We investigated Ca2+ handling in isolated brain synaptic and non‐synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non‐synaptic mitochondria from 2‐ and 12‐month‐old YAC128 mice had larger Ca2+ uptake capacity than mitochondria from YAC18 and wild‐type FVB/NJ mice. Synaptic mitochondria from 12‐month‐old YAC128 mice had further augmented Ca2+ capacity compared with mitochondria from 2‐month‐old YAC128 mice and age‐matched YAC18 and FVB/NJ mice. This increase in Ca2+ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12‐month‐old YAC128 mice. We speculate that this may happen because of mHtt‐mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca2+‐induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca2+ followed by recovery to near resting levels. Following recovery of cytosolic Ca2+, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca2+, suggesting similar Ca2+ release and, consequently, Ca2+ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca2+ handling in brain mitochondria of YAC128 mice.
Triptans, 5-HT1B/ID agonists, act on peripheral and/or central terminals of trigeminal ganglion neurons (TGNs) and inhibit the release of neurotransmitters to second-order neurons, which is considered as one of key mechanisms for pain relief by triptans as antimigraine drugs. Although high-voltage activated (HVA) Ca2+ channels contribute to the release of neurotransmitters from TGNs, electrical actions of triptans on the HVA Ca2+ channels are not yet documented.
Results
In the present study, actions of zolmitriptan, one of triptans, were examined on the HVA Ca2+ channels in acutely dissociated rat TGNs, by using whole-cell patch recording of Ba2+ currents (IBa) passing through Ca2+ channels. Zolmitriptan (0.1–100 μM) reduced the size of IBa in a concentration-dependent manner. This zolmitriptan-induced inhibitory action was blocked by GR127935, a 5-HT1B/1D antagonist, and by overnight pretreatment with pertussis toxin (PTX). P/Q-type Ca2+ channel blockers inhibited the inhibitory action of zolmitriptan on IBa, compared to N- and L-type blockers, and R-type blocker did, compared to L-type blocker, respectively (p < 0.05). All of the present results indicated that zolmitriptan inhibited HVA P/Q- and possibly R-type channels by activating the 5-HT1B/1D receptor linked to Gi/o pathway.
Conclusion
It is concluded that this zolmitriptan inhibition of HVA Ca2+ channels may explain the reduction in the release of neurotransmitters including CGRP, possibly leading to antimigraine effects of zolmitriptan. 相似文献
The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+]i) in the target cells, which activates the Ca2+/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+]i and NO production. The current study assessed whether and how glutamate drives Ca2+-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+]i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain. 相似文献
Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.
Methods
Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted.
Results
Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.
Conclusions
Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential. 相似文献
Huntington's disease (HD) is caused by an expansion of CAG repeats in the HTT gene, leading to expression of mutant huntingtin (mHTT) and selective striatal neuronal loss, frequently associated with mitochondrial dysfunction and decreased support of brain-derived neurotrophic factor (BDNF). New neurons derived from the subventricular zone (SVZ) are apparently not able to rescue HD pathological features. Thus, we analyzed proliferation, migration and differentiation of adult SVZ-derived neural stem/progenitor cells (NSPC) from mild (6 month-old (mo)) and late (10 mo) symptomatic HD YAC128 mice expressing full-length (FL)-mHTT versus age-matched wild-type (WT) mice. SVZ cells derived from 6 mo YAC128 mice exhibited higher migratory capacity and a higher number of MAP2 + and synaptophysin + cells, compared to WT cells; MAP2 labeling was enhanced after exposure to BDNF. However, BDNF-evoked neuronal differentiation was not observed in 10 mo YAC128 SVZ-derived cells. Interestingly, 6 mo YAC128 SVZ-derived cells showed increased intracellular Ca2+ levels in response to KCl, which was potentiated by BDNF, evidencing the presence of differentiated neurons. In contrast, KCl depolarization-induced intracellular Ca2+ increase in 10 mo YAC128 SVZ-derived cells was shown to be increased only in BDNF-treated YAC128 SVZ-derived cells, suggestive of decreased differentiation capacity. In addition, BDNF-untreated NSPC from 10 mo YAC128 mice exhibited lower mitochondrial membrane potential and increased mitochondrial Ca2+ accumulation, in relation with NSPC from 6 mo YAC128 mice. Data evidence age-dependent reduced migration and decreased acquisition of a neuronal phenotype, accompanied by decreased mitochondrial membrane potential in SVZ-derived cells from YAC128 mice through HD symptomatic phases. 相似文献
Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Mg2+ and Ca2+. Pertussis toxin treatment abolished this glutamate-induced [Ca2+]i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg2+, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca2+]i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg2+ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine2 (5-HT2) receptor signaling. EAAs inhibited 5-HT2 receptor-mediated intracellular Ca2+ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca2+. In addition, this inhibitory effect on 5-HT2 receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT2 receptor-mediated [Ca2+]i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca2+ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C. 相似文献
The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca2+ concentration ([Ca2+]i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca2+]i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT2 receptor that is different from the 5-HT2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT2A and 5-HT2B receptors, but not for the 5-HT2C receptor, identifying the 5-HT2 receptor activated by fluoxetine as the 5-HT2B receptor, the most recently cloned 5-HT2 receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [3H]mesulergine, a ligand for all three 5-HT2 receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT2B-mediated effects are initially down-regulated and subsequently up-regulated. 相似文献
Summary Human 5-HT3 receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na+ were Li+, 1.16; K+, 1.04; Rb+, 1.11; Cs+ 1.11; NMDG+, 0.04; Ca2+, 0.49, and Mg2+, 0.37. The permeability sequence of the alkali metal cations was Li+ > Rb+ = Cs+ > K+ > Na+. Increased external concentrations of Ca2+ or Mg2+ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT3 receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT3 receptors between species may explain interspecies differences in pharmacological properties. 相似文献
Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na+-oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca2+-mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion. 相似文献
Huntington’s disease (HD) is an autosomal neurodegenerative disease. Its manifestations is selective degeneration of medium-sized spiny neurons (MSN) in the striatum. The specificity of the vulnerability of these GABAergic MSNs can be explained by abnormal protein accumulation, excitotoxicity, mitochondrial dysfunction, and failure of trophic control, among other dysfunctions. In this study, we used in vitro and in vivo models of HD to study the effects of GABAergic neuron stimulation on the cellular protein degradation machinery. We administered the GABAB receptor agonist, baclofen, to wild-type or mutant huntingtin-expressing striatal cells (HD19 or HD43). Chymotrypsin-like proteasome activity and cell viability were significantly increased in the mutant huntingtin-expressing striatal cells (HD43) after GABAB receptor agonist treatment. In addition, we systemically administered baclofen to a HD model containing the entire human huntingtin gene with 128 CAG repeats (YAC128). Chymotrypsin-like proteasome activity was significantly increased in YAC128 transgenic mice after baclofen administration. Baclofen-injected mutant YAC128 mice also showed significantly reduced numbers of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum. Baclofen markedly improved behavioral abnormalities in mutant YAC128 mice as determined by the rotarod performance test. These data indicate that stimulation of GABAergic neurons with the GABAB receptor agonist, baclofen, enhances ubiquitin-proteasome system (UPS) function and cell survival in in vitro and in vivo models of HD. 相似文献
Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis. 相似文献
AbstractThe interaction of SCH 23390 with dopamine (DA) and serotonin (5-HT) systems has been examined in vivo and in vitro. Like selective 5-HT2 blockers, SCH 23390 inhibited in vivo [3H]spiperone binding in the rat frontal cortex (ID50: 1.5 mg/kg) without interacting at D2 sites. SCH 23390 was equipotent to cinanserin and methysergide. In vitro, SCH 23390 inhibited [3H]ketanserin binding to 5-HT2 sites (IC50 = 30 nM). Biochemical parameters linked to DA and 5-HT were not changed excepted in striatum where SCH 23390 increased HVA and DOPAC. In the L-5-HTP syndrome model, SCH 23390 clearly showed antagonism of 5-HT2 receptors. SCH 23390 had weak affinity for 5-HT1B (IC50 = 0.5 μM), 5-HT1A (IC50 = 2.6 μM) and α;1-adenergic receptors (IC50 = 4.4 μM). 相似文献
1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2C) and as cAMP and intracellular Ca2+ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [3H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca2+ changes were determined using the ratiometric method of intracellular Ca2+ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [3H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca2+ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca2+ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca2+ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca2+ levels. This suggests that 5-HT may utilize intracellular Ca2+ to regulate 5-HT uptake. 相似文献
Abstract: The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-d -glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 µM Co2+/Cd2+ or by 1 µMω-conotoxin MVIIC/1 µMω-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 µM mCPBG was substantially reduced by 10 µM Co2+/Cd2+ and was completely blocked by 1 µM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant. 相似文献
It is shown that agmatine inhibits L-type Ca2+ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I1Rs) rather than α2-adrenoceptors (α2-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca2+ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca2+ concentration ([Ca2+]in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca2+]in; at 500 μM-2 mM, [Ca2+]in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca2+]in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I1Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca2+]in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I1Rs and ryanodine receptors (RyRs), respectively, [Ca2+]in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I1R agonist rilmenidine increased [Ca2+]in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca2+]in, does not stimulate I1Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I1Rs and involve RyRs. 相似文献
Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y2 receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y2R-PLC-IP3 pathway to evoke Ca2+ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca2+]I; this effect was partly inhibited by P2Y2R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca2+]i was significantly lower than that after treatment with P2Y2R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca2+ triggers Ca2+ release from ER Ca2+ stores. Baseline [Ca2+]i declined after treatment with the Ca2+ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca2+ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca2+ triggers Ca2+ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca2+ signals indirectly via the P2Y2R-PLC-IP3 pathway. 相似文献
The role of trans-sarcolemma membrane electron efflux in the α-adrenergic control of Ca2+ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca2+ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 μM for each agonist. Activation of the rate of ferricyanide reduction by both 10 μM methoxamine and 10 μM phenylephrine was blocked by the α-adrenergic antagonist, phenoxybenzamine, but not by the β-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the α-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the α-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca2+ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl2 significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca2+ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by α-adrenergic agonists resulted from a change in the apparent Vmax, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that α-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca2+. The data suggest that α-agonist-mediated changes in electron efflux may lead to Ca2+ influx. 相似文献
Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin. 相似文献