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1.
J Biol Chem ; 285(49): 38251-9, 2010 Dec 03.
Article in English | MEDLINE | ID: mdl-20870729

ABSTRACT

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.


Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Lysosomes/enzymology , Membrane Glycoproteins/metabolism , Nucleotidyltransferases/metabolism , Pancreas, Exocrine/enzymology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1/genetics , Animals , Calcium Signaling/drug effects , Cell Line , Cholagogues and Choleretics/pharmacology , Cholecystokinin/pharmacology , Lysosomes/genetics , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , NADP/analogs & derivatives , NADP/biosynthesis , NADP/genetics , Nucleotidyltransferases/genetics , Rats
2.
PLoS One ; 4(8): e6770, 2009 Aug 26.
Article in English | MEDLINE | ID: mdl-19707551

ABSTRACT

BACKGROUND: Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS: SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE: These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE.


Subject(s)
Hippocampus/physiopathology , Kv1.3 Potassium Channel/physiology , Microglia/physiology , Status Epilepticus/physiopathology , Animals , Mice , Mice, Inbred C57BL
3.
Curr Biol ; 16(19): 1931-7, 2006 Oct 10.
Article in English | MEDLINE | ID: mdl-17027490

ABSTRACT

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.


Subject(s)
Calcium Signaling , Calcium/metabolism , Endocytosis/physiology , Acetylcholine/physiology , Animals , Cells, Cultured , Cholecystokinin/physiology , Cyclic ADP-Ribose/physiology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/physiology , Inositol 1,4,5-Trisphosphate/physiology , Lysosomes/metabolism , Lysosomes/physiology , NADP/analogs & derivatives , NADP/physiology , Secretory Vesicles/metabolism , Secretory Vesicles/physiology
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