Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, ...the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.
Full text
Available for:
BFBNIB, NUK, PNG, UL, UM, UPUK
The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against ...the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations Ca2+ i in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic–pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.
Sigma-1 receptors (Sig-1Rs) are integral ER membrane proteins. They bind diverse ligands, including psychoactive drugs, and regulate many signaling proteins, including the inositol ...1,4,5-trisphosphate receptors (IP3Rs) that release Ca2+ from the ER. The endogenous ligands of Sig-1Rs are unknown. Phospholipase D (PLD) cleaves phosphatidylcholine to choline and phosphatidic acid (PA), with PA assumed to mediate all downstream signaling. We show that choline is also an intracellular messenger. Choline binds to Sig-1Rs, it mimics other Sig-1R agonists by potentiating Ca2+ signals evoked by IP3Rs, and it is deactivated by metabolism. Receptors, by stimulating PLC and PLD, deliver two signals to IP3Rs: IP3 activates IP3Rs, and choline potentiates their activity through Sig-1Rs. Choline is also produced at synapses by degradation of acetylcholine. Choline uptake by transporters activates Sig-1Rs and potentiates Ca2+ signals. We conclude that choline is an endogenous agonist of Sig-1Rs linking extracellular stimuli, and perhaps synaptic activity, to Ca2+ signals.
Display omitted
•Choline, but not its metabolites, binds to Sigma-1 receptors•Choline potentiates IP3-evoked Ca2+ release by activating Sigma-1 receptors•Bradykinin stimulates Ca2+ release by stimulating formation of IP3 and choline•Choline uptake by a specific transporter potentiates IP3-evoked Ca2+ release
Sigma-1 receptors respond to diverse stimuli and regulate many signaling proteins. Brailoiu et al. show that choline is an endogenous agonist of Sigma-1 receptors. Choline links receptors and cholinergic synaptic activity, through Sigma-1 receptors, to enhanced Ca2+ release through IP3 receptors.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX
and OX
, Gq-coupled GPCRs. We examined the effect of a selective OX
agonist, ...OXA (17-33) on cytosolic calcium concentration, Ca
, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased Ca
in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX
receptors antagonist. In Ca
-free saline, the OXA (17-33)-induced increase in Ca
was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP
) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the Ca
response elicited by OXA (17-33). Cocaine potentiated the increase in Ca
by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX
via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is ...abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca2+ release from acidic organelles via two-pore channels (TPCs). It has been difficult to ...resolve this trigger event from its amplification via endoplasmic reticulum Ca2+ stores, fuelling speculation that archetypal intracellular Ca2+ channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca2+ influx independent of ryanodine receptors and that it activates a Ca2+-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca2+ channel.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
GPR55, an atypical cannabinoid receptor activated by lysophosphatidylinositol (LPI) has been involved in various physiological and pathological processes. We examined the effect of GPR55 activation ...on rat brain microvascular endothelial cells (RBMVEC), an essential component of the blood–brain barrier (BBB). GPR55 was detected in RBMVEC by western blot and immunocytochemistry. Treatment of RBMVEC with LPI increased cytosolic Ca2+ concentration, Ca2+i, in a concentration-dependent manner; the effect was abolished by the GPR55 antagonist, ML-193. Repetitive application of LPI induced tachyphylaxis. LPI-induced increase in Ca2+i was not sensitive to U-73122, a phospholipase C inhibitor, but was abolished by the blockade of voltage-gated Ca2+ channels or in Ca2+-free saline, indicating that Ca2+ influx was involved in this response. LPI induced a biphasic change in RBMVEC membrane potential: a fast depolarization followed by a long-lasting hyperpolarization. The hyperpolarization phase was prevented by apamin and charibdotoxin, inhibitors of small- and intermediate-conductance Ca2+-activated K+ channels (KCa). Immunofluorescence studies indicate that LPI produced transient changes in tight and adherens junctions proteins and F-actin stress fibers. LPI decreased the electrical resistance of RBMVEC monolayer assessed with Electric Cell-Substrate Impedance Sensing (ECIS) in a dose-dependent manner. In vivo studies indicate that systemic administration of LPI increased the permeability of the BBB, assessed with Evans Blue method. Taken together, our results indicate that GPR55 activation modulates the function of endothelial cells of brain microvessels, produces a transient reduction in endothelial barrier function and increases BBB permeability.
•GPR55, atypical cannabinoid receptor activated by LPI, is expressed in rat brain microvascular endothelial cells (RBMVEC).•In RBMVC, LPI increased cytosolic Ca2+ and produced a fast depolarization followed by a long-lasting hyperpolarization.•LPI disrupted tight and adherens junctions proteins and altered RBMVEC barrier function assessed with ECIS.•In vivo studies, reveal for the first time that LPI via GPR55 activation increases BBB permeability.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We found that the selective stimulation of the intracellular, transmembrane G protein–coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in ...normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.
Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert ...their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood-brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP3, respectively. Here, we establish Ca2+ signals between the two sources as fundamental ...building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated.
Display omitted
•Lysosomal TPC2 channels evoke local and global Ca2+ signals involving the ER•ER-localized IP3 receptor channels locally couple to TPC2•TPC2 sensitizes IP3 receptors in an agonist-selective manner•Crosstalk regulates physiological Ca2+ signals and Ca2+-dependent function
Yuan et al. investigate organelle crosstalk in the genesis of cytosolic Ca2+ signals. They resolve a continuum of Ca2+ transients from local elementary events driven by lysosomal TPC2 channels, through Ca2+-coupled events driven by IP3 receptors on the neighboring endoplasmic reticulum, to functionally relevant global signals during physiological cell stimulation.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
PDF
