Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine.

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i 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 (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.

Assessment of Blood-Brain Barrier Permeability Using Miniaturized Fluorescence Microscopy in Freely Moving Rats.

We report here the method of visualization of brain microcirculation and assessment of blood-brain barrier (BBB) permeability changes using the miniature integrated fluorescence microscope (i.e., miniscope) technology in awake, freely moving rats. The imaging cannula is implanted in the brain area of interest of anesthetized adult rats. After recovery and habituation, sodium fluorescein, a low-molecular-weight tracer, is injected i.v. Fluorescence intensity in the vicinity of microvessels, as an indicator of BBB permeability, is then recorded in vivo via the miniscope for extended periods of time. The method can be used to assess the changes in BBB permeability produced by pharmacologic agents; in this case, the drug of interest is administered after sodium fluorescein. An increase in the sodium fluorescein extravasation in brain microcirculation demonstrates an increase in BBB permeability. The method described here allows a high-resolution visualization of real-time changes in BBB permeability in awake, freely moving rats.

1-Chromonyl-5-Imidazolylpentadienone Demonstrates Anti-Cancer Action against TNBC and Exhibits Synergism with Paclitaxel.

Curcumin has been well studied for its anti-oxidant, anti-inflammatory, and anti-cancer action. Its potential as a therapy is limited due to its low bioavailability and rapid metabolism. To overcome these challenges, investigators are developing curcumin analogs, nanoparticle formulations, and combining curcumin with other compounds or dietary components. In the present study, we used a 1-chromonyl-5-imidazolylpentadienone named KY-20-22 that contains both the pharmacophore of curcumin and 1,4 benzopyrone (chromone) moiety typical for flavonoids, and also included specific moieties to enhance the bioavailability. When we tested the in vitro effect of KY-20-22 in triple-negative breast cancer (TNBC) cell lines, we found that it decreased the cell survival and colony formation of MDA-MB-231 and MDA-MB-468 cells. An increase in mitochondrial reactive oxygen species was also observed in TNBC cells exposed to KY-20-22. Furthermore, KY-20-22 decreased epithelial-mesenchymal formation (EMT) as evidenced by the modulation of the EMT markers E-cadherin and N-cadherin. Based on the fact that KY-20-22 regulates interleukin-6, a cytokine involved in chemotherapy resistance, we combined it with paclitaxel and found that it synergistically induced anti-proliferative action in TNBC cells. The results from this study suggested that 1-chromonyl-5-imidazolylpentadienone KY-20-22 exhibited anti-cancer action in MDA-MB-231 and MDA-MB-468 cells. Future studies are required to evaluate the anti-cancer ability and bioavailability of KY-20-22 in the TNBC animal model.

Direct evidence of bradycardic effect of omega-3 fatty acids acting on nucleus ambiguus.

Docosahexaenoic acid (DHA) an omega-3 polyunsaturated fatty acid, is an agonist of FFA1 receptor. DHA administration reduces the heart rate via unclear mechanisms. We examined the effect of DHA on neurons of nucleus ambiguus that provide the parasympathetic control of heart rate. DHA produced a dose-dependent increase in cytosolic Ca2+ concentration in cardiac-projecting nucleus ambiguus neurons; the effect was prevented by GW1100, a FFA1 receptor antagonist. DHA depolarized cultured nucleus ambiguus neurons via FFA1 activation. Bilateral microinjection of DHA into nucleus ambiguus produced bradycardia in conscious rats. Our results indicate that DHA decreases heart rate by activation of FFA1 receptor on cardiac-projecting nucleus ambiguus neurons.

Acute cocaine administration alters permeability of blood-brain barrier in freely-moving rats- Evidence using miniaturized fluorescence microscopy.

BACKGROUND: Cocaine has a variety of negative effects on the central nervous system, including reports of decreased barrier function of brain microvascular endothelial cells. However, few studies have directly shown the effects of cocaine on blood-brain barrier (BBB) function in vivo. The miniature integrated fluorescence microscope (i.e., miniscope) technology was used to visualize cocaine-induced changes in BBB permeability in awake, freely-moving rats. METHODS: The miniscope was implanted in the prefrontal cortex of adult male rats. After recovery and acclimation, rats received an injection of cocaine (5-20 mg/kg ip) 15 minutes following iv infusion of sodium fluorescein, a low molecular weight tracer. Fluorescence intensity was recordedin vivo via the miniscope for 30 minutes or 24 hours post cocaine administration and served as an indicator of BBB permeability. RESULTS: Results demonstrate that cocaine increased the sodium fluorescein extravasation in brain microcirculation in a dose-dependent manner 30 minutes, but not 24 hours after administration. CONCLUSION: We report for the first time using direct visualization of brain microcirculation with the miniscope technology in awake, freely-moving rats, that acute cocaine administration produced a transient increase in the BBB permeability.

GPR55-mediated effects on brain microvascular endothelial cells and the blood-brain barrier.

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.

Choline Is an Intracellular Messenger Linking Extracellular Stimuli to IP3-Evoked Ca2+ Signals through Sigma-1 Receptors.

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.

Effects of Platelet-Activating Factor on Brain Microvascular Endothelial Cells.

Platelet-activating factor (PAF) is a potent phospholipid mediator that exerts various pathophysiological effects by interacting with a G protein-coupled receptor. PAF has been reported to increase the permeability of the blood-brain barrier (BBB) via incompletely characterized mechanisms. We investigated the effect of PAF on rat brain microvascular endothelial cells (RBMVEC), a critical component of the BBB. PAF produced a dose-dependent increase in cytosolic Ca2+ concentration; the effect was prevented by the PAF receptor antagonist, WEB2086. The effect of PAF on cytosolic Ca2+ was abolished in Ca2+-free saline or in the presence of L-type voltage-gated Ca2+ channel inhibitor, nifedipine, indicating that Ca2+ influx is critical for PAF-induced increase in cytosolic Ca2+. PAF produced RBMVEC depolarization; the effect was inhibited by WEB2086. In cells loaded with [(4-amino-5-methylamino-2',7'-difluoro-fluorescein)diacetate] (DAF-FM), a nitric oxide (NO)-sensitive fluorescent dye, PAF increased the NO level; the effect was prevented by WEB2086, nifedipine or by l-NAME, an inhibitor of NO synthase. Immunocytochemistry studies indicate that PAF reduced the immunostaining of ZO-1, a tight junction-associated protein, increased F-actin fibers, and produced intercellular gaps. PAF produced a decrease in RBMVEC monolayer electrical resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS), indicative of a disruption of endothelial barrier function. In vivo studies indicate that PAF increased the BBB permeability, assessed with sodium fluorescein and Evans Blue methods, via PAF receptor-dependent mechanisms, consequent to Ca2+ influx and increased NO levels. Our studies reveal that PAF alters the BBB permeability by multiple mechanisms, which may be relevant for central nervous system (CNS) inflammatory disorders.

Modulation of cardiac vagal tone by bradykinin acting on nucleus ambiguus.

Bradykinin (BK), a component of the kallikrein-kininogen-kinin system exerts multiple effects via B1 and B2 receptor activation. In the cardiovascular system, bradykinin has cardioprotective and vasodilator properties. We investigated the effect of BK on cardiac-projecting neurons of nucleus ambiguus, a key site for the parasympathetic cardiac regulation. BK produced a dose-dependent increase in cytosolic Ca2+ concentration. Pretreatment with HOE140, a B2 receptor antagonist, but not with R715, a B1 receptor antagonist, abolished the response to BK. A selective B2 receptor agonist, but not a B1 receptor agonist, elicited an increase in cytosolic Ca2+ similarly to BK. Inhibition of N-type voltage-gated Ca2+ channels with ω-conotoxin GVIA had no effect on the Ca2+ signal produced by BK, while pretreatment with ω-conotoxin MVIIC, a blocker of P/Q-type of Ca2+ channels, significantly diminished the effect of BK. Pretreatment with xestospongin C and 2-aminoethoxydiphenyl borate, antagonists of inositol 1,4,5-trisphosphate receptors, abolished the response to BK. Inhibition of ryanodine receptors reduced the BK-induced Ca2+ increase, while disruption of lysosomal Ca2+ stores with bafilomycin A1 did not affect the response. BK produced a dose-dependent depolarization of nucleus ambiguus neurons, which was prevented by the B2 receptor antagonist. In vivo studies indicate that microinjection of BK into nucleus ambiguus elicited bradycardia in conscious rats via B2 receptors. In summary, in cardiac vagal neurons of nucleus ambiguus, BK activates B2 receptors promoting Ca2+ influx and Ca2+ release from endoplasmic reticulum, and membrane depolarization; these effects are translated in vivo by bradycardia.

HIV Tat excites D1 receptor-like expressing neurons from rat nucleus accumbens.

BACKGROUND: HIV-1 infection and drug abuse are frequently co-morbid and their association greatly increases the severity of HIV-1-induced neuropathology. While nucleus accumbens (NAcc) function is severely perturbed by drugs of abuse, little is known about how HIV-1 infection affects NAcc. METHODS: We used calcium and voltage imaging to investigate the effect of HIV-1 trans-activator of transcription (Tat) on rat NAcc. Based on previous neuronal studies, we hypothesized that Tat modulates intracellular Ca2+ homeostasis of NAcc neurons. RESULTS: We provide evidence that Tat triggers a Ca2+ signaling cascade in NAcc medium spiny neurons (MSN) expressing D1-like dopamine receptors leading to neuronal depolarization. Firstly, Tat induced inositol 1,4,5-trisphsophate (IP3) receptor-mediated Ca2+ release from endoplasmic reticulum, followed by Ca2+ and Na+ influx via transient receptor potential canonical channels. The influx of cations depolarizes the membrane promoting additional Ca2+ entry through voltage-gated P/Q-type Ca2+ channels and opening of tetrodotoxin-sensitive Na+ channels. By activating this mechanism, Tat elicits a feed-forward depolarization increasing the excitability of D1-phosphatidylinositol-linked NAcc MSN. We previously found that cocaine targets NAcc neurons directly (independent of the inhibition of dopamine transporter) only when IP3-generating mechanisms are concomitantly initiated. When tested here, cocaine produced a dose-dependent potentiation of the effect of Tat on cytosolic Ca2+. CONCLUSION: We describe for the first time a HIV-1 Tat-triggered Ca2+ signaling in MSN of NAcc involving TRPC and depolarization and a potentiation of the effect of Tat by cocaine, which may be relevant for the reward axis in cocaine-abusing HIV-1-positive patients.

Effects of VPAC1 activation in nucleus ambiguus neurons.

The pituitary adenylyl cyclase-activating polypeptide (PACAP) and its G protein-coupled receptors, PAC1, VPAC1 and VPAC2 form a system involved in a variety of biological processes. Although some sympathetic stimulatory effects of this system have been reported, its central cardiovascular regulatory properties are poorly characterized. VPAC1 receptors are expressed in the nucleus ambiguus (nAmb), a key center controlling cardiac parasympathetic tone. In this study, we report that selective VPAC1 activation in rhodamine-labeled cardiac vagal preganglionic neurons of the rat nAmb produces inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization, membrane depolarization and activation of P/Q-type Ca2+ channels. In vivo, this pathway converges onto transient reduction in heart rate of conscious rats. Therefore we demonstrate a VPAC1-dependent mechanism in the central parasympathetic regulation of the heart rate, adding to the complexity of PACAP-mediated cardiovascular modulation.

Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca2+ concentration in RBMVEC via Ca2+ release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca2+ influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

Modulation of Calcium Entry by the Endo-lysosomal System.

Endo-lysosomes are acidic organelles that besides the role in macromolecules degradation, act as intracellular Ca(2+) stores. Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+)-mobilizing second messenger, produced in response to agonist stimulation, activates Ca(2+)-releasing channels on endo-lysosomes and modulates a variety of cellular functions. NAADP-evoked signals are amplified by Ca(2+) release from endoplasmic reticulum, via the recruitment of inositol 1,4,5-trisphosphate and/or ryanodine receptors through a Ca(2+)-induced Ca(2+)- release (CICR) mechanism. The endo-lysosomal Ca(2+) channels activated by NAADP were recently identified as the two-pore channels (TPCs). In addition to TPCs, endo-lysosomes express another distinct family of Ca(2+)- permeable channels, namely the transient receptor potential mucolipin (TRPML) channels, functionally distinct from TPCs. TPCs belong to the voltage-gated channels, resembling voltage-gated Na(+) and Ca(2+) channels. TPCs have important roles in vesicular fusion and trafficking, in triggering a global Ca(2+) signal and in modulation of the membrane excitability. Depletion of acidic Ca(2+) stores has been shown to activate store-operated Ca(2+) entry in human platelets and mouse pancreatic ß-cells. In human platelets, Ca(2+) influx in response to acidic stores depletion is facilitated by the tubulin-cytoskeleton and occurs through non-selective cation channels and transient receptor potential canonical (TRPC) channels. Emerging evidence indicates that activation of intracellular receptors, situated on endo-lysosomes, elicits canonical and non-canonical signaling mechanisms that involve CICR and activation of non-selective cation channels in plasma membrane. The ability of endo-lysosomal Ca(2+) stores to modulate the Ca(2+) release from other organelles and the Ca(2+) entry increases the diversity and complexity of cellular signaling mechanisms.

Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV.

Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor-inositol 1,4,5-trisphosphate-transient receptor potential canonical channel pathways.

Cocaine promotes addictive behavior primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens (nAcc); however, additional mechanisms are continually emerging. Sigma-1 receptors (σ1Rs) are known targets for cocaine, yet the mechanisms underlying σ1R-mediated effects of cocaine are incompletely understood. The present study examined direct effects of cocaine on dissociated nAcc neurons expressing phosphatidylinositol-linked D1 receptors. Endoplasmic reticulum-located σ1Rs and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) were targeted using intracellular microinjection. IP3 microinjection robustly elevated intracellular Ca(2+) concentration, [Ca(2+)]i. While cocaine alone was devoid of an effect, the IP3-induced response was σ1R-dependently enhanced by cocaine co-injection. Likewise, cocaine augmented the [Ca(2+)]i increase elicited by extracellularly applying an IP3-generating molecule (ATP), via σ1Rs. The cocaine-induced enhancement of the IP3/ATP-mediated Ca(2+) elevation occurred at pharmacologically relevant concentrations and was mediated by transient receptor potential canonical channels (TRPC). IP3 microinjection elicited a slight, transient depolarization, further converted to a greatly enhanced, prolonged response, by cocaine co-injection. The cocaine-triggered augmentation was σ1R-dependent, TRPC-mediated and contingent on [Ca(2+)]i elevation. ATP-induced depolarization was similarly enhanced by cocaine. Thus, we identify a novel mechanism by which cocaine promotes activation of D1-expressing nAcc neurons: enhancement of IP3R-mediated responses via σ1R activation at the endoplasmic reticulum, resulting in augmented Ca(2+) release and amplified depolarization due to subsequent stimulation of TRPC. In vivo, intra-accumbal blockade of σ1R or TRPC significantly diminished cocaine-induced hyperlocomotion and locomotor sensitization, endorsing a physio-pathological significance of the pathway identified in vitro.

Irisin evokes bradycardia by activating cardiac-projecting neurons of nucleus ambiguus.

Irisin is a newly identified hormone induced in muscle and adipose tissues by physical activity. This protein and its encoding gene have been identified in the brain; in addition, the precursor for irisin, FNDC5, can cross the blood-brain barrier. The fact that irisin is secreted during exercise together with the lower resting heart rate in athletes prompted us to investigate the effect of irisin on cardiac-projecting vagal neurons of nucleus ambiguus, a key regulatory site of heart rate. In vitro experiments in cultured nucleus ambiguus neurons indicate that irisin activates these neurons, inducing an increase in cytosolic Ca(2+) concentration and neuronal depolarization. In vivo microinjection of irisin into the nucleus ambiguus promotes bradycardia in conscious rats. Our study is the first to report the effects of irisin on the neurons controlling the cardiac vagal tone and to link a myokine to a cardioprotective role, by modulating central cardiovascular regulation.

The Lysophosphatidylinositol Receptor GPR55 Modulates Pain Perception in the Periaqueductal Gray.

Emerging evidence indicates the involvement of GPR55 and its proposed endogenous ligand, lysophosphatidylinositol (LPI), in nociception, yet their role in central pain processing has not been explored. Using Ca(2+) imaging, we show here that LPI elicits concentration-dependent and GPR55-mediated increases in intracellular Ca(2+) levels in dissociated rat periaqueductal gray (PAG) neurons, which express GPR55 mRNA. This effect is mediated by Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptors and by Ca(2+) entry via P/Q-type of voltage-gated Ca(2+) channels. Moreover, LPI depolarizes PAG neurons and upon intra-PAG microinjection, reduces nociceptive threshold in the hot-plate test. Both these effects are dependent on GPR55 activation, because they are abolished by pretreatment with ML-193 [N-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)-phenyl)-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide], a selective GPR55 antagonist. Thus, we provide the first pharmacological evidence that GPR55 activation at central levels is pronociceptive, suggesting that interfering with GPR55 signaling in the PAG may promote analgesia.

G protein-coupled estrogen receptor-mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells.

G protein-coupled estrogen receptor (GPER) is a relatively recently identified non-nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca(2+) concentration, [Ca(2+)](i), nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G-1, a selective GPER agonist, or extracellular administration of 17-ß-estradiol and tamoxifen, increased [Ca(2+)](i) in RBMVEC. The effect of G-1 on [Ca(2+)](i) was abolished in Ca(2+) -free saline or in the presence of a L-type Ca(2+) channel blocker. G-1 increased nitric oxide production in RBMVEC; the effect was prevented by NG-nitro-l-arginine methyl ester. G-1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate-conductance Ca(2+) -activated K(+) channels, apamin, and charibdotoxin. GPER-mediated responses were sensitive to G-36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G-1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature. Activation of the G protein-coupled estrogen receptor (GPER) in rat brain microvascular endothelial cells (RBMVEC) increases [Ca(2+)](i) by promoting Ca(2+) influx. The increase in [Ca(2+)](i) leads to membrane hyperpolarization, nitric oxide (NO) production, and to cytoskeletal changes and increased cell stiffness. Our results unravel the mechanisms underlying GPER-mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease.