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

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 (KATP) 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 KATP 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.

Cytoskeletal Transport, Reorganization, and Fusion Regulation in Mast Cell-Stimulus Secretion Coupling

Mast cells are well known for their role in allergies and many chronic inflammatory diseases. They release upon stimulation, e.g., via the IgE receptor, numerous bioactive compounds from cytoplasmic secretory granules. The regulation of granule secretion and its interaction with the cytoskeleton and transport mechanisms has only recently begun to be understood. These studies have provided new insight into the interaction between the secretory machinery and cytoskeletal elements in the regulation of the degranulation process. They suggest a tight coupling of these two systems, implying a series of specific signaling effectors and adaptor molecules. Here we review recent knowledge describing the signaling events regulating cytoskeletal reorganization and secretory granule transport machinery in conjunction with the membrane fusion machinery that occur during mast cell degranulation. The new insight into MC biology offers novel strategies to treat human allergic and inflammatory diseases targeting the late steps that affect harmful release from granular stores leaving regulatory cytokine secretion intact.

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization.

The Adrenal Medulla Modulates Mechanical Allodynia in a Rat Model of Neuropathic Pain

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not affect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chromaffin cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerve.

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

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 (KATP) channels, plasma membrane depolarisation, Ca2+ influx, and finally the exocytosis of insulin granules (Ashcroft et al., 1984; Cook and Hales, 1984). However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the KATP channel alone may not be sufficient to explain all β-cell responses to glucose elevation (Henquin, 1998; Seghers et al., 2000). Here we show using total internal reflection fluorescence (TIRF) microscopy (Axelrod, 1981) that glucose as well as the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in β-cells (Johnson and Misler, 2002; Masgrau et al., 2003), 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). This is the first demonstration of elemental Ca2+ signals in response to NAADP, although they have been suspected (Davis et al., 2020). 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 (Parker et al., 1996; Parker and Ivorra, 1990), and ryanodine receptor mediated sparks (Cheng et al., 1993). 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.

Fresh and cultured mouse islets differ in their response to nutrient stimulation

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs–Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

Synaptotagmin-7 is a calcium sensor for facilitation at the splanchnic-chromaffin cell synapse

The splanchnic-chromaffin cell synapse is the site at which stimulus-secretion coupling in the adrenal medulla is regulated. However, since the discovery that acetylcholine underlies chemical signaling at this synapse, attention has been disproportionately placed on postsynaptic chromaffin cell function. As a result, the determinants of Ca2+-sensing and exocytosis from splanchnic nerves remain poorly understood. This study shows, for the first time, that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7) is expressed within the neurons that innervate the adrenal medulla. In synapses that lack Syt7, evoked excitatory postsynaptic currents (EPSCs) are smaller in amplitude and decay with more rapid kinetics than wild-type synapses stimulated in an identical manner. EPSCs in Syt7-deficient adrenal slices also fail to facilitate, which is ordinarily a robust property of these synapses. These data are the first to implicate a role for Syt7 in regulating short-term synaptic plasticity in the peripheral nervous system.