolarisation and muscarinic M, receptor subtype evoked release of noradrenaline [lo]. Acute pretreatment with the PKC activating phorbol ester 12-0tetradecanoylphorbol13-acetate (TPA) enhanced noradrenaline release [ I I], whereas addition of the selective PKC inhibitors Ro 31 -7549 and Ro 31 -8220 inhibited noradrenaline release, indicating that PKC plays an important role in the release of noradrenaline from SH-SYSY. The first step in the investigation of the role of annexins in release of noradrenaline from SH-SY5Y was to determine which annexins were present in the cell. Annexins were isolated from SH-SYSY utilising the reversible calcium dependent binding properties of annexins to membrane phospholipids [ 121. These isolated annexins were separated on a two dimensional SDS PAGE gel and analysis of protein molecular weight and isoelectric points enabled the tentative identification of annexins l , l l , lV,V and VI by comparison with bovine lung annexins [12]. These results were confirmed by Western blot analysis of SH-SY5Y total homogenate. SH-SY5Y cells were harvested in Laemmli buffer and proteins separated by 12.5% SDS PAGE [I31 before transfer to polyvinylidene difluoride membranes. After blocking with 5% non fat milk solution, membranes were incubated with monoclonal antibodies to annexins I,II.IV and VI or with an affinity purified polyclonal antibody to annexin V. Immunolabelled proteins were identified by a peroxidase conjugated secondary antibody amplified by the addition of extra peroxidase using a Biotinyl Tyramide Streptavidin-Peroxidase system (BLAST from NEN Du Pont). with 4 chloro I napthol as a peroxidase substrate [14]. Immunoblotting showed the presence of annexins I,II,IV,V and VI in SH-SYSY (Fig I . ) . The annexins I,II,IV V and VI, detected in SH-SYSY have also been detected in the chromaffin cell [7], however only annexin II has been implicated in exocytotic regulation. It is of interest that the neuroblastoma cell line expresses a much broader range of annexins than neurones in mammalian brain, where only annexin VI is detected [15]. Perhaps the increased expression reflects the transformed phenotype and its capacity for continual growth. c 9 7

The adenosine receptors present on the plasma membrane of chromaffin cells are of the A2b subtype.

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.

Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.

Alamethicin channel permeation by Ca 2+, Mn 2+ and Ni 2+ in bovine chromaffin cells

Alamethicin causes a concentration-dependent increase of (Ca 2+), in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Ca 2+ (234 ± 37 nM; n=4) increased to a maximum of 2347±791 nM ( n=3) with 100 μg/ml alamethicin. In the presence of 1 nM Ca 2+ the increase reached a plateau within about 2–5 s. This increase was due to Ca 2+ entry into chromaffin cells, since in the absence of Ca 2+ alamethicin did not modify [Ca 2+]. This contrasts with ionomycin (1 μM) which produced a Ca 2+ transient even in the absence of Ca 2+, Mn 2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2-fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn 2+ which was drastically increased by cell depolarization by K + (50 nM) addition. This suggests that Mn 2+ is able to permeate voltage-dependent Ca 2+ channels. Ni 2+ uptake into either resting of K +-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni 2+ was decreased by 50 mM K +. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.