Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca²⁺ stores that release Ca²⁺ in response to the second messengers IP₃ and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca²⁺ released from acidic organelles by NAADP subsequently recruits IP₃ or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca²⁺ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca²⁺ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca²⁺-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca²⁺ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca²⁺ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca²⁺ oscillations and waves.     

Arginine Thiazolidine Carboxylate Stimulates Insulin Secretion through Production of Ca2+-Mobilizing Second Messengers NAADP and cADPR in Pancreatic Islets

Oxothiazolidine carboxylic acid is a prodrug of cysteine that acts as an anti-diabetic agent via insulin secretion and the formation of the Ca²⁺-mobilizing second messenger, cyclic ADP-ribose (cADPR). Here we show that a hybrid compound, arginine thiazolidine carboxylate (ATC), increases cytoplasmic Ca²⁺ in pancreatic β-cells, and that the ATC-induced Ca²⁺ signals result from the sequential formation of two Ca²⁺-mobilizing second messengers: nicotinic acid adenine dinucleotide phosphate (NAADP) and cADPR. Our data demonstrate that ATC has potent insulin-releasing properties, due to the additive action of its two components; thiazolidine carboxylate (TC) and _L-arginine. TC increases glutathione (GSH) levels, resulting in cAMP production, followed by a cascade pathway of NAADP/nitric oxide (NO)/cGMP/cADPR synthesis. _L-arginine serves as the substrate for NO synthase (NOS), which results in cADPR synthesis via cGMP formation. Neuronal NOS is specifically activated in pancreatic β-cells upon ATC treatment. These results suggest that ATC is an ideal candidate as an anti-diabetic, capable of modulating the physiological Ca²⁺ signalling pathway to stimulate insulin secretion.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL-8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that IL-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca²⁺ signaling as well as cell migration, which was completely blocked by bafilomycin A1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. IL-8 or exogenous cADPR, but not NAADP, increased intracellular cAMP levels. cGMP analog, 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate, increased both cADPR and NAADP production, whereas the cAMP analog, 8-(4-chlorophenylthio)-cAMP, increased only NAADP production, suggesting that cAMP is essential for IL-8-induced NAADP formation. Furthermore, activation of Rap1, a downstream molecule of Epac, was required for IL-8-induced NAADP formation in LAK cells. Taken together, our data suggest that IL-8-induced NAADP production is mediated by CD38 activation through the actions of cAMP/Epac/protein kinase A/Rap1 in LAK cells and that NAADP plays a key role in Ca²⁺ signaling of IL-8-induced LAK cell migration.     

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells

The Ca²⁺ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca²⁺ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca²⁺ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca²⁺ release (but not cADPR- or IP₃-evoked Ca²⁺ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of NAADP-evoked Ca²⁺ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca²⁺ release without depleting acidic Ca²⁺ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca²⁺ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP₃-dependent Ca²⁺ signaling with potential therapeutic value.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

cADPR stimulates SERCA activity in Xenopus oocytes

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca²⁺.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation

Ca²⁺ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca²⁺-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca²⁺ transients followed by sustained Ca²⁺ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca²⁺ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca²⁺ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca²⁺ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca²⁺ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively.     

Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca²⁺ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H₂O₂ or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H₂O₂, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca²⁺ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca²⁺. To identify direct modulators of TRPM2, we have studied the effects of H₂O₂, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H₂O₂ (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K_0.5 = 104 and 35 μm). H₂O₂, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

SECOND MESSENGERS AND THE REGULATION OF CA 2+ FLUXES BY CA 2+ -MOBILIZING AGONISTS IN RAT LIVER

Knowledge of the mechanism of action of Ca²⁺-mobilizing agonists in liver has progressed considerably following the discovery that their interaction with specific receptors on the plasma membrane is accompanied by the hydrolysis of PIP₂ and the generation of the second messengers diacylglycerol and IP₃, for the activation of protein kinase C and the mobilization of intracellular Ca²⁺, respectively. Although the second messenger functions of diacylglycerol and IP₃ in these actions seem well established, it is not yet clear how the agonists are able to regulate Ca²⁺ influx across the plasma membrane, an event which is crucial for those actions of the agonists which are dependent on the maintenance of an elevated level of cytosolic Ca²⁺, Whilst there is evidence for the existence of more than one pathway for Ca²⁺ influx in liver, it appears that in each instance the Ca²⁺ influx process is regulated differently to the Ca²⁺ influx through the volage-sensitive Ca²⁺ channels that is known to occur in excitable tissues. At present it is not clear whether any of the Ca²⁺ influx pathways in liver is regulated by direct coupling to the agonist receptor mechanism on the outer surface of the plasma membrane, or whether the regulation involves the production of some second messenger(s). However, indirect evidence from a number of tissues appears to favour the involvement of both IP₃ and IP₄ in the regulation of Ca²⁺ influx. The mechanism by which IP₃ and IP₄ may regulate Ca²⁺ influx remains to be established, but it has been proposed that Ca²⁺ entry into the cell occurs through a pathway connecting the plasma membrane and the endoplasmic reticulum, following the release of intracellular Ca²⁺ from the lumen of the endoplasmic reticulum. Although it is not yet known whether glucagon (or cyclic AMP) activates the same pathway for Ca²⁺ influx as Ca²⁺-mobilizing agonists, the marked potentiation by cyclic AMP of the Ca²⁺ influx induced by Ca²⁺-mobilizing agonists has provided a powerful system with which to study the regulation of Ca²⁺ influx in liver. Whether this Ca²⁺ influx process occurs through some ion exchange mechanism (such as Ca²⁺/Na⁺ exchange) remains to be determined. Results from this study suggests that the Ca²⁺ influx is inhibited by neomycin, acidic pH, and a depolarization of the plasma membrane. The observation that cyclic AMP synergistically potentiates the influx of Ca²⁺ induced by Ca²⁺-mobilizing agonists, that this influx appears to correlate with the reported ability of these agonists to induce PIP₂ hydrolysis and accumulation of IP₃, and that cyclic AMP synergistically potentiates the production of IP₄ by vasopressin, are all consistent with the notion that IP₃ and IP₄ are involved in regulating Ca²⁺ influx. Whilst little is known about the Ca²⁺ transport process itself, these studies coupled with the recent finding that Ca²⁺ influx into the liver cell can occur through different pathways, seem set to lead to a better understanding of this important process in the near future.     

Departure gate of acidic Ca2+ confirmed

More potent, but less known than IP₃ that liberates Ca²⁺ from the ER, NAADP releases Ca²⁺ from acidic stores. The notion that TPC channels mediate this Ca²⁺ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P₂‐activated Na⁺ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P₂. They attribute the failure of others to observe TPC‐dependent NAADP‐induced Ca²⁺ release in vivo to inadequate mouse models that retain partial TPC function.     

NaCl-elicited,vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells

High environmental salt elicits an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) in plants, which is generated by extracellular Ca²⁺ influx and Ca²⁺ release from intracellular stores, such as vacuole and endoplasmic reticulum. This study aimed to determine the physiological mechanisms underlying Ca²⁺ release from vacuoles and its role in ionic homeostasis in Populus euphratica. In vivo Ca²⁺ imaging showed that NaCl treatment induced a rapid elevation in [Ca²⁺]_cyt, which was accompanied by a subsequent release of vacuolar Ca²⁺. In cell cultures, NaCl-altered intracellular Ca²⁺ mobilization was abolished by antagonists of inositol (1, 4, 5) trisphosphate (IP₃) and cyclic adenosine diphosphate ribose (cADPR) signaling pathways, but not by slow vacuolar (SV) channel blockers. Furthermore, the NaCl-induced vacuolar Ca²⁺ release was dependent on extracellular ATP, extracellular Ca²⁺ influx, H₂O₂, and NO. In vitro Ca²⁺ flux recordings confirmed that IP₃, cADPR, and Ca²⁺ induced substantial Ca²⁺ efflux from intact vacuoles, but this vacuolar Ca²⁺ flux did not directly respond to ATP, H₂O₂, or NO. Moreover, the IP₃/cADPR-mediated vacuolar Ca²⁺ release enhanced the expression of salt-responsive genes that regulated a wide range of cellular processes required for ion homeostasis, including cytosolic K⁺ maintenance, Na⁺ and Cl⁻ exclusion across the plasma membrane, and Na⁺/H⁺ and Cl⁻/H⁺ exchanges across the vacuolar membrane.     

CD38-mediated Ca2+ Signaling Contributes to Angiotensin II-induced Activation of Hepatic Stellate Cells: ATTENUATION OF HEPATIC FIBROSIS BY CD38 ABLATION*

CD38 is a type II glycoprotein that is responsible for the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), Ca²⁺-mobilizing second messengers. The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis because these cells are the main producers of extracellular matrix proteins in the liver. Recent evidence indicates that the renin-angiotensin system plays a major role in liver fibrosis. In this study, we showed that angiotensin II (Ang II) evoked long lasting Ca²⁺ rises and induced NAADP or cADPR productions via CD38 in HSCs. Inositol 1,4,5-trisphosphate as well as NAADP-induced initial Ca²⁺ transients were prerequisite for the production of cADPR, which was responsible for later sustained Ca²⁺ rises in the Ang II-treated HSCs. Ang II-mediated inositol 1,4,5-trisphosphate- and NAADP-stimulated Ca²⁺ signals cross-talked in a dependent manner with each other. We also demonstrated that CD38 plays an important role in Ang II-induced proliferation and overproduction of extracellular matrix proteins in HSCs, which were reduced by an antagonistic cADPR analog, 8-bromo-cADPR, or in CD38^−/− HSCs. Moreover, we presented evidence to implicate CD38 in the bile duct ligation-induced liver fibrogenesis; infiltration of inflammatory cells and expressions of α-smooth muscle actin, transforming growth factor-β1, collagen αI(1), and fibronectin were reduced in CD38^−/− mice compared with those in CD38^+/+ mice. These results demonstrate that CD38-mediated Ca²⁺ signals contribute to liver fibrosis via HSCs activation, suggesting that intervention of CD38 activation may help prevent hepatic fibrosis.     

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons

Calcium ions (Ca²⁺) released from inositol trisphosphate (IP₃)-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP₃ receptor (IP₃R) antagonists represent an important tool for dissociating these consequences of IP₃ generation and IP₃R-dependent internal Ca²⁺ release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca²⁺ sources. In this study, we have described the actions of the IP₃R and store-operated Ca²⁺ channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca²⁺ release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 μM) sufficient for attenuating or blocking IP₃-mediated internal Ca²⁺ release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca²⁺-dependent potassium (K⁺) conductances.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils

The Ca²⁺-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca²⁺ acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H₂O₂, which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC₅₀) of 1 μM. Potentiation by Ca²⁺ is dose-dependent with an EC₅₀ of 300 nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100 nM), which significantly shifts the EC₅₀ for cADPR from 44 to 3 μM and for NAADP from 95 to 1 μM. TRPM2 activation by ADPR can be suppressed by AMP with an IC₅₀ of 10 μM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100 μM H₂O₂ enables subthreshold concentrations of ADPR (100 nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.     

The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.     

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilizing second messenger which triggers Ca²⁺ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca²⁺ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca²⁺ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca²⁺ release. In mammalian cell homogenates, incubation with low concentrations of [³²P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [³²P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [³²P]5-N₃-NAADP.