Depletion of caffeine-sensitive calcium store results in diminution of ATP-induced metabotropic calcium responses in rat neocortical neurons

ATP receptor-mediated changes in the Ca²⁺ concentration were recorded from neurons of the sensorimotor cortex in brain slices from 3-week-old rats. To measure the cytoplasmic concentration of Ca²⁺, slices were incubated with Fura-2/AM, and a microfluorimetry system was focused on an individual cell. Possible glutamatergic signals resulting from ATP-evoked glutamate release were excluded. After elimination of calcium from the extracellular solution, the first ATP-induced [Ca²⁺] _i transient decreased to 62±9% of a similar response in the normal solution, suggesting the participation of metabotropic purinoreceptor-triggered Ca release in transient generation. Depletion of the caffeine-sensitive calcium store results in diminution of ATP-induced [Ca²⁺] _i transient in the Ca²⁺-free solution by 31.4±7.0% (P<0.01). This may indicate that in pyramidal neurons of the sensorimotor cortex InsP₃- and Ca-induced Ca-releases demonstrate noticeable functional interaction. Nevertheless, there is no single compartment in the endoplasmic reticulum bearing both IICR and CICR channels.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.     

Calcium Release From the Internal Stores as a Possible Target for Antinociceptive Treatment in Rats with Experimentally-Induced Diabetes

Distal neuropathy is the most common complication of diabetes mellitus, and it is highly important to reveal the cellular mechanisms leading to its development. In our experiments, neurons of control and streptozotocin-treated diabetic rats were examined. Changes in the intracellular free calcium concentrations ([Ca²⁺] _i ) were fluorometrically measured in primary and secondary nociceptive (dorsal root ganglion, DRG, and dorsal horn, DH, respectively) neurons. The [Ca²⁺] _i elevation was induced by different agents, which can release calcium from the endoplasmic reticulum (ER) calcium stores. The amplitudes of calcium elevation induced by application of caffeine and ionomicine in DRG and DH neurons of diabetic rats were significantly lower, as compared with the control. Application of ATP and glutamate to a Ca-free extracellular solution induced calcium release from the IP₃-sensitive store in DH neurons. Release of calcium from the IP₃-sensitive ER calcium stores became significantly smaller in neurons from diabetic rats. Taken together, these data indicate that significant changes in the calcium regulating mechanisms of the ER develop under diabetes conditions.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca²⁺]_i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca²⁺]_i) was monitored using Fura-2-based ([Ca²⁺]_i) microfluorimetry. The ET-triggered ([Ca²⁺]_i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-free extracellular solution and the ET-triggered [Ca²⁺]_i elevation was blocked by 500 nM thapsigargin indicating that the [Ca²⁺]_i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca²⁺]_i increase in Ca²⁺-free solution was shorter in duration. Restoration of normal extracellular [Ca²⁺] briefly after the ET application induced a second [Ca²⁺]_i increase indicating the presence of a secondary Ca²⁺ influx which prolongs the Ca²⁺ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca²⁺ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca²⁺ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ET_B receptors which induce the generation of intracellular [Ca²⁺]_i signals by activation of Ca²⁺ release from InsP₃-sensitive intracellular stores followed by a secondary Ca²⁺ influx.     

Glutamate- and GABA-mediated neuron–satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging

In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular Ca²⁺ ([Ca²⁺]_i) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation with glutamate and its agonist and/or the antagonist of the GABA_A receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor (mGluR) agonist t-ACPD evoked [Ca²⁺]_i increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca²⁺]_i increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca²⁺]_i, suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than that without bicuculline (107.3 ± 93.4 vs. 231.8 ± 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in both neurons and satellite cells of NG. In conclusion, the levels of [Ca²⁺]_i of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron–glia interaction in the nodose ganglion may regulate sensory information from visceral organs.     

Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice

Abstract: The time course of the decline in energy levels during an in vitro ischemia-like condition was compared with changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod-2. During the on-line monitoring of [Ca²⁺]_i, the slices were perfused with an in vitro ischemia-like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region-specific and acute [Ca²⁺]_i elevations were observed in CA1 ～4 min after onset of the in vitro ischemia-like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca²⁺]_i. ATP levels in all subregions gradually decreased before the acute [Ca²⁺]_i elevation. Concomitant with the acute [Ca²⁺]_i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high-energy-charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca²⁺]_i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region-specific increase in [Ca²⁺]_i, which activates a Ca²⁺-ATP-driven pump and produces a subsequent fall in neuronal ATP content.     

Activation of protein kinase C inhibits ATP-induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Extracellular ATP elicits transient elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoblasts through interaction with more than one subtype of cell surface P₂-purinoceptor. Elevation of [Ca²⁺]_i arises, at least in part, by release of Ca²⁺ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca²⁺]_i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca²⁺, ATP (100 μM) induced transient elevation of [Ca²⁺]_i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-β-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca²⁺]_i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca²⁺ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4β-phorbol 12, 13-dibutyrate, but not by the control compounds 4α-phorbol or 4α-phorbol 12, 13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca²⁺]_i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P_2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P_2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P_2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley-Liss, Inc.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca²⁺]_i) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca²⁺]_i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca²⁺]_i after application at the doses of 30 nM (n?=?34, p?<?0.01), 100 nM (n?=?41, p?<?0.001) and 300 nM (n?=?46, p?<?0.001). The stimulatory effect of OT (300 nM) on [Ca²⁺]_i was persistent in Ca²⁺-free conditions (n?=?56, p?<?0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca²⁺]_i (n?=?28, p?<?0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.     

The steady-state force–Ca<Superscript>2+</Superscript> relationship in intact lobster (<Emphasis Type="Italic">Homarus americanus</Emphasis>) cardiac muscle

The heart of the decapod crustacean is activated by regular impulse bursts from the cardiac ganglion. The cardiac pump function depends on ganglionic burst frequency, burst duration, and burst impulse frequency. Here, we activated isolated lobster cardiac ostial muscle (Orbicularis ostii muscle, OOM) by stimulus trains in vitro in order to characterize the response of the contractile apparatus to [Ca²⁺]_i . We employed stimulus trains that generate a steady state between the [Ca²⁺]_i and force in order to estimate the Ca²⁺ sensitivity of myofilaments. Force and [Ca²⁺]_i transients were simultaneously recorded using a silicon strain gauge and the fluorescence of iontophoretically microinjected fura-2 salt. We examined the effects of tetanus duration (TD), the interval between trains, and 6 M cyclopiazonic acid, an inhibitor of the SR Ca²⁺ pump, on the steady-state force–[Ca²⁺]_i relationship. The instantaneous force–[Ca²⁺]_i relationships appeared sigmoidal (EC₅₀ and Hill coefficient, 98.8±32.7 nM and 2.47±0.20, mean ± SD, respectively), as did the curves superimposed after 500 ms following the start of stimulation, indicating that the force–[Ca²⁺]_i relationship had reached a steady state at that time. Also, the maximum activated force (F_max) was estimated using the steady-state force–[Ca²⁺]_i relationship. Prolonged stimulus trains, decreasing the interval between recurrent trains from 5 to 2.5 s, and cyclopiazonic acid each increased the measured EC₅₀ without changing F_max. The EC₅₀ correlated strongly with averaged [Ca²⁺]_i over time. We conclude that the steady-state force–[Ca²⁺]_i relationships in the OOM indicate cooperation between force generation and Ca²⁺ binding by the myofilaments. Our data also suggest the existence of a novel Ca²⁺-dependent mechanism which reduces Ca²⁺ sensitivity and accelerates relaxation of lobster cardiac muscle myofilaments.Communicated by L.C.-H. Wang     

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca²⁺ indicator dye Fura-2 was used to measure Ca²⁺ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca²⁺ responses were evoked by kainic acid (KA), α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D -aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca²⁺]_i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca²⁺ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca²⁺]_i were carried by Ca²⁺-permeable receptor channels. Significantly smaller changes in [Ca²⁺]_i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca²⁺ free-EGTA–buffered media, including l -cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclopentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca²⁺]_i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca²⁺]_i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca²⁺ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca²⁺]_i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca²⁺]_i to increase in NM neurons. Presumably, each route represents a means by which Ca²⁺ can alter cellular processes. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 321–337, 1998     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced diabetes mellitus in rat: Effect of cholecystokinin-octapeptide

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca²⁺) and magnesium (Mg²⁺) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg^–1, I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg^–1 urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca²⁺ and Mg²⁺ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 ± 2.42 mg dl^–1 (n= 44) and >500 mg dl^–1 (n= 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 ± 0.05 ul min^–1 (n= 10) and 1.28 ± 0.16 ul min^–1 (n= 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca²⁺]_i in control and diabetic fura-2-loaded acinar cells was 109.40 ± 15.41 nM (n= 15) and 130.62 ± 17.66 nM (n= 8), respectively. CCK-8 (10^–8M) induced a peak response of 436.55 ± 36.54 nM (n= 15) and 409.31 ± 34.64 nM (n= 8) in control and diabetic cells, respectively. Basal [Mg²⁺]_i in control and diabetic magfura-2-loaded acinar cells was 0.96 ± 0.06 nM (n= 18) and 0.86 ± 0.04 nM (n= 10). In the presence of CCK-8 (10^–8) [Mg²⁺]_i in control and diabetic cells was 0.80 ± 0.05 nM (n= 18) and 0.60 ± 0.02 nM (n= 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca²⁺ and Mg²⁺ homeostasis. (Mol Cell Biochem 261: 83–89, 2004)