Regulation of intracellular Ca concentration by interleukin-1β in rat cortical synaptosomes: an age-related study

The pro-inflammatory cytokine interleukin-1β (IL-1β) is released by cells during injury and stress, and increased neuronal expression of IL-1β is a feature of age-related neurodegeneration. We have recently reported that IL-1β has a biphasic effect on the K⁺-induced rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cortical synaptosomes, exerting an inhibitory effect on the K⁺-induced rise in [Ca²⁺]_i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K⁺-induced rise in [Ca²⁺]_i at higher (100 ng/mL) concentrations. In the present study, we observed that the K⁺-induced rise in [Ca²⁺]_i was inhibited to a similar extent by the lower concentration of IL-1β in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1β, resulting in a marked elevation in [Ca²⁺]_i. We propose that the age-related increase in neuronal concentration of IL-1β promotes a dramatic elevation in [Ca²⁺]_i following membrane depolarization, thereby altering Ca²⁺ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.     

Sphingolipid derivatives modulate intracellular Ca2+ in rat synaptosomes

Sphingosylphosphorylcholine (SPC) induces a rapid increase of intracellular Ca2+ concentration in isolated synaptosomes. This effect is dose-dependent and is also dependent on extracellular Ca2+. Sphingosine (SPH) has a smaller effect and treatment with psychosine (PSY) is ineffective, which suggests that phosphorylation of the 1-carbon of SPH is required for the SPC to act as a Ca2+ release agonist in synaptosomes. Experiments performed in the presence of heparin or ryanodine indicate that SPC-elicited Ca2+ release is not mediated by IP3 or ryanodine receptors. Finally, our results show that the effect of SPC on Ca2+ concentration is nimodipine-sensitive, suggesting that SPC possibly activates a specific sphingolipid-gated Ca2+ channel in synaptosomes.     

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca²⁺ or Mg²⁺ ([Ca²⁺]_i, [Mg²⁺]_i) on Ca current (I _Ca) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg²⁺]_i an increase in [Ca²⁺]_i enhancedI _Ca whereas at lower [Mg²⁺]_i I _Ca was suppressed. The increase inI _Ca caused by Ca²⁺ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5-triphosphate analogue ,-methyleneadenosine 5-triphosphate did not prevent the Ca²⁺-inducedI _Ca increase.I _Ca was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg²⁺]_i was lowered from 1.0 × 10^–3 to 1.0 × 10^–6 M at a [Ca²⁺]_i of 10^–8 M. The concentration response relation for inhibition of Ca channels by [Mg²⁺]_i is modulated by [Ca²⁺]_i. To account for the experimental results it is postulated that competitive binding of Ca²⁺ or Mg²⁺ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.     

Regulation of intracellular pH by Ca2+ - activated proton channel

Background and aim: We have characterized a membrane current associated with a decrease in pHi, which can be induced by either elevating intracellular calcium or extracellular application of methylmercury (a potent agent in elevating intracellular Ca²⁺ concentration) in the alveolar macrophages bathing in the impermeant bilateral cesium aspartate solution.

Results: Decreasing pHi and elevating [Ca²⁺]i profoundly enhanced, but H-7 (a broad-spectrum kinase inhibitor), W-7 (a selective calmodulin antagonist) and KN-93 (a calmodulin kinase II inhibitor) inhibited the currents.

Conclusion: These results indicate that rat alveolar macrophages possess a calcium-activated and pHi-sensitive proton channel which can be phosphorylated and activated by calmodulin kinase II.     

Measurement of intracellular Ca2+ concentration using Indo-1 during simultaneous flash photolysis to release Ca2+ from DM-nitrophen

We have constructed a modular instrument to measure intracellular [Ca²⁺] ([Ca²⁺]_i) in single isolated cells while simultaneously imposing step changes in [Ca²⁺]_i using caged Ca²⁺. By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd: YAG) laser, the instrument can operate with low maintained illumination to measure [Ca²⁺]_i using a ratiometric Ca²⁺-sensitive fluorophore and also activate the release of Ca²⁺ from a caged-Ca²⁺ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca²⁺ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca²⁺]_i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.     

Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons.

The ratiometric indicators 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pHi) and calcium ([Ca2+]i) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37 degrees C. Under both HCO3-/CO2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a triphasic change in pHi consisting of an initial fall in pHi, a subsequent rise in pHi in the continued absence of O2 and, finally, a further rise in pHi upon the return to normoxia, which recovered towards preanoxic steady-state pHi values if the duration of the anoxic insult was < or = 5 min. In parallel experiments performed on sister cultures, anoxia of 3, 5 or 10 min duration evoked rises in [Ca2+]i which, in all cases, commenced after the start of the fall in pHi, reached a peak at or just following the return to normoxia and then declined towards preanoxic resting levels. Removal of external Ca2+ markedly attenuated increases in [Ca2+]i, but failed to affect the pHi changes evoked by 5 min anoxia. The latency from the start of anoxia to the start of the increase in pHi observed during anoxia was increased by perfusion with media containing either 2 mM Na+, 20 mM glucose or 1 microM tetrodotoxin. Because each of these manoeuvres is known to delay the onset and/or attenuate the magnitude of anoxic depolarization, the results suggest that the rise in pHi observed during anoxia may be consequent upon membrane depolarization. This possibility was also suggested by the findings that Zn2+ and Cd2+, known blockers of voltage-dependent proton conductances, reduced the magnitude of the rise in pHi observed during anoxia. Under HCO3-/CO2-free conditions, reduction of external Na+ by substitution with N-methyl-D-glucamine (but not Li+) attenuated the magnitude of the postanoxic alkalinization, suggesting that increased Na+/H+ exchange activity contributes to the postanoxic rise in pHi. In support, rates of pHi recovery from internal acid loads imposed following anoxia were increased compared to control values established prior to anoxia in the same neurons. In contrast, rates of pHi recovery from acid loads imposed during anoxia were reduced, suggesting the possibility that Na+/H+ exchange is inhibited during anoxia. We conclude that the steady-state pHi response of cultured rat hippocampal neurons to transient anoxia is independent of changes in [Ca2+]i and is characterized by three phases which are determined, at least in part, by alterations in Na+/H- exchange activity and, possibly, by a proton conductance which is activated during membrane depolarization.     

Dopamine D1/5 receptor-mediated long-term potentiation of intrinsic excitability in rat prefrontal cortical neurons: Ca2+-dependent intracellular signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.     

Control of intracellular Ca2+ activity in rat myometrium.

Four fractions enriched, respectively, in plasma membrane (PM), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria were isolated from estrogen-dominated rat myometrium. Ca2+ uptake by these fractions was studied in order to estimate the relative potential of the corresponding organelles for controlling intracellular Ca2+ activity. Ca2+ uptake properties of the PM, SER, and RER fractions were similar except that potentiation by oxalate was in the order RER greater than or equal SER greater than PM. However, studies with the ionophores X-537A and A23187 suggested that Ca2+ was transported into the lumen of membrane vesicles of all these fractions. Unlike that of skeletal muscle sarcoplasmic reticulum, Ca2+ uptake by the myometrial fractions was not supported by high-energy compounds other than ATP. Mitochondria took up much less Ca2+ at low, and much more Ca2+ at high, free Ca2+ concentrations than did the other fractions. The amount of Ca2+ taken up in 30 s from a 1 muM free Ca2+ solution in the presence of ATP was similar for all fractions. These results suggested that mitochondria may act as an important Ca2+ control system in rat myometrium when the intracellular Ca2+ concentration is near 1 muM or higher, whereas the PM, SER, and RER may be of major importance at Ca2+ levels of 0.3 muM or lower.     

Protein kinase Calpha modulates depolarizaton-evoked changes of intracellular Ca2+ concentration in a rat pheochromocytoma cell line

Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca(2+) and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKCalpha, a cPKC, could be activated by depolarization in a neuroendocrine cell line and whether activation occurred on a time scale that modulated the depolarization-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCalpha labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca(2+)](i) changes were monitored with fura-2. Two point mutations that render PKCalpha inactive, D187A in the Ca(2+) binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca(2+)](i) signal. A mutation that modulates membrane insertion (W58G) and two mutations of an autophosphorylation site (S657A, S657E) had no effect on the kinetics of the [Ca(2+)](i) signal. We conclude that in PC12 cells, Ca(2+) entry through VGCC rapidly activates PKCalpha, and that PKCalpha can modulate the Ca(2+) signal on a physiologically relevant time scale. Point mutations of PKCalpha can be used as specific and potent modulators of the PKC signaling pathway.     

Multiple P2 receptors contribute to a transient increase in intracellular Ca2+ concentration in ATP-stimulated rat brown adipocytes

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca(2+) concentration ([Ca(2+)](i)), which arises primarily from a release of Ca(2+) from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca(2+)](i) elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 microM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca(2+)](i) elevation mediated through store-operated Ca(2+) entry. The inhibitory effect of ATP was antagonized by PPADS with IC(50) of 0.7 microM. In the presence of PPADS at concentrations of more than 5 microM, the ATP-induced [Ca(2+)](i) elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca(2+)](i) elevation was reduced in a concentration-dependent manner by PPADS with an IC(50) of 200 microM. In contrast, the ATP-induced [Ca(2+)](i) elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca(2+)](i) responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca(2+) stores, which is probably followed by the activation of store-operated Ca(2+) entry. Extracellular ATP, however, would inhibit this Ca(2+) entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca(2+)](i) elevation in response to extracellular ATP.     

Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells

Changes in membrane potential and cytosolic free Ca2+ concentrations, [Ca2+]i, in response to L-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K+ concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4-(3)). The [Ca2+]i as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K+ and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K+ induced rise in [Ca2+]i but did not significantly affect the responses produced by NMDA, quisqualate and kainate, suggesting that the increase in intracellular Ca2+ in response to glutamate receptor agonists is primarily due to Ca2+ influx through receptor-coupled ion channels.     

Modulation of a Ca2+-dependent K+-current by intracellular cAMP in rat thalamocortical relay neurons

Voltage-activated calcium channels in thalamic neurons are considered important elements in the generation of thalamocortical burst firing during periods of electroencephalographic synchronization. A potent counterpart of calcium-mediated depolarization may reside in the activation of calcium-dependent potassium conductances. In the present study, thalamocortical relay cells that were acutely dissociated from the rat ventrobasal thalamic complex (VB) were studied using whole-cell patch-clamp techniques. The calcium-dependent potassium-current (I_K(Ca)) was evident as a slowly activating component of outward current sensitive to the calcium ions (Ca²⁺)-channel blocker methoxyverapamil (10 μM) and to substitution of external calcium by manganese. The I_K(Ca) was blocked by tetraethylammonium chloride (1 mM) and iberiotoxin (100 nM), but not apamin (1 μM). In addition, isolated VB neurons were immunopositive to anti-α_(913–926) antibody, a sequence-directed antibody to the α-subunit of “big” Ca²⁺-dependent K⁺-channel (BK_Ca) channels. Activators of the adenylyl cyclase cyclic adenosine monophosphate (cAMP) system, such as forskolin (20 μM), dibutyryl-cAMP (10 mM) and 3-isobutyl-1-methylxanthine (500 μM), selectively and reversibly suppressed I_K(Ca). These results suggest that a rise in intracellular cAMP level leads to a decrease in a calcium-dependent potassium conductance presumably mediated via BK_Ca type channels, thereby providing an additional mechanism by which neurotransmitter systems are able to control electrogenic activity in thalamocortical neurons and circuits during various states of electroencephalographic synchronization and de-synchronization.     

Increase of intracellular Ca by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat

Astrocytes express purinergic receptors that are involved in glial–neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed “astroglia” throughout. Astroglia expressed predominantly P2X_4,6,7 as well as P2Y_1,2 receptor-subtypes. Less intensive immunostaining was also found for P2X₅ and P2Y_4,6,13,14 receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca²⁺ ([Ca²⁺]_i). Of the agonists tested, only the P2X_1,3 receptor-selective α,β-methylene ATP was ineffective. Experiments with Ca²⁺-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca²⁺-ATPase, indicated that the [Ca²⁺]_i response to most nucleotides, except for ATP and 2′,3′-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca²⁺ from intracellular stores. A Gprotein–mediated release of Ca²⁺ is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y_1,2,6,14 and probably also P2Y₄) receptors. Several unidentified P2X receptors, including P2X₇, may also be present, although they appear to only moderately participate in the regulation of [Ca²⁺]_i. The rise of [Ca²⁺]_i is due in this case to the transmembrane flux of Ca²⁺ via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca²⁺]_i of cultured astroglia and thereby may play an important role in cell-to-cell signaling.     

GABAA-receptor-mediated increase in intracellular Ca2+ concentration in the regenerating retina of adult newt

We used optical recording with the Ca(2+)-sensitive dye, fura-2, in living slice preparations from the newt retina at different stages of regeneration. gamma-Aminobutyric acid (GABA) induced pronounced [Ca(2+)](i) rise in progenitor cells and differentiating ganglion cells in the 'intermediate' stage of retinal regeneration. This [Ca(2+)](i) rise became less pronounced at the beginning of synapse formation in the late regenerating retina. At the late period of the late regenerating retina with the IPL thickness comparable to that of the control retina, GABA-induced [Ca(2+)](i) rise became undetectable or sometimes a small decrease in [Ca(2+)](i) was observed in regenerated ganglion cells. In contrast, N-methyl-d-aspartate (NMDA)-induced [Ca(2+)](i) rise appeared in premature ganglion cells and became prominent gradually as the regeneration proceeded. The [Ca(2+)](i) rise to GABA was mediated by GABA(A) receptors. This was shown by inhibition of GABA-induced Ca(2+) response with the preincubation of the GABA(A) receptor antagonist, bicuculline. The [Ca(2+)](i) rise due to GABA was suppressed in the absence of extracellular Ca(2+) or in the presence of the L-type voltage-gated Ca(2+) channel blocker, verapamil, suggesting that Ca(2+) may be entered through L-type Ca(2+) channels. Transient appearance of [Ca(2+)](i) rise to GABA during regeneration and origin of GABA-induced [Ca(2+)](i) rise were similar to those in the developing retina [J. Neurobiol. 24 (1993) 1600]. These similarities may suggest that common mechanisms may control neurogenesis and/or synaptogenesis during development and regeneration.     

Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones.

The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BK(Ca)) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca2+]i). These BK(Ca) channels had a conductance of approximately 200 pS and were charybdotoxin- and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK(Ca) channel activity, which was inhibited by 10 microM ryanodine. Met-enkephalin (10 microM) evoked a similar increase in BK(Ca) channel activity, which was dependent on the presence of extracellular Ca2+ and inhibited by either ryanodine (10 microM) or naloxone (1 microM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+]i. Met-enkephalin-induced mobilization of intracellular Ca+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.     

灌流液镁浓度对测定谷氨酸引起的培养神经细胞内钙浓度的影响

Ｍｇ２＋对谷氨酸（ｇｌｕ）受体亚型Ｎ－甲基－Ｄ－门冬氨酸（ＮＭＤＡ）受体通道的阻断作用，早在８０年代就已由电生理实验证实，此后通过荧光测定细胞内游离钙浓度［Ｃａ２＋］ｉ得到进一步证实。令人困惑的是，在研究ｇｌｕ对离休神经细胞作用的文献中，所用缓冲液的...