Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Opioid-Induced Increase in [Ca2+]i in ND8-47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein-Coupled δ-Opioid Receptors and Desensitized by Chronic Exposure to Opioid

Abstract: δ-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) in ND8-47 cells by activating dihydropyridine-sensitive Ca²⁺ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d -Ser²,Leu⁵]enkephalin-Thr (DSLET)-induced increase in [Ca²⁺]_i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca²⁺]_i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [³H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits α_i2, α_i3, α_s, and α_q were detected in ND8-47 cell membranes by western blot; α_o and α_i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α-subunits. These results suggest that the DSLET-induced increase in [Ca²⁺]_i is mediated through pertussis toxin-sensitive G proteins (probably G_i2 or G_i3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.     

Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: implication for epilepsy

Although the role of Na⁺ in several aspects of Ca²⁺ regulation has already been shown, the exact mechanism of intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase resulting from an enhancement in the persistent, non‐inactivating Na⁺ current (I_Na,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na⁺ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na⁺ concentration ([Na⁺]_i) and biphasic [Ca²⁺]_i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca²⁺ response was tetrodotoxin‐ and extracellular Ca²⁺‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca²⁺]_i rise was the net result of Ca²⁺ influx through voltage‐gated Ca²⁺ channels and mitochondrial Ca²⁺ sequestration. The robust second phase in addition involved reverse operation of the Na⁺–Ca²⁺ exchanger and mitochondrial Ca²⁺ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na⁺ current and [Ca²⁺]_i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I_Na,P. Describing the magnitude, temporal pattern and sources of Ca²⁺ increase induced by I_Na,P may provide novel targets for antiepileptic drug therapy.     

Ca2+ Release from Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) was examined in primary cultured neurons from rat frontal cortices using the Ca²⁺-sensitive fluorescent indicator fura-2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca²⁺]_i in a concentration-dependent manner (100 μM to 1 mM). These four AD-induced [Ca²⁺]_i increases were not altered by the absence of external Ca²⁺ or by the presence of La³⁺ (30 μM), suggesting that these ADs provoked intracellular Ca²⁺ mobilization rather than Ca²⁺ influx. All four ADs increased inositol 1,4,5-trisphosphate (IP₃) contents by 20–60% in the cultured cells. The potency of the IP₃ production by these ADs closely correlated with the AD-induced [Ca²⁺]_i responses. Pretreatment with neomycin, an inhibitor of IP₃ generation, significantly inhibited amitriptyline- and imipramine-induced [Ca²⁺]_i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP₃ generation and mobilize the intracellular Ca²⁺, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline-induced [Ca²⁺]_i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores and that the responses are pertussis toxin-insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.     

Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108-15) Cells

Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P₂ purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P₂U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca²⁺]_i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca²⁺]_i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca²⁺]_i elicited by ATP occurred concomitantly with the hydrolysis off [³²P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca²⁺]_i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP^4-) in the medium and not with the concentration of magnesium-ATP (MgATP^2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca²⁺]_i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca²⁺]_i. In cells labeled with [³²P]P_i or [¹⁴C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [¹⁴C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P₂ nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A₂ and D.     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

Reversal of the calcium pump in human red cells

Summary Human red cells containing low ATP and high P_i concentrations were suspended in media with and without 2mm Ca²⁺, and the incorporation of (³²P)P_i into ATP was measured. There was some incorporation whatever the medium, but in every experiment there was an extra incorporation when the cells were in the Ca²⁺-containing medium. This extra incorporation was abolished by the ionophore A23187, which collapses the Ca²⁺ concentration gradient across the membranes, or by LaCl₃, which blocks the Ca²⁺ pump. Starved and phosphate-loaded cells also show an uptake of Ca²⁺ which is not apparent in fresh cells. Results are consistent with the idea that Ca²⁺-dependent incorporation of P_i into ATP is catalyzed by the Ca²⁺ pump using energy derived from the Ca²⁺ concentration gradient.     

Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas

The data presented here describe ratio-imaging of in intracellular free calcium (Ca²⁺_i) during the self-incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura-2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial-temporal alterations in Ca²⁺_i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca²⁺]_i) in the SI response. Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip-focused gradient of Ca^2+bf_i, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non-growing pollen tubes did not exhibit this tip-focused gradient. Basal levels of Ca²⁺_i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low-level fluctuations +/? 50 nM observed. Challenge with incompatible S proteins resulted in S-specific, rapid and dramatic alterations in [Ca²⁺]_i within a few seconds of challenge. Increases in [Ca²⁺]_i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca²⁺]_i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip-focused gradient was observed, which following some fluctuation, was reduced to basal levels within ～1 min. Our data suggest that some of these alterations in [Ca²⁺]_i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca²⁺]_i in the subapical/shank region are very rapid, because tip [Ca²⁺]_i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca²⁺]_i at the pollen tube tip.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

大黄素影响巨噬细胞升高[Ca2+]i 和释放TNF-α的作用特征

为了研究大黄素（emodin)对正常的和细菌脂多糖（LPS）刺激的大鼠腹腔巨噬细胞（PMφ）释放肿瘤坏死因子α（TNF－α）和升高[Ca^2 ]i的影响,应用L929细胞系和MTT法检测TNF－α量,同时用激光共焦扫描显微术检测单细胞[Ca^2 ]i变化动力学。结果显示大黄素能轻度促进正常PMφ释放TNF－α,并发现大黄素诱发PMφ[Ca^2 ]i变化呈振荡波模式。大黄紫显著抑制LPS刺激PMφ过度释放TNF－α和升高[Ca^2 ]i,10^-5mol/L大黄素抑制了10mg/L LPS刺激的TNF－α峰值的50％和[Ca^2 ]i峰值的68％。LPS诱发MPφ[Ca^2 ]i变化呈现高幅值的“平台期”,大黄素使之转变为低幅值的波动变化。以上结果说明,大黄素对PMφ释放TNF－α和升高[Ca^2 ]i表现出的双向调节作用之间有一定的相关性,大黄素对LPS诱发的[Ca^2 ]i升高的调制,可能是抑制LPS刺激PMφ释放TNF－α的信号传导通路中的重要环节。     

Phospholipase A2 Stimulation by Methyl Mercury in Neuron Culture

Abstract: In primary prelabeled cultures of cerebellar granule cells, methyl mercury (MeHg) induced a concentration- and time-dependent release of [³H]arachidonic acid. MeHg-induced [³H]arachidonate release was partially dependent on the extracellular Ca²⁺ concentration. MeHg at 10–20 µM also stimulated basal ⁴⁵Ca²⁺ uptake after 20 min of incubation at 37°C, and at 10 µM inhibited K⁺ depolarization-stimulated uptake. MeHg stimulated [³H]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A₂ (PLA₂) activation preceded cytotoxicity, but at higher concentrations of MeHg such dissociation was not evident. Inhibition of MeHg-induced PLA₂ activation by 100 µM mepacrine failed to modify cytotoxicity. MeHg-induced lipoperoxidation, measured as the production of thiobarbituric acid-reacting products, was inhibited by α-tocopherol without inhibition of [³H]arachidonate release. The absence of α-tocopherol inhibition of MeHg-induced arachidonate release precludes a causal role for lipoperoxide-induced PLA₂ activation in this system. Moreover, MeHg induced an increased susceptibility of unilamellar vesicles to exogenous PLA₂ in the presence of low Ca²⁺ concentrations without evidence of lipid peroxidation. [³H]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relatively high proportional incorporation was found in the combined phosphatidylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the phosphatidylinositol fraction, indicating a preferential turnover in this phospholipid species in the presence of MeHg.     

ZUR ENERGIEBEDÜRFTIGEN Ca2+-AKKUMULATION IN MITOCHONDRIEN AUS RATTENHIRN

—(1) The properties of a preparation of functional intact rat brain mitochondria are described and the resulting fraction is enzymatically characterized. (2) These mitochondria are able to accumulate Ca²⁺ in a respiration-dependent reaction without additions of P_i and/or adenine nucleotides. (3) As criteria the increased acidity of the incubation medium accompanying the energy-dependent Ca²⁺ accumulation, its inhibition by rotenon and Antimycin A, the acceleration of respiration and the redox change of the pyridine nucleotides were recorded and Ca²⁺ accumulation by the mitochondria was determined by complexemetric methods. (4) The maximum Ca²⁺ accumulation by rat brain mitochondria amounts to only 25 per cent of that by rat liver mitochondria under similar conditions (on the base per mg protein); after addition of 1 mm -ADP (without P_i) the comparable value was about 50 per cent.     

Effect of P2Y Agonists on Adenosine Transport in Cultured Chromaffin Cells

Abstract: Adenosine transport in cultured chromaffin cells was inhibited by purinergic P_2y-receptor agonists without significant changes in the affinity constant, the values being between 1 ± 0.4 and 1.6 ± 0.6 μM. The V_max parameter was modified significantly, being 40 ± 1.0, 26 ± 5.0, 32 ± 3.0, and 22 ± 4.7 pmol/10⁶ cells/min for control, adenosine-5′-O-(2-thiodiphosphate), 5′-adenylylimidodiphosphate, and P¹,P⁴-di(adenosine-5′-) tetraphosphate (Ap₄A) (100 μM for every effector), respectively. Ap₄A, a physiological ligand for P_2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the activation of protein kinase C (PKC). Experiments of [Ca²⁺]_i measurement with the fura-2 technique showed that P_2y agonists, as well as bradykinin, were able to increase [Ca²⁺]_i, this effect being independent of the presence of extracellular Ca²⁺. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca²⁺ mobilization in chromaffin cells, exhibited a behavior similar to that of P_2y agonists in adenosine transport inhibition (39%). P_2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P_2y-purinergic receptors mediated via Ca²⁺ mobilization and PKC activation.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.