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  • Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells.

    8 December 2017

    How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle selection") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively select the ER Ca2+ store, whereas cholecystokinin additionally recruits a lysosome-related organelle. Similarly, in a pancreatic beta cell line MIN6, acetylcholine selects only the ER, whereas glucose mobilizes Ca2+ from a lysosome-related organelle. We also show that the key to organelle selection is the agonist-specific coupling messenger(s) such that the ER is selected by recruitment of inositol 1,4,5-trisphosphate (or cADP-ribose), whereas lysosome-related organelles are selected by NAADP.

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

    8 December 2017

    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.

  • Regulation of L-type calcium channel and delayed rectifier potassium channel activity by p21-activated kinase-1 in guinea pig sinoatrial node pacemaker cells.

    8 December 2017

    Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p(21)-activated kinase-1 (Pak1), a serine-threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca(2+) current (I(CaL)) and delayed rectifier potassium current (I(K)) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of I(CaL) and I(K); (3) inhibition of protein phosphatase 2A increases the enhancement of I(K) and I(CaL) by isoproterenol in Ad-Pak1-infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.

  • Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca2+ spiking in mouse pancreatic acinar cells.

    8 December 2017

    Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

  • Modulation of endoplasmic reticulum Ca2+ store filling by cyclic ADP-ribose promotes inositol trisphosphate (IP3)-evoked Ca2+ signals.

    12 December 2017

    In addition to its well established function in activating Ca(2+) 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 Ca(2+) into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca(2+) signals via changes in ER Ca(2+) store content, by imaging Ca(2+) liberation through inositol trisphosphate receptors (IP(3)R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca(2+)] was transiently elevated by applying voltage-clamp pulses to induce Ca(2+) influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca(2+) signals evoked by strong photorelease of IP(3), and increased numbers of local Ca(2+) puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca(2+) elevation alone, indicating that they did not arise through direct actions of cADPR or Ca(2+) on the IP(3)R, 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 Ca(2+) may modulate IP(3)R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.

  • 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.

    12 December 2017

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-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 Ca(2+) signals by discharging acidic Ca(2+) 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 Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) 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 Ca(2+) release from lysosomal stores in pancreatic acinar cells.

  • Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate.

    12 December 2017

    The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.

  • Three-dimensional imaging of direct-written photonic structures.

    5 December 2017

    Third-harmonic generation microscopy has been used to analyze the morphology of photonic structures created using the femtosecond laser direct-write technique. Three-dimensional waveguide arrays and waveguide-Bragg gratings written in fused-silica and doped phosphate glass were investigated. A sensorless adaptive-optical system was used to correct the optical aberrations occurring in the sample and microscope system, which had a lateral resolution of less than 500 nm. This nondestructive testing method creates volume reconstructions of photonic devices and reveals details invisible to other linear microscopy and index profilometry techniques.

  • Adaptive optics for structured illumination microscopy.

    8 December 2017

    We implement wave front sensor-less adaptive optics in a structured illumination microscope. We investigate how the image formation process in this type of microscope is affected by aberrations. It is found that aberrations can be classified into two groups, those that affect imaging of the illumination pattern and those that have no influence on this pattern. We derive a set of aberration modes ideally suited to this application and use these modes as the basis for an efficient aberration correction scheme. Each mode is corrected independently through the sequential optimisation of an image quality metric. Aberration corrected imaging is demonstrated using fixed fluorescent specimens. Images are further improved using differential aberration imaging for reduction of background fluorescence.