Konosuke Kumakura

Calmodulin and lipid binding to synaptobrevin regulates calcium‐dependent exocytosis

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP‐25. Calcium influx is thought to control this process via Ca2+‐binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C‐terminal domain of VAMP (VAMP77–90), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild‐type VAMP77–90, but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre‐incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca2+‐dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT‐resistant (Q76V, F77W) VAMP, but additional targeted mutations in VAMP77–90 abolished its ability to rescue release. The calmodulin‐ and phospholipid‐binding domain of VAMP 2 is thus required for Ca2+‐dependent exocytosis, possibly to regulate SNARE complex assembly.

Site of docking and fusion of insulin secretory granules in live MIN6 β cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy

To determine the site of insulin exocytosis in the pancreatic β cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 β cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-β-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 β cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

Quantitative analysis of exocytosis directly visualized in living chromaffin cells

Chromaffin cells isolated from the bovine adrenal medulla were observed under a Nomarski microscope through a CCD camera and an image processor. Exocytotic events of individual granules including fusion, extrusion, swelling, omega-figure formation, and membrane retrieval were visualized in individual cells stimulated by acetylcholine or K-rich solution. Initial steps were quicker than 16 ms, and the membrane retrieval was slower than 1-60 s. These findings provided a light microscopic proof for the exocytosis hypothesis as well as a basis for quantification of hormonal release. The technique was used to demonstrate significant secretion induced by a muscarinic agonist.

Regulatory Role of the GTP‐Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

Abstract: To elucidate the possible involvement of GTP‐binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guano‐sine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), and antibodies against the G proteins (Gi and Go) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine‐evoked catecholamine release. High K+‐, histamine‐, or γ‐aminobutyric acid‐evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10−4M acetylcholine was decreased significantly in PTX‐treated cells. In digitonin‐permeabilized cells, PTX pretreatment resulted in a decrease of the half‐maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP‐γ‐S (a nonhydrolyzable GTP analogue) inhibited Ca2+‐dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the α subunit of the PTX‐sensitive G protein, Go. Treatment with similar antibodies against the α subunit of Gi had no effect. These findings suggest that Go directly controls the Ca2+‐triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.

Secretory function of adrenal chromaffin cells cultured on polypyrrole films

Polypyrrole (PPy) is a conducting polymer and is obtained electrochemically on an electrode such as indium-tin oxide (ITO). In this study, in order to develop a novel cell-culture system which makes it possible to communicate with cultured mammalian cells, bovine adrenal chromaffin cells were cultured on PPy-coated ITO plates for 7 days and the influence of PPy-coating on the cell functions was investigated. Since the chromaffin cells synthesize and secrete catecholamines such as adrenaline and noradrenaline, the amount of synthesized and released catecholamines from the chromaffin cells cultured on PPy-coating and ITO itself were measured. The cells on the PPy-coated ITO plate could be kept in culture, without any significant changes in morphology and in the secretory responsiveness to acetylcholine as compared with those of the cells cultured on collagen. On the contrary, the cells on the ITO plate lost the responsiveness, while the amount of catecholamines synthesized was affected little by both PPy and ITO surfaces. It is suggested that PPy supports the secretory function of the chromaffin cells when they are cultured on it. This paper describes that PPy films are applicable as a polymer-modified electrode which support the cell function without collagen.

Endothelin-3 stimulates the release of catecholamine from cortical and striatal slices of the rat

Endothelin-3 (ET-3) evoked the release of dopamine/noradrenaline from cortical slices and dopamine from striatal slices in a concentration-dependent manner. This action peaked slowly and was long-lasting in real-time monitoring, being different from the high K(+)-evoked response. The striatal response to 10 microM of ET-3 was reduced by extracellular Ca2+ depletion to 40% of control and by Ca2+ antagonists, especially nifedipine and flunarizine, to 40% of control. Our findings suggest that ET has a physiological significance in the brain as a neuromodulator for catecholaminergic transmission.

Stimulatory Action of γ‐Aminobutyric Acid on Catecholamine Secretion from Bovine Adrenal Chromaffin Cells Measured by a Real‐Time Monitoring System

Abstract: The present study was designed to evaluate the role of γ‐aminobutyric acid (GABA) in the secretory function of cultured chromaffin cells using the method of realtime monitoring. GABA evoked the secretion of catecholamines (CA) from adrenal chromaffin cells in a dose‐dependent manner. Bicuculline 10‐−5M inhibited the stimulatory action of GABA. Diazepam 5 × 10‐−6 and 2.5 × 10‐−5M facilitated the secretory response evoked by 7 × 10‐−5M GABA by 22% and 96%, respectively, which was antagonized by Ro 15–1788. This finding suggests that GABA‐benzodiazepine receptor coupling can function in the secretion of CA from the adrenal chromaffin cells in a manner similar to that observed in the brain. GABA‐evoked release of CA was reduced by 1 μM nifedipine to 16% of control, suggesting the involvement of voltage‐sensitive Ca2+ channels in the mechanisms of the CA‐releasing action of GABA in these cells. From these findings, the involvement of GABAergic mechanisms in the regulation of adrenal medullary function can be proposed.

Exocytosis: The Chromaffin Cell As a Model System

Abstract: Neurons and neuroendocrine cells release transmitters and hormones by exocytosis of secrctory vesicles or granules. Among the cell models that have provided insight into the molecular machinery underlying the successive steps of exocytosis, adrenal chromaffin cells have taken a prominent place. Thus, most of the molecular players that orchestrate the formation, targeting, docking, and fusion of secrctory granules have been identified in chromaffin cells. By offering the opportunity to combine the use of recent biophysical techniques allowing single‐vesicle resolution and specific biochemical modifications in the protein machinery involved in exocytosis, chromaffn cells remain a powerful model to address new and still open questions in the field of secretion.

Inhibition of Ca2+-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells

Abstract To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K + -evoked catecholamine (CA) release. The IC 50 for high K + -evoked CA release was 1μM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca 2+ -dependent exocytosis was inhibited in a dose-dependent manner (IC 50 , 1μM). These findings suggest the implication of MLCK in the Ca 2+ -triggered process in the machinery of exocytosis.

Real-Time Monitoring of the Secretory Function of Cultured Adrenal Chromaffin Cells

A system to discriminate the real‐time dynamics of the secretory function in cultured adrenal chromaffin cells, using a cell bed perfusion technique and an amperometric detector, was established. Examination of basal conditions revealed that the electrode potential and flow rate are crucial factors for monitoring precise dynamics of the secretory process. Stimulation of the cells either with acetylcholine (ACh) or with high K* concentration caused a transient current response. The current responses showed concentration dependence for both stimuli, and also showed a high correlation with the amount of catecholamines (CA) in the respective peak fraction of perfusate. Either prolonged cholinergic stimulation or maintained depolarization produced a transient response, which is not attributable to a depletion of releasable storage of CA as indicated by double‐stimulation experiments. Stimulation with high K* concentration evoked an additional release of CA even after the cellular response to prolonged ACh was inactivated, whereas maintained depolarization with high K* produced both facilitatory and inhibitory effects on the cell responsiveness to ACh. Most probably the transient natures of the secretory responses to ACh and to high K* are mediated by different mechanisms. All the results suggest that the direct monitoring is profitable for studies on the regulatory mechanisms of the secretory function.