Facilitation Of Secretion Research Articles

Formation of the Full SNARE Complex Eliminates Interactions of Its Individual Protein Components with the Kv2.1 Channel

Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.

Bidirectional Modulation of Exocytosis by Angiotensin II Involves Multiple G-Protein-Regulated Transduction Pathways in Chromaffin Cells

Angiotensin II (AngII) receptors couple to a multitude of different types of G-proteins resulting in activation of numerous signaling pathways. In this study we examined the consequences of this promiscuous G-protein coupling on secretion. Chromaffin cells were voltage-clamped at -80 mV in perforated-patch configuration, and Ca(2+)-dependent exocytosis was evoked with brief voltage steps to +20 mV. Vesicle fusion was monitored by changes in membrane capacitance (DeltaC(m)), and released catecholamine was detected with single-cell amperometry. Ca(2+) signaling was studied by recording voltage-dependent Ca(2+) currents (I(Ca)) and by measuring intracellular Ca(2+) ([Ca(2+)](i)) with fura-2 AM. AngII inhibited I(Ca) (IC(50) = 0.3 nm) in a voltage-dependent, pertussis toxin (PTX)-sensitive manner consistent with G(i/o)-protein coupling to Ca(2+) channels. DeltaC(m) was modulated bi-directionally; subnanomolar AngII inhibited depolarization-evoked exocytosis, whereas higher concentrations, in spite of I(Ca) inhibition, potentiated DeltaC(m) fivefold (EC(50) = 3.4 nm). Potentiation of exocytosis by AngII involved activation of phospholipase C (PLC) and Ca(2+) mobilization from internal stores. PTX treatment did not affect AngII-dependent Ca(2+) mobilization or facilitation of exocytosis. However, protein kinase C (PKC) inhibitors decreased the facilitatory effects but not the inhibitory effects of AngII on stimulus-secretion coupling. The AngII type 1 receptor (AT1R) antagonist losartan blocked both inhibition and facilitation of secretion by AngII. The results of this study show that activation of multiple types of G-proteins and transduction pathways by a single neuromodulator acting through one receptor type can produce concentration-dependent, bi-directional regulation of exocytosis.

Recent developments in teleost renal transport

The teleost renal tubule continues to be a valuable tool with which to investigate epithelial membrane transport mechanisms and factors that control them. Several newer aspects of fish renal transport are reviewed, and hypothetical models incorporating these findings are proposed. Fluorescence microscopy has revealed a new Na-independent organic anion (OA) transport process and new information about the classical Na-dependent transporter. With the latter, OA enters the cytosol in exchange for a-ketoglutarate (αKG) at the basolateral membrane (BLM). Larger molecular weight OAs enter independently of (αKG and exit to the lumen on a brush-border membrane (BBM) ATP-dependent transporter. The Na-dependent OA transporter is inhibited by activation of protein kinase C (PKC). A primary messenger is dopamine whereas adrenergic stimulation increases OA secretion. Avid sulfate secretion by the proximal tubule involves two anion exchangers in series. Recent studies implicate carbonic anhydrase (CA) in facilitation of secretion through dehydroxylation of bicarbonate entering from the lumen in exchange for cellular sulfate. Hydroxyl ions resulting from this catalysis exchange for interstitial sulfate. Factors that activate PKC stimulate phosphate (Pi) secretion; those that activate protein kinase A stimulate Pi reabsorption. The flounder Na-coupled Pi transporter has been cloned (NaPi-II), kinetically characterized, and localized mainly to the BLM of proximal segment II (PII) and BBM of collecting duct. Morphology and ion distribution studies strongly implicate aggregation of Mg in cytosolic vesicles as a step in Mg secretion. These were found in PII and certain collecting duct cells. J. Exp. Zool. 283:653–661, 1999. © 1999 Wiley-Liss, Inc.

Recent developments in teleost renal transport

The teleost renal tubule continues to be a valuable tool with which to investigate epithelial membrane transport mechanisms and factors that control them. Several newer aspects of fish renal transport are reviewed, and hypothetical models incorporating these findings are proposed. Fluorescence microscopy has revealed a new Na-independent organic anion (OA) transport process and new information about the classical Na-dependent transporter. With the latter, OA enters the cytosol in exchange for a-ketoglutarate (αKG) at the basolateral membrane (BLM). Larger molecular weight OAs enter independently of (αKG and exit to the lumen on a brush-border membrane (BBM) ATP-dependent transporter. The Na-dependent OA transporter is inhibited by activation of protein kinase C (PKC). A primary messenger is dopamine whereas adrenergic stimulation increases OA secretion. Avid sulfate secretion by the proximal tubule involves two anion exchangers in series. Recent studies implicate carbonic anhydrase (CA) in facilitation of secretion through dehydroxylation of bicarbonate entering from the lumen in exchange for cellular sulfate. Hydroxyl ions resulting from this catalysis exchange for interstitial sulfate. Factors that activate PKC stimulate phosphate (Pi) secretion; those that activate protein kinase A stimulate Pi reabsorption. The flounder Na-coupled Pi transporter has been cloned (NaPi-II), kinetically characterized, and localized mainly to the BLM of proximal segment II (PII) and BBM of collecting duct. Morphology and ion distribution studies strongly implicate aggregation of Mg in cytosolic vesicles as a step in Mg secretion. These were found in PII and certain collecting duct cells. J. Exp. Zool. 283:653–661, 1999. © 1999 Wiley-Liss, Inc.

Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion

Although microtubules appear necessary to maintain mucin granule transport in intestinal goblet cells, the role of microfilaments in mucus secretion is unknown. To determine the functional significance of microfilaments in goblet cell secretion, fluorescent cytochemistry of microfilaments and autoradiographic studies on granule movement were performed on rabbit intestinal goblet cells, with and without the actin depolymerizing agents, cytochalasin D (cyto D), and dihydro-cytochalasin B (dihydro B). In normal goblet cells, cytochemical localization of F-actin with NBD-phallacidin demonstrated their restriction to the apical surface of the goblet cell. Visualization of the goblet cell apical surface by electron microscopy revealed the presence of a thin layer of cytoplasm overlying the granule mass. Treatment with cyto D and dihydro B eliminated NBD-phallacidin staining of the apical cell surface. Quantitative analysis of baseline granule translocation demonstrated that treatment with cyto D and dihydro B resulted in dramatic acceleration of granule movement through goblet cells. This cellular response results from an increase in baseline secretion and facilitation of secretion of newly synthesized mucins, not stimulation of an accelerated secretory event. These data imply that actin filaments fulfill a barrier function in baseline secretion by hindering granule access to the plasma membrane; once the granule contacts the plasma membrane, exocytosis occurs. Secretion is balanced by the translocation of subjacent granules. In contrast, an accelerated secretory event is not triggered by plasma membrane access alone; this event requires a regulatory signal. We hypothesize that, unlike accelerated secretion, baseline secretion is constitutive, with exocytosis limited solely by the physical constraint of secretory granule access to the apical plasma membrane.

Cationic environment and dynamics of insulin secretion. III. Effect of the absence of potassium.

The absence of extracellular K+ modifies glucose-induced insulin secretion. The dynamics of these changes was studied in isolated rat islets perifused with K+-deprived media containing various Na+ concentrations. Omission of K+ from the perifusate during stimulation with 300 mg glucose/100 ml did not cause significant changes in IRI secretion, when Na+ concentration was normal (149 mM Na+). If external Na+ was partially substituted (24 mM Na+) by choline, a decrease in the amplitude of the second phase of release was noted. Both phases were markedly depressed, when Na+ was reduced to 2 mM. Removal of K+ for 25 min before glucose stimulation resulted in an acceleration and potentiation of the early phase of release, contrasting with the progressive inhibition of the late one. This inhibition was slowly reversible 10 min after K+ reintroduction. Under the same experimental conditions, decrease of Na+ concentration to 24 mM slowed and inhibited the first phase of secretion, and depressed further the second phase, without alteration of the dynamics of secretion. Glucose oxidation by islets was reduced by 35% after 2 h of incubation in a K+-free medium; this inhibition was more pronounced (65%) after 1 h, when the islets were previously perifused for 60 min in the absence of K+. From these results, it is suggested that changes in glucose-induced secretion observed in the absence of K+, are secondary to the intracellular modifications of Na+ and K+ concentrations due to the decreased activity of the Na+/K+) pump. They seem to be the result of two antagonistic effects: a facilitation of secretion by Na+ accumulation and an inhibition by K+ depletion.