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Glucose and NAADP trigger elementary intracellular β-cell Ca²⁺ signals.

ABSTRACT: Pancreatic β-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in β-cells leads to closure of cell membrane ATP-gated potassium (K_ATP) channels, plasma membrane depolarisation, Ca²⁺ influx, and finally the exocytosis of insulin granules. However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the K_ATP channel alone may not be sufficient to explain all β-cell responses to glucose elevation. We have previously proposed that NAADP-evoked Ca²⁺ release is an important step in stimulus-secretion coupling in pancreatic β-cells. Here we show using total internal reflection fluorescence (TIRF) microscopy that glucose as well as the Ca²⁺ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in β-cells, lead to highly localised elementary intracellular Ca²⁺ signals. These were found to be obscured by measurements of global Ca²⁺ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). Building on our previous work demonstrating that NAADP-evoked Ca²⁺ release is an important step in stimulus-secretion coupling in pancreatic β-cells, we provide here the first demonstration of elementary Ca²⁺ signals in response to NAADP, whose occurrence was previously suspected. Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca²⁺ signalling events, inositol trisphosphate (IP₃) receptor mediated blips, and ryanodine receptor mediated quarks. We propose that a mechanism based on these highly localised intracellular Ca²⁺ signalling events mediated by NAADP may initially operate in β-cells when they respond to elevations in blood glucose.

SUBMITTER: Heister PM

PROVIDER: S-EPMC8140081 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Project description:Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.

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Glucose and NAADP trigger elementary intracellular β-cell Ca²⁺ signals.

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Dataset Information

Glucose and NAADP trigger elementary intracellular β-cell Ca2+ signals.

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OmicsDI is part of the ELIXIR infrastructure

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Glucose and NAADP trigger elementary intracellular β-cell Ca²⁺ signals.