Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells

G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.     

Activation of alpha adrenergic and muscarinic receptors modifies early glucose suppression of cytoplasmic Ca2+ in pancreatic β-cells

Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca²⁺ ([Ca²⁺]_i) below baseline in pancreatic β-cells. The period of [Ca²⁺]_i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α_2A and muscarinic M₃ receptors affects the initial [Ca²⁺]_i response to increase of glucose from 3 to 20 mM in β-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca²⁺]_i during 90–120 s followed by rise due to opening of voltage-dependent Ca²⁺ channels. The period of [Ca²⁺]_i decrease was prolonged by activation of the α_2A adrenergic receptors (1 μM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M₃ receptors (0.1 μM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10–100 μM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca²⁺]_i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca²⁺]_i regulates the subsequent β-cell response to glucose.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca²⁺]_i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca²⁺]_i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca²⁺]_i transient in osteoblasts. Fluorescent dyes DiBAC₄(3) and fura-2 AM were respectively used to detect membrane potential and [Ca²⁺]_i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca²⁺]_i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca²⁺]_i transients. Replacing extracellular Na⁺ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca²⁺]_i transients. However, voltage-activated K⁺ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca²⁺ or blocking of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca²⁺]_i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca²⁺ mobilization wherein FSS activated SACs to promote Na⁺ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca²⁺ influx though L-VSCCs switched on [Ca²⁺]_i response in osteoblasts.     

Mechanism of dopamine D2 receptor-induced Ca2 + release in PC-12 cells

Intracellular Ca^2 + levels are tightly regulated in the neuronal system. The loss of Ca^2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca^2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca^2 + release followed by a delayed Ca^2 + release. The delayed Ca^2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca^2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca^2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca^2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca^2 + release, decreased ATP-evoked IP₃R-mediated Ca^2 + release, and impaired subsequent Ca^2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP₃R-mediated Ca^2 + release in PC-12 cells. In schizophrenia, Ca^2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca^2 + levels as well as dopamine signaling.     

Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Cholesterol enrichment of rabbit platelets enhances the Ca2 + entry pathway induced by platelet-derived secondary feedback agonists

AimsHypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca^2 + mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂).Main methodsIn vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca^2 + mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB₂ from platelets were measured by a luciferin–luciferase ATP assay system and a TXB₂ ELISA Kit, respectively.Key findingsCholesterol enrichment of rabbit platelets significantly enhanced Ca^2 + mobilization induced by thrombin, accompanying an augmented Ca^2 + entry. The augmentation of Ca^2 + entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB₂ induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca^2 +]_i induced by ADP or U46619, a TXA₂ mimetic, was significantly enhanced.SignificanceThese results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca^2 + mobilization via ADP/TXA₂-dependent augmentation of the Ca^2 + entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.     

Pulsed electromagnetic field enhances brain-derived neurotrophic factor expression through L-type voltage-gated calcium channel- and Erk-dependent signaling pathways in neonatal rat dorsal root ganglion neurons

Although pulsed electromagnetic field (PEMF) exposure has been reported to promote neuronal differentiation, the mechanism is still unclear. Here, we aimed to examine the effects of PEMF exposure on brain-derived neurotrophic factor (Bdnf) mRNA expression and the correlation between the intracellular free calcium concentration ([Ca²⁺]_i) and Bdnf mRNA expression in cultured dorsal root ganglion neurons (DRGNs). Exposure to 50 Hz and 1 mT PEMF for 2 h increased the level of [Ca²⁺]_i and Bdnf mRNA expression, which was found to be mediated by increased [Ca²⁺]_i from Ca²⁺ influx through L-type voltage-gated calcium channels (VGCCs). However, calcium mobilization was not involved in the increased [Ca²⁺]_i and BDNF expression, indicating that calcium influx was one of the key factors responding to PEMF exposure. Moreover, PD098059, an extracellular signal-regulated kinase (Erk) inhibitor, strongly inhibited PEMF-dependant Erk1/2 activation and BDNF expression, indicating that Erk activation is required for PEMF-induced upregulation of BDNF expression. These findings indicated that PEMF exposure increased BDNF expression in DRGNs by activating Ca²⁺- and Erk-dependent signaling pathways.     

Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2+ signaling in HEK293 cells

Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca^2 + ([Ca^2 +]_i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca^2 +]_i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3 μM AA did not impact the basal [Ca^2 +]_i but significantly suppressed the thapsigargin-induced Ca^2 + release and Ca^2 + influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3 μM AA on PM fluidity and agonist-induced Ca^2 + signal. However, AA at high concentration, 30 μM, caused robust release and entry of Ca^2 + accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 μM AA-induced Ca^2 + influx by 45%, Gd^3 + at concentration of 10 μM could completely abolish both release and entry of Ca^2 + induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca^2 + signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca^2 +]_i response, while Gd^3 +-sensitive ‘pores’ in PM/ER play an important role in AA-induced Ca^2 + signal in HEK293 cells.     

Receptor-mediated Ca2 + and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin

A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300 kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca^2 +]_i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin–EGFP revealed that Ca^2 + elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60 s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca^2 +]_i elevation and PKC activation. To understand the mechanism for Ca^2 + mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670–694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin–EYFP and PKA RII–ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca^2 + and PKC.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca²⁺]_i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm² for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity^TM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca²⁺]_i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca²⁺]_i compared to statically cultured cells. The maximum increases of PI uptake and [Ca²⁺]_i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca²⁺]_i waves in shear cultured HUVECs was reduced compared to the statically cultured cells.     

Involvement of plasma membrane Ca2+ channels,IP3 receptors,and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct

In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29 ± 0.009 Hz (n = 43) and an amplitude of 14.6 ± 1.25 mg (n = 29). SRCs completely disappeared following removal of extracellular Ca²⁺ using a solution containing 5 mM EGTA. Application of the non-specific Ca²⁺ channel blockers Co²⁺, Ni²⁺, and Cd²⁺ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca²⁺ entry through plasma membrane Ca²⁺ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca²⁺ by locking ryanodine receptor (RyR)-Ca²⁺ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30–50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP₃R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca²⁺-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca²⁺ influx through plasma membrane Ca²⁺ channels and Ca²⁺ release from RyRs play an essential role in pacing SRCs and that Ca²⁺ release from IP₃Rs may play a role in modulating the amplitude of SRCs, probably via activation of PLC.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.     

BAPTA-AM,an intracellular calcium chelator,inhibits RANKL-induced bone marrow macrophages differentiation through MEK/ERK,p38 MAPK and Akt,but not JNK pathways

To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca²⁺ concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca²⁺]_i was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1 h after RANKL-inducing, and the increased response of [Ca²⁺]_i was completely abolished and sustained to at least 8 h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca²⁺]_i which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

F281, synthetic agonist of the sigma-2 receptor,induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.     

Increase of intracellular Ca2+ by adenine and uracil nucleotides in human midbrain-derived neuronal progenitor cells

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca²⁺]_i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca²⁺-free external medium moderately decreased, whereas a depletion of the intracellular Ca²⁺ storage sites by cyclopiazonic acid markedly depressed the [Ca²⁺]_i transients induced by either ATP or UTP. Further, the P2Y₁ receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y_1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y_1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y₂ receptor-activation and thereby causes [Ca²⁺]_i transients by stimulating a P2Y₁-like receptor.