Ion Channels in Secretory Granules of the Pancreas: Molecular Identification and Their Role in Regulated Secretion

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (α)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3)peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.

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Actomyosin II contractility expels von Willebrand factor from Weibel–Palade bodies during exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.201011119 ◽

2011 ◽

Vol 194 (4) ◽

pp. 613-629 ◽

Cited By ~ 79

Author(s):

Thomas D. Nightingale ◽

Ian J. White ◽

Emily L. Doyle ◽

Mark Turmaine ◽

Kimberly J. Harrison-Lavoie ◽

...

Keyword(s):

Von Willebrand Factor ◽

Secretory Granules ◽

Myosin Ii ◽

Human Endothelial Cells ◽

Spatiotemporal Resolution ◽

Regulated Secretion ◽

Experimental Systems ◽

Von Willebrand ◽

Willebrand Factor ◽

Extracellular Environment

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel–Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament–based prevention and promotion of specific exocytic steps during regulated secretion.

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Regulated secretion of atrial natriuretic factor from cultured ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.1.h282 ◽

1993 ◽

Vol 264 (1) ◽

pp. H282-H285 ◽

Cited By ~ 4

Author(s):

C. E. Irons ◽

C. A. Sei ◽

C. C. Glembotski

Keyword(s):

Transit Time ◽

Atrial Natriuretic Factor ◽

Ventricular Myocytes ◽

Secretory Granules ◽

Subcellular Fractionation ◽

Neonatal Rat ◽

Cell Type ◽

Regulated Secretion ◽

Natriuretic Factor ◽

Atrial Natriuretic

We have investigated endothelin (ET)-regulated secretion of atrial natriuretic factor (ANF) from primary neonatal rat ventricular myocytes, where hormone release is thought to be constitutive. In a dose-dependent, nifedipine-sensitive manner, ET acutely enhanced ANF release by two- to fivefold over control cultures within 15 min of agonist exposure, demonstrating that ventricular myocytes display a primary characteristic of a regulated secretory cell type. Unlike atrial cultures, ET enhanced ANF release during the first 30 min of exposure; thereafter, secretion rates returned to control levels. KCl, however, effectively enhanced ANF release only during the first 15 min of exposure. Subcellular fractionation of ventricular culture homogenates did not reveal atrial-type dense secretory granules, and pulse-chase labeling experiments showed that the transit time of newly synthesized ANF was short in ventricular myocytes [time required for half of labeled ANF to be released from cells (t1/2) = 0.5-1.5 h) compared with atrial myocytes (t1/2 = 4 h). These results suggest that, whereas ventricular myocytes possess some of the characteristics of a constitutively secreting cell type (e.g., few, if any, dense secretory granules and rapid transit time for newly synthesized hormone); however, they also display the capacity for regulated secretion of ANF in response to the physiological agonist ET.

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Expression of pro-Muclin in pancreatic AR42J cells induces functional regulated secretory granules

AJP Cell Physiology ◽

10.1152/ajpcell.00099.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1169-C1178 ◽

Cited By ~ 9

Author(s):

Robert C. De Lisle ◽

Oxana Norkina ◽

Eileen Roach ◽

Donna Ziemer

Keyword(s):

Secretory Granules ◽

Tail Domain ◽

Hormonal Stimulation ◽

Pancreatic Cell ◽

Regulated Secretion ◽

Ar42j Cells ◽

Cargo Protein ◽

Constitutive Secretion ◽

Poorly Differentiated ◽

Sorting Receptor

It is not clear how protein cargo is sorted to and retained in forming regulated secretory granules (RSG). Here, the sulfated mucin-type glycoprotein pro-Muclin was tested for its ability to induce RSG in the poorly differentiated rat pancreatic cell line AR42J. AR42J cells express RSG content proteins, but they fail to make granules. Adenovirus-pro-Muclin-infected AR42J cells store amylase, accumulate RSG, and respond to hormonal stimulation by secreting the stored protein. Expression of pro-Muclin combined with the inducing effect of dexamethasone resulted in a significant enhancement of the efficiency of regulated secretion. The effect of pro-Muclin was a strong decrease in constitutive secretion compared with dexamethasone-induction alone. A pro-Muclin construct missing the cytosolic tail domain was less effective at improving the efficiency of regulated secretion compared with the full-length construct. Increased expression of cargo (using adenovirus amylase) also modestly enhanced regulated secretion, indicating that part of pro-Muclin's effect may be due to increased expression of cargo protein. Overall, the data show that pro-Muclin acts as a sorting receptor that can induce RSG, and that its cytosolic tail is important in this process.

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Membrane insertion of chromogranin B for granule maturation in regulated secretion

10.1101/2019.12.28.890053 ◽

2019 ◽

Author(s):

Gaya P. Yadav ◽

Haiyuan Wang ◽

Joke Ouwendijk ◽

Mani Annamalai ◽

Stephen Cross ◽

...

Keyword(s):

Plasma Membranes ◽

Secretory Granules ◽

Neuroendocrine Cells ◽

Membrane Insertion ◽

List Type ◽

Chromogranin B ◽

Regulated Secretion ◽

Anion Conductance ◽

Altered Glucose ◽

Granule Maturation

ABSTRACTRegulated secretion serves responses to specific stimuli in eukaryotes. An anion conductance was found essential for maturation and acidification of secretory granules four decades ago, but its genetic identity was unknown. We now demonstrate that chromogranin B (CHGB), an obligate granule protein, constitutes the long-sought anion channel. High-pressure freezing immuno-electron microscopy and biochemical assays showed native CHGB in close proximity to secretory granule membranes, and its membrane-bound and soluble forms both reconstituted Cl- channels. Release of secretory granules delivered CHGB clusters to plasma membranes, which dominate whole-cell anion conductance. Intragranular pH measurements and cargo maturation assays found that CHGB channels supported proinsulin - insulin conversion and dopamine-loading in neuroendocrine cells. β-cells from Chgb-/- mice exhibited significant granule deacidification, accounting for hyperproinsulinemia, altered glucose-tolerance response and lower dopamine concentration in chromaffin granules in these animals. Membrane insertion of well-conserved CHGB is thus indispensable for granule maturation in exocrine, endocrine and neuronal cells.HighlightsNative CHGB is amphipathic and distributes in the lumen and membranes of secretory granules with contrastingly different destinies and functions.Native CHGB, once delivered to cell surface via granule exocytosis, dominates anion conductance in plasma membranes.CHGB channels facilitate granule acidification and cargo maturation in cultured and primary neuroendocrine cells.CHGB channels from bovine, rat and mouse cells all serve the long-missing, intra-organellar anion shunt pathway in the secretory granules for regulated secretion.

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Multiple Sorting Systems for Secretory Granules Ensure the Regulated Secretion of Peptide Hormones

Traffic ◽

10.1111/tra.12029 ◽

2012 ◽

Vol 14 (2) ◽

pp. 205-218 ◽

Cited By ~ 10

Author(s):

Meng Sun ◽

Tsuyoshi Watanabe ◽

Hiroki Bochimoto ◽

Yuko Sakai ◽

Seiji Torii ◽

...

Keyword(s):

Secretory Granules ◽

Peptide Hormones ◽

Regulated Secretion

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Synaptotagmin II Negatively Regulates Ca2+-triggered Exocytosis of Lysosomes in Mast Cells

Journal of Experimental Medicine ◽

10.1084/jem.189.10.1649 ◽

1999 ◽

Vol 189 (10) ◽

pp. 1649-1658 ◽

Cited By ~ 80

Author(s):

Dana Baram ◽

Roberto Adachi ◽

Ora Medalia ◽

Michael Tuvim ◽

Burton F. Dickey ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cathepsin D ◽

Cell Biology ◽

Secretory Granules ◽

Triggered Release ◽

Lysosomal Fraction ◽

Regulated Secretion ◽

Processed Form

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcεRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II–regulated secretion from lysosomes may play an important role in mast cell biology.

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Differential sorting behavior for soluble and transmembrane cargoes at the trans-Golgi network in endocrine cells

10.1101/797134 ◽

2019 ◽

Author(s):

Blake H. Hummer ◽

Drew Maslar ◽

Margarita Soltero Gutierrez ◽

Noah F. de Leeuw ◽

Cedric S. Asensio

Keyword(s):

Endocrine Cells ◽

Secretory Granules ◽

Complex Model ◽

Secreted Proteins ◽

Secretory Pathways ◽

Regulated Secretion ◽

Trans Golgi Network ◽

Differential Behavior ◽

Golgi Network ◽

Kinetics Of

AbstractRegulated secretion of neuropeptides and peptide hormones by secretory granules (SGs) is central to physiology. Formation of SGs occurs at the trans-Golgi network (TGN) where their soluble cargo aggregates to form a dense core, but the mechanisms controlling the sorting of regulated secretory cargoes (soluble and transmembrane) away from constitutively secreted proteins remain unclear. Optimizing the use of the retention using selective hooks (RUSH) method in (neuro-)endocrine cells, we now quantify TGN budding kinetics of constitutive and regulated secretory cargoes. We further show that, by monitoring two cargoes simultaneously, it becomes possible to visualize sorting to the constitutive and regulated secretory pathways in real-time. Further analysis of the localization of SG cargoes immediately after budding from the TGN revealed that, surprisingly, the bulk of two studied transmembrane SG cargoes (phogrin and VMAT2) does not sort directly onto SGs during budding, but rather exit the TGN into non-regulated vesicles to get incorporated to SGs at a later step. This differential behavior of soluble and transmembrane cargoes suggests a more complex model of SG biogenesis than anticipated.

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VAMP8/Endobrevin as a General Vesicular SNARE for Regulated Exocytosis of the Exocrine System

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0974 ◽

2007 ◽

Vol 18 (3) ◽

pp. 1056-1063 ◽

Cited By ~ 70

Author(s):

Cheng-Chun Wang ◽

Hong Shi ◽

Ke Guo ◽

Chee Peng Ng ◽

Jie Li ◽

...

Keyword(s):

Salivary Glands ◽

Secretory Granules ◽

Acinar Cells ◽

Sweat Glands ◽

Lacrimal Glands ◽

Regulated Secretion ◽

Syntaxin 4 ◽

Pancreatic Exocrine ◽

Carbonic Anhydrase Vi ◽

Immunohistochemical Studies

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.

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Mitochondrial Channels: Ion Fluxes and More

Physiological Reviews ◽

10.1152/physrev.00021.2013 ◽

2014 ◽

Vol 94 (2) ◽

pp. 519-608 ◽

Cited By ~ 175

Author(s):

Ildiko Szabo ◽

Mario Zoratti

Keyword(s):

Ion Channels ◽

Molecular Identification ◽

Cell Biology ◽

Energy Supply ◽

Integrative Approach ◽

Mitochondrial Channels ◽

Electrophysiological Properties ◽

Molecular Identity ◽

Substantial Progress ◽

Entire Cell

The field of mitochondrial ion channels has recently seen substantial progress, including the molecular identification of some of the channels. An integrative approach using genetics, electrophysiology, pharmacology, and cell biology to clarify the roles of these channels has thus become possible. It is by now clear that many of these channels are important for energy supply by the mitochondria and have a major impact on the fate of the entire cell as well. The purpose of this review is to provide an up-to-date overview of the electrophysiological properties, molecular identity, and pathophysiological functions of the mitochondrial ion channels studied so far and to highlight possible therapeutic perspectives based on current information.

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