Alterations In Intracellular Calcium Concentration Research Articles

Antileukemic activity of novel adenosine derivatives

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

Inhibition of ATP-Induced Ca2+ Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca(2+) mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca(2+)]i). ATP, an algesic agent, caused [Ca(2+)]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM-1 microM for 5 min) inhibited ATP-induced [Ca(2+)]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca(2+) response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 microM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 microM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 microM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca(2+)]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca(2+)]i transients in cultured DRG neurons.

Alterations of intracellular calcium concentration and nitric oxide generation in pulmonary artery endothelium after subarachnoid hemorrhage of the rabbit

The present study was designed to investigate whether endothelial intracellular calcium concentration ([Ca 2+] i), endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) generation altered in association with impaired endothelium-dependent relaxation (EDR) in pulmonary artery (PA) specimens from experimental subarachnoid hemorrhage (SAH) rabbits. Injecting non-heparinized autologous arterial blood into cisterna magna induced the SAH. Simultaneous measurements of endothelial [Ca 2+] i and isometric tension of PA specimens were performed using fura 2. The subjects included normal control rabbits (group N), SAH rabbits with normal EDR (group A) and with impaired EDR (group B). When treated with 10 − 7 M acetylcholine (ACh), endothelial [Ca 2+] i was significantly lower in group B (74.1 ± 8.5 nM) than that in groups A (153.0 ± 28.0 nM, p < 0.05) and N (184.8 ± 27.8 nM, p < 0.01). Basal and ACh-stimulated cyclic GMP productions as a marker of NO generation were also significantly ( p < 0.005) decreased in group B as compared to those in the other two groups. Meanwhile, there were no differences in eNOS activity per se among the three groups. These results suggest that the attenuated endothelial [Ca 2+] i elevation leads to the impaired NO generation in PA endothelium, which in turn impairs the EDR and possibly increases the vascular resistance of PA following SAH.

Evidence that injury-induced changes in hippocampal neuronal calcium dynamics during epileptogenesis cause acquired epilepsy.

Alterations in hippocampal neuronal Ca(2+) and Ca(2+)-dependent systems have been implicated in mediating some of the long-term neuroplasticity changes associated with acquired epilepsy (AE). However, there are no studies in an animal model of AE that directly evaluate alterations in intracellular calcium concentration ([Ca(2+)](i)) and Ca(2+) homeostatic mechanisms (Ca(2+) dynamics) during the development of AE. In this study, Ca(2+) dynamics were evaluated in acutely isolated rat CA1 hippocampal, frontal, and occipital neurons in the pilocarpine model by using [Ca(2+)](i) imaging fluorescence microscopy during the injury (acute), epileptogenesis (latency), and chronic-epilepsy phases of the development of AE. Immediately after status epilepticus (SE), hippocampal neurons, but not frontal and occipital neurons, had significantly elevated [Ca(2+)](i) compared with saline-injected control animals. Hippocampal neuronal [Ca(2+)](i) remained markedly elevated during epileptogenesis and was still elevated indefinitely in the chronic-epilepsy phase but was not elevated in SE animals that did not develop AE. Inhibiting the increase in [Ca(2+)](i) during SE with the NMDA channel inhibitor MK801 was associated in all three phases of AE with inhibition of the changes in Ca(2+) dynamics and the development of AE. Ca(2+) homeostatic mechanisms in hippocampal neurons also were altered in the brain-injury, epileptogenesis, and chronic-epilepsy phases of AE. These results provide evidence that [Ca(2+)](i) and Ca(2+)-homeostatic mechanisms are significantly altered during the development of AE and suggest that altered Ca(2+) dynamics may play a role in the induction and maintenance of AE and underlie some of the neuroplasticity changes associated with the epileptic phenotype.

Localization of calcitonin gene-related peptide in cardiomyocytes: comparison of neonatal and dedifferentiating cells to adult myocytes

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the l-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the l-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.

Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice.

Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factor nuclear factor of activated T cells (NFAT). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, hypertrophy of mature myofibers, and fiber type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists about the functional role of calcineurin signaling in each of these processes. Here we evaluated the molecular phenotypes of skeletal muscle from both calcineurin Aalpha and calcineurin Abeta gene-targeted mice. Calcineurin Aalpha was observed to be the predominant catalytic isoform expressed in nearly all skeletal muscles examined. Neither calcineurin Aalpha or Abeta null mice showed any gross growth-related alterations in skeletal muscle, nor was fiber size or number altered in glycolytic/fast muscle types. In contrast, both calcineurin Aalpha and Abeta gene-targeted mice demonstrated an alteration in myofiber number in the soleus, an oxidative/slow-type muscle. More significantly, calcineurin Aalpha and Abeta gene-targeted mice showed a dramatic down-regulation in the oxidative/slow fiber type program in multiple muscles (both slow and fast). Associated with this observation, NFAT-luciferase reporter transgenic mice showed significantly greater activity in slow fiber-containing muscles than in fast. However, only calcineurin Aalpha null mice showed a defect in NFAT nuclear occupancy or NFAT-luciferase transgene activity in vivo. Collectively, our results suggest that calcineurin signaling plays a critical role in regulating skeletal muscle fiber type switching but not hypertrophy. Our results also suggest that fiber type switching occurs through an NFAT-independent mechanism.

The Relationship between Pulsatile Secretion and Calcium Dynamics in Single, Living Gonadotropin-Releasing Hormone Neurons

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1–43 fluorescence to monitor changes of secretion in individual GT1–7 cells and then combined this approach with simultaneous measurement of intracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mm) or N-methyl-d-aspartate (NMDA, 100μ m) predictably evoked dynamic increases of both FM1–43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that[ Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons.

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1-43 fluorescence to monitor changes of secretion in individual GT1-7 cells and then combined this approach with simultaneous measurement ofintracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100 microM) predictably evoked dynamic increases of both FM1-43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

Development and characterisation of a glutamate-sensitive motor neurone cell line.

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.

Lipid second messengers derived from glycerolipids and sphingolipids, and their role in smooth muscle function.

The processes that link activation of an external receptor to the internal mechanisms that elicit a physiological response have been the subject of extensive investigation. It has been established that rather than just being an inert barrier to protect the cell from environmental damage, there are populations of phospholipids located within the plasma membrane that act as a reservoir for signalling molecules and when a receptor binds its appropriate activating ligand a chain of events is initiated which leads to the breakdown of these lipids and the release of second messengers. Such processes are rapid enough for physiological responses to be effected. The purpose of this review is to examine the profile of lipid second messengers derived from glycerophospholipids and sphingolipids. In the former class are included phosphoinositide and phosphatidylcholine and the latter includes sphingomyelin. Hydrolysis of such parent compounds is mediated by phospholipases and the profile of metabolites appears to be agonist specific and modulated by a number of mechanisms including heterotrimeric G-protein subunits, small G-proteins, alterations in intracellular calcium concentration, protein kinase C and tyrosine kinases. The recent interest in sphingolipids, particularly in vascular smooth muscle cells, has been provoked by the observation that ceramide and sphingoid base formation is observed in response to vasoconstrictor hormones.

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

By using a FURA2 ratio imaging method, the intracellular free calcium concentration was investigated in cultured mice neuroblastoma cells under the influence of an amplitude-modulated (AM) field (5 kHz sine wave AM 16 Hz sinusoidal 800 V/m and 80 V/m), as well as of electric field pulses (300-ms unipolar pulses of 1000 V/m and 800 V/m, 5 pulses during 10 s and 50 pulses during 100 s). An increase in free intracellular calcium was found in about 50% of cells after field application, whereas in control experiments only about 20% of the cells showed similar increases. However, this effect depended on the amount of UV irradiation used for excitation of FURA2 fluorescence. Experiments with 1/30 to former total illumination no longer demonstrated an increase in control cells or in cells treated with AM fields. The number of cells showing calcium increase after the application of pulsed fields was reduced significantly. Therefore, the UV light itself, applied as double flashes for the fluorescence measurement, activates the cellular calcium regulation. These findings offer a possible explanation for the low reproducibility of field effects found in different laboratories, in which investigations were performed with different equipment using different intensities of UV excitation.

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

Induction of CYP1A1 by GABA Receptor Ligands

The induction of CYP1A1 is mediated via the aromatic hydrocarbon (Ah) receptor. Studies from our laboratory show CYP1A1 induction by picrotoxin and phenobarbital which prompted us to examine if other ligands of the γ-aminobutyric acid (GABA) receptor could also induce CYP1A1. Here we report the nuclear translocation of the Ah receptor and its DNA binding activity to radiolabeled double-stranded synthetic xenobiotic response elements (XREs) in nuclear extracts, increased accumulation of CYP1A1 mRNA, and alterations in intracellular calcium concentrations in cells exposed to GABA receptor ligands.

Intracellular Ca2+ mobilization in cultured human mucosal microvascular endothelial cells from the nasal inferior turbinate.

Human mucosal microvascular endothelial cells (HMMECs) were isolated and cultured from the mucosa of nasal inferior turbinates. These cells were identified by human factor-VIII antigen and UEA-I. We also investigated alterations in intracellular calcium concentration [Ca2+]i, in HMMECs using the Ca(2+)-sensitive dye fura-2. Depolarization by high K+ (150 mM) induced no changes in [Ca2+]i in HMMECs, thereby indicating the lack of voltage-dependent Ca2+ channels in these cells. Extracellular ATP increased [Ca2+]i in HMMECs both in the presence and absence of extracellular Ca2+. These results suggest that ATP may induce a release of Ca2+ from intracellular stores in HMMECs. Adenosine induced no changes in [Ca2+]i in HMMECs. It is thus likely that HMMECs obtained from the nasal inferior turbinate may carry P2-purinoceptors but not P1-purinoceptors.

Factors Affecting the Production of Insoluble Elastin in Aortic Organ Cultures

Aortic organ and cell culture systems have made important contributions to the understanding of the synthesis and accumulation of elastin in arterial tissues. However, methods used to quantitate production of elastin have been diverse, and evaluations of different culture conditions have not been made. In this study we describe a convenient and reproducible organ culture system for the assay of insoluble elastin synthesis. Under optimum incubation conditions, insoluble elastin is produced in organ culture at rates approximating in vivo accumulation rates. The assay is stable under a wide variety of incubation conditions, although elastin production is sensitively inhibited by acidification of the incubation medium. Alterations in intracellular calcium concentration had little effect on elastin production. In contrast, manipulations of sodium ion concentration, including treatment with ouabain or monensin, depressed elastin production. Except at high doses, pharmacological or humoral agents, including non-steroidal anti-inflammatory agents, alpha and beta adrenergic agonists, cyclic nucleotides, theophylline or aminophylline, and histamine, generally had only small effects on elastin production in this organ culture system.

In vitro effects of thyroxine on the mechanical properties of erythrocytes

In vitro effects of thyroxine on erythrocyte deformability and mechanical fragility were observed. Deformability of erythrocytes was improved in a dose dependent manner by thyroxine. Mechanical hemolysis was found to be lower if thyroxine was included in erythrocyte suspensions at concentrations close to the physiological levels (10 −9M). These changes might be related to the alterations of intracellular calcium concentration, as in the erythrocyte suspensions containing 10 −9M thyroxine, intracellular calcium concentration was found to be 30 times lower than the control suspensions which did not contain thyroxine. Thyroxine also reduced the mechanical hemolysis ratio in calcium loaded cells. These observations suggest that thyroxine might play some role in the regulation of the mechanical properties of erythrocytes which might be mediated via the effects on calcium metabolism.

Effects of calcium on vasopressin-mediated cyclic adenosine monophosphate formation in cultured rat inner medullary collecting tubule cells. Evidence for the role of intracellular calcium.

We explored the effects of alterations in extracellular and intracellular calcium concentration on arginine vasopressin (AVP)-stimulated cAMP formation in cultured rat inner medullary collecting tubule cells. cAMP formation remains constant at extracellular calcium concentrations between 0.5 and 4.0 mM, which did not change intracellular calcium. Maneuvers that alter intracellular calcium concentration are associated with marked changes in cAMP generation. EGTA decreases intracellular calcium and enhances AVP-stimulated cAMP formation, while increasing cellular calcium with 2 microM A23187 decreases AVP-stimulated cAMP formation in the presence, but not in the absence, of extracellular calcium. The changes in cAMP formation observed when intracellular calcium is altered are associated with reciprocal changes in prostaglandin E2 (PGE2) synthesis. Despite greater than 95% inhibition of PGE2 synthesis with 5 microM meclofenamic acid, the changes in cAMP formation accompanying alterations in intracellular calcium concentration are still evident. These studies suggest that intracellular calcium critically influences AVP-stimulated cAMP formation. It does so by a mechanism independent of PG that is probably mediated by a direct effect of the cation on the adenylate cyclase complex.

Cholinergic stimulation of glucose transport in human erythrocytes

The effects of cholinergic stimulation on glucose equilibrium exchange rate have been studied in human erythrocytes. Carbamylcholine increases the V of equilibrium exchange by 20% but has no significant effect on K m. The cholinergic effect is abolished by the muscarinic antagonist atropine or by alterations in intracellular calcium concentrations induced by the calcium ionophore A23187.

Effects of the calcium ionophore A23187 on dispersed bovine parathyroid cells.

Dispersed bovine parathyroid cells were employed to assess the effects of the divalent cation ionophore A23187 on parathyroid function. In the presence of ionophore, both basal parathyroid hormone (PTH) release and isoproterenol-stimulated cAMP accumulation were inhibited by significantly lower concentrations of calcium (half-maximal inhibition at 0.7–0.8 mmvs. 1.0 mm and 1.3–1.4 vs. 1.7–2.0 mm calcium, respectively). After preincubation with ionophore, there was also an alteration in the relationship between agonist-stimulated cAMP accumulation and PTH release similar to that observed previously with elevated extracellular calcium concentrations; with A23187, a given increase in secretion rate was associated with nearly a 10-fold greater increase in cAMP accumulation. A23187 effected a highly significant increase in the rate of efflux of 45Ca from dispersed cells. The uptake of 45Ca, in contrast, was decreased slightly by ionophore. A predominant effect of ionophore on calcium efflux was also suggested by a significant reduction in cellular 45Ca after the addition of A23187 to cells at apparent equilibrium with calcium in the medium. The present results support the hypothesis that alterations in intracellular calcium concentration play a role in the inhibitory effects of medium calcium on 1) basal PTH release, 2) agonist-stimulated cAMP accumulation, and 3) the relationship of agonist-stimulated secretion to cAMP accumulation. Direct studies of 45Ca uptake and efflux, however, suggest that the effect of A23187 on these parameters results predominantly from mobilization of intracellular calcium stores rather than influx of extracellular calcium. (Endocrinology106: 133, 1980)