Browse
Submit Data
Databases
API
Help
Login

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

ABSTRACT: alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

PROVIDER: S-EPMC1170414 | BioStudies | 1998-01-01T00:00:00Z

REPOSITORIES: biostudies

ACCESS DATA

Json Xml

Similar Datasets

The alpha-latrotoxin mutant LTXN4C enhances spontaneous and evoked transmitter release in CA3 pyramidal neurons.

Project description:Alpha-latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX(N4C) to selectively study the receptor-mediated actions of LTX. LTX(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX(N4C) is reversible and is not attenuated by La3+ that is known to block LTX pores. On the other hand, LTX(N4C) action, which requires extracellular Ca2+, is inhibited by thapsigargin, a drug depleting intracellular Ca2+ stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca2+ release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca2+ indicator directly demonstrate that LTX(N4C) increases presynaptic, but not dendritic, free Ca2+ concentration; this Ca2+ rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX(N4C) involves mobilization of Ca2+ from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca2+ release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis.

| S-EPMC6741093 | BioStudies

Calcium current inactivation rather than pool depletion explains reduced exocytotic rate with prolonged stimulation in insulin-secreting INS-1 832/13 cells.

Project description:Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (?Cm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ?Cm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ?10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

| S-EPMC4126658 | BioStudies

Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin.

Project description:Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (alpha-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant alpha-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, alpha-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.

1996-01-01 | S-EPMC1217025 | BioStudies

Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic ?-Cell Line.

Project description:In glucose-stimulated insulin secretion (GSIS) of pancreatic ?-cells, the rise of free cytosolic Ca2+ concentration through voltage-gated calcium channels (VGCCs) triggers the exocytosis of insulin-containing granules. Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic ?-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.

| S-EPMC7290496 | BioStudies

Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors.

Project description:The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, alpha-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTX(N4C) induced intracellular Ca2+ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.

| S-EPMC2649109 | BioStudies

Dynamics of Ca2+ and guanosine 5'-[gamma-thio]triphosphate action on insulin secretion from alpha-toxin-permeabilized HIT-T15 cells.

Project description:The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond to Ca2+ after prolonged perfusion at low Ca2+ (run-down), nor to depletion of a particular secretory-granule pool. It was rather due to desensitization of the secretory machinery to Ca2+ that was not reversed by selective inhibition of the Ca2+/calmodulin-dependent kinase II with KN-62. However, an intermediate Ca2+ concentration (2 microM) increased insulin secretion to stable level without causing any desensitization. Imposed oscillations of Ca2+ (0.1-10 microM) produced phasic oscillations of insulin secretion, but did not prevent desensitization to Ca2+. Poorly hydrolysable GTP analogues increased insulin secretion at low Ca2+, whereas they strongly inhibited Ca(2+)-induced insulin secretion. By contrast, GTP did not affect basal secretion, and slightly increased Ca(2+)-evoked secretion. These results indicate the following. (1) Oscillations of insulin secretion are tightly coupled to cytosolic Ca2+ oscillations. (2) Oscillations of Ca2+ do not prevent high-Ca(2+)-induced desensitization to Ca2+; this result does not support the idea of a greater efficiency of oscillations compared with sustained Ca2+ rises in triggering exocytosis. (3) Activation of G-proteins modulates exocytosis in a bimodal manner.

1994-01-01 | S-EPMC1137112 | BioStudies

Somatostatin inhibition of Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells.

Project description:Somatostatin inhibited Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells, albeit with decreased sensitivity relative to intact cells. The inhibitory action required the presence of GTP, whereas GDP could not substitute for GTP. Pertussis-toxin treatment before cell permeabilization abolished the inhibition of secretion. Thus somatostatin, by activating a G-protein, interferes with exocytosis distal to the generation of soluble intracellular messengers.

1990-01-01 | S-EPMC1131711 | BioStudies

Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic ?-Cells.

Project description:The regulation of insulin biosynthesis and secretion in pancreatic ?-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic ?-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.

| S-EPMC7370021 | BioStudies

Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.

Project description:Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 ?m free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

| S-EPMC6618444 | BioStudies

alpha-Latrotoxin releases calcium in frog motor nerve terminals.

Project description:alpha-Latrotoxin (alpha-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca(2+). Although alpha-LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that alpha-LTX causes transmitter release by mobilizing intracellular Ca(2+) in frog motor nerve terminals. Transmitter release was measured electrophysiologically and with the vesicle marker FM1-43; presynaptic ion concentration dynamics were measured with fluorescent ion-imaging techniques. We report that alpha-LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca(2+). Neither the blockade of Ca(2+) release nor the depletion of Ca(2+) from endoplasmic reticulum affected Ca(2+) signals produced by alpha-LTX. The Ca(2+) source is likely to be mitochondria, because the effects on Ca(2+) mobilization of CCCP (which depletes mitochondrial Ca(2+)) and of alpha-LTX are mutually occlusive. The release of mitochondrial Ca(2+) is partially attributable to an increase in intracellular Na(+), suggesting that the mitochondrial Na(+)/Ca(2+) exchanger is activated. Effects of alpha-LTX were not blocked when Ca(2+) increases were reduced greatly in saline lacking both Na(+) and Ca(2+) and by application of intracellular Ca(2+) chelators. Therefore, although increases in intracellular Ca(2+) may facilitate the effects of alpha-LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca(2+)-independent alpha-LTX mechanisms or uses of alpha-LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca(2+), which itself can trigger exocytosis.

| S-EPMC6773046 | BioStudies

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data