Calcium store depletion potentiates a phosphodiesterase inhibitor- and dibutyryl cGMP-evoked calcium influx in rat pituitary GH3 cells

AbstractA role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 μM-1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 μM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre-incubation of cells with 1 μM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 μM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores

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

Funder: University of Oxford Department of PharmacologyFunder: UK Medical Research CouncilFunder: Wellcome TrustPancreatic 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

TPC: the NAADP discovery channel?

Abstract The Ca 2 + -mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20 years and yet we still cannot fully agree on the identity of its target Ca 2 + -release channel. In spite of some recent robust challenges to the idea that two-pore channels (TPCs) represent the elusive "NAADP receptor", evidence continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story. Acidic Ca 2 + stores Ca 2 + mobilization from intracellular stores is an ancient and conserved signal transduction pathway from plants to animals, and while the study of the endoplasmic reticulum (ER) Ca 2 + store has dominated this arena for decades, another organelle Ca 2 + store family -the acidic Ca 2 + stores -is stepping out from the shadow of its bigger brother and assuming an importance in its own right. The so-called acidic Ca 2 + stores encompass a spectrum of organelles with an acidic lumen such as endosomes, lysosomes, secretory vesicles and lysosome-related organelles (that include vacuoles, secretory lysosomes and yolk platelets) [1]. That these organelles are key for diverse cellular functions is without doubt, that they also store and release Ca 2 + may be less familiar but arguably just as important. How Ca 2 + is sequestered into these acidic vesicles is unclear for higher organisms but, by analogy with the better understood plants and yeast systems [2], it is anticipated to involve some form of Ca NAADP, a unique messenger The archetypal ER Ca 2 + store is mobilized by IP 3 or cADPR and, in a comparable manner, acidic Ca 2 + stores are mobilized by their own unique second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate) [1]. Key words: NAADP, TPC, Ca 2 + , lysosome, endosome. Abbreviations: ER, endoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate; pHL, luminal pH; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; RyRs, ryanodine receptors; TPCs, two-pore channels; TRPML1, mucolipin-1. 1 To whom correspondence should be addressed (email Anthony.morgan@pharm. ox.ac.uk). Coupled to a burgeoning list of extracellular stimuli, NAADP plays a unique role and is physiologically important in contexts as broad as fertilization, immunology, angiogenesis, cardiac function and autophagy [9]. However, the identity of the Ca 2 + -release channel targeted by NAADP has long been sought and debated, with several candidate channels proposed over the years including ryanodine receptors (RyRs), mucolipin-1 (TRPML1), TRPM2 and the two-pore channel family (TPCs) [1]. TPCs have received the lion's share of recent attention owing to a substantial body of evidence linking it to NAADP, as we shall briefly discuss. . This was a tantalizing development because the vacuole is the acidic Ca 2 + store in plants, and pre-empted a cluster of papers in 2009 that proposed that the animal orthologues of TPC were endo-lysosomal Ca 2 + -release channels activated by NAADP i.e. TPCs were the long sought after "NAADP receptors" [13][14][15]. TPCs: a new channel family Building on this foundation, different laboratories subsequently appeared to confirm and extend these observations using Ca 2 + imaging and/or electrophysiology and revealed that TPCs recapitulated the properties expected of the "NAADP receptor": they were permeable to Ca 2 + (or Ca 2 + surrogates), associated with NAADP-binding sites [7] and were activated by NAADP (in mammalian cells, with the characteristic bell-shaped concentration-response curve) [16]. In view of this compelling body of evidence, it came as something of a surprise in late 2012/early 2013 when this premise was challenged by a couple of high profile papers that asserted that TPCs had nothing to do with either NAADP or Ca 2 + : TPCs were described as lipid-regulated Na + channel

TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+-release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.Publisher PDFPeer reviewe

An emerging role for NAADP-mediated Ca2+ signaling in the pancreatic beta-cell

Several recent reports, including one in this journal, have reignited the debate about whether the calcium-mobilizing messenger, nicotinic adenine nucleotide diphosphate (NAADP) plays a central role in the regulation of calcium signalling in pancreatic β-cell. These studies have highlighted a role for NAADP-induced Ca(2+) mobilization not only in mediating the effects of the incretin, GLP-1 and the autocrine proliferative effects of insulin, but also possibly a fundamental role in glucose-mediated insulin secretion in the pancreatic β-cell

Oxytocin Influences Male Sexual Activity via Non-synaptic Axonal Release in the Spinal Cord

Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion—a localized volume transmission—to reach oxytocin receptors on GRP neurons and facilitate male sexual function

'Click cyclic ADP-ribose':A neutral second messenger mimic

Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approach to design drug-like signalling pathway modulators