Characterization of NAADP-mediated calcium signaling in human spermatozoa

Ca²⁺ signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca²⁺ signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca²⁺ and pH. Ca²⁺ fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca²⁺] increases in human sperm even in the absence of extracellular Ca²⁺. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.     

Acidic NAADP-sensitive Calcium Stores in the Endothelium: AGONIST-SPECIFIC RECRUITMENT AND ROLE IN REGULATING BLOOD PRESSURE*

Accumulating evidence implicates nicotinic acid adenine dinucleotide phosphate (NAADP) in the control of Ca²⁺-dependent functions. Little, however, is known concerning its role in the vascular endothelium, a major regulator of blood pressure. Here, we show that NAADP acetoxymethyl ester (NAADP-AM), a cell-permeant NAADP analog, increases cytosolic Ca²⁺ concentration in aortic endothelial cells. We demonstrate that these signals and those evoked by acetylcholine are blocked by disrupting acidic organelles with bafilomycin A1. In contrast, Ca²⁺ signals in response to thrombin are only partially inhibited by bafilomycin A1 treatment, and those to ATP were insensitive, suggesting that recruitment of acidic stores is agonist-specific. We further show that NAADP-evoked Ca²⁺ signals hyperpolarize endothelial cells and generate NO. Additionally, we demonstrate that NAADP dilates aortic rings in an endothelium- and NO-dependent manner. Finally, we show that intravenous administration of NAADP-AM into anesthetized rats decreases mean arterial pressure. Our data extend the actions of NAADP to the endothelium both in vitro and in vivo, pointing to a previously unrecognized role for this messenger in controlling blood pressure.     

Two Pore Channel 2 (TPC2) Inhibits Autophagosomal-Lysosomal Fusion by Alkalinizing Lysosomal pH

Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca²⁺ mobilizing messengers, elicits Ca²⁺ release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca²⁺ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca²⁺ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca²⁺ signals sensitive to inhibitors of both acidic Ca²⁺ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca²⁺ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca²⁺ transients were recorded both as increases of the free cytosolic Ca²⁺ concentration and as decreases of the sarcoplasmic luminal Ca²⁺ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca²⁺ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.     

Ca2+ Release Events in Cardiac Myocytes Up Close: Insights from Fast Confocal Imaging

The spatio-temporal properties of Ca²⁺ transients during excitation-contraction coupling and elementary Ca²⁺ release events (Ca²⁺ sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca²⁺ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca²⁺ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca²⁺ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca²⁺]_i. 2-D imaging of Ca²⁺ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca²⁺ entry through surface membrane Ca²⁺ channels and subsequent activation of Ca²⁺-induced Ca²⁺ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca²⁺ entry could be detected that was followed by SR Ca²⁺ release after an additional 3 ms delay. Maximum Ca²⁺ release was observed 4 ms after the beginning of release. The timing of Ca²⁺ entry and release was confirmed by simultaneous [Ca²⁺]_i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca²⁺ release events that fused into a peripheral ring of elevated [Ca²⁺]_i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca²⁺ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca²⁺ transient. In summary, ultra-fast confocal imaging allows investigation of Ca²⁺ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.     

NAADP controls cross-talk between distinct Ca2+ stores in the heart

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.     

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.     

The endo-lysosomal system as an NAADP-sensitive acidic Ca(2+) store: role for the two-pore channels

Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca²⁺ that is tapped by the Ca²⁺-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca²⁺ release from this acidic Ca²⁺ store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family.     

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.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

The Ecto-enzyme CD38 Is a Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthase That Couples Receptor Activation to Ca2+ Mobilization from Lysosomes in Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca²⁺ signals by discharging acidic Ca²⁺ stores and leading to digestive enzyme secretion. From cells derived from CD38^−/− mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca²⁺-signaling pathways in these cells by restoring Ca²⁺ mobilization from lysosomes during CCK-induced Ca²⁺ signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca²⁺ release from lysosomal stores in pancreatic acinar cells.     

Convergent regulation of the lysosomal two‐pore channel‐2 by Mg2+, NAADP,PI(3,5)P2 and multiple protein kinases

Lysosomal Ca²⁺ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca²⁺ signaling was the discovery of the two‐pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P₂ activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg²⁺ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg²⁺ specifically inhibited TPC2 outward current, whereas lysosomal Mg²⁺ partially inhibited both outward and inward currents in a lysosomal lumen pH‐dependent manner. Under controlled Mg²⁺, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P₂. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP‐mediated Ca²⁺ release in intact cells is regulated by Mg²⁺, PI(3,5)P₂, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP‐mediated Ca²⁺ signaling and link this pathway to Mg²⁺ homeostasis and MAP kinases, pointing to roles for lysosomal Ca²⁺ in cell growth, inflammation and cancer.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca²⁺-handling. We investigated the effects of ascending aortic banding (AoB) on Ca²⁺-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca²⁺-release at the cell edge whereas Ca²⁺ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na⁺/Ca²⁺ exchanger, SR Ca²⁺ ATPase or phospholamban. Mathematical modeling indicated that [Ca²⁺]_i transients at the cell center were accounted for by simple centripetal diffusion of Ca²⁺ released at the cell edge. In contrast, caffeine (10 mM) induced Ca²⁺ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca²⁺-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca²⁺ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca²⁺-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca²⁺-release.     

The Calcium-mobilizing Messenger Nicotinic Acid Adenine Dinucleotide Phosphate Participates in Sperm Activation by Mediating the Acrosome Reaction

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca²⁺ from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn²⁺ quenching of luminal fura-2 and ⁴⁵Ca²⁺ to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca²⁺. We propose a new model for acrosome reaction signaling in which Ca²⁺ influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca²⁺ to drive acrosomal exocytosis.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

Role of acidic stores in secretory epithelia

There is growing evidence that intracellular calcium plays a primary role in the pathophysiology of the pancreas in addition to its crucial importance in major physiological functions. Pancreatic acinar cells have a remarkably large amount of Ca²⁺ stored in both the endoplasmic reticulum (ER) and the acidic stores. The vast majority of the classical ER Ca²⁺ store is located in the basal part of the acinar cells with extensions protruding into the apical area, however, the acidic stores are exclusively located in the secretory granular area of the cells. Both types of Ca²⁺ store respond to all three intracellular Ca²⁺ messengers – inositol trisphosphate (InsP₃), cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The two stores interact with each other via calcium-induced calcium release; however, they can be separated using pharmacological tools. The ER relies on sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) that can be blocked by the specific inhibitor thapsigargin. The acidic store requires a low pH that can be modified by blocking vacuolar H⁺-ATPase.