Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca²⁺) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca²⁺]_i and significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K⁺- and 4-AP-induced Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) completely suppressed the enhancements of [Ca²⁺]_i and Ca²⁺-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K⁺- or 4-AP-induced glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through P- and N-type Ca²⁺ channels.     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Appearance of Depolarization- and Maitotoxin-Induced [Ca2+]i Elevation in Single LAN-1 Human Neuroblastoma Cells on Exposure to Retinoic Acid

Abstract: LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca²⁺]_i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca²⁺]_i elevation elicited by 55 mM K⁺. Maitotoxin, a putative activator of voltage-sensitive Ca²⁺ channels, did not evoke an elevation of [Ca²⁺]_i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca²⁺]_i when superfused in retinoic acid-treated cells. Both high K⁺- and maitotoxin-induced [Ca²⁺]_i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd³⁺, an inorganic Ca²⁺-entry blocker, and by the snail toxin ω-conotoxin GVIA, which interacts with the N sub-type of voltage-sensitive Ca²⁺ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel sub-type, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca²⁺]_i due to Ca²⁺ influx elicited by membrane depolarization. K⁺-induced [Ca²⁺]_i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K⁺-induced increase of [Ca²⁺]_i was still present. In conclusion, the results of the present study demonstrated that retinoic acid-induced differentiation of LAN-1 cells, which lack a high K⁺-evoked [Ca²⁺]_i increase in the undifferentiated state, induces the functional expression of an ω-conotoxin GVIA-sensitive, dihydropyridine-insensitive N-type voltage-sensitive Ca²⁺ channel that can be activated by maitotoxin and negatively modulated by protein kinase C.     

The Difference in the Effect of Glutamate and NO Synthase Inhibitor on Free Calcium Concentration and Na+,K+-ATPase Activity in Synaptosomes from Various Brain Regions

The significant increase of free calcium concentration ([Ca²⁺]_i) was found in rat cerebral cortex synaptosomes and hippocampal crude synaptosomal fraction after their exposure to glutamate. But no change of [Ca²⁺]_i was revealed in cerebellar synaptosomes, the slight increase of [Ca²⁺]_i in striatal synaptosomes was not significant. The presence of N^g-nitro-L-arginine methyl ester (L-NAME) in the incubation medium practically prevented the increase of [Ca²⁺]_i initiated by glutamate in cerebral cortex synaptosomes, but not in hippocampal ones. The significant diminution of [Ca²⁺]_i in the presence of this inhibitor was shown in striatal synaptosomes exposed to glutamate. Na⁺,K⁺-ATPase activity is significantly lower in cerebral cortex, striatal and hippocampal synaptosomes exposed to glutamate. L-NAME prevented the inactivation of this enzyme by glutamate. In cerebellar synaptosomes the tendency to the decrease of enzymatic activity in the presence of L-NAME was on the contrary noticed. Thus, the data obtained provide evidence of the protective effect of NO synthase inhibitor in brain cortex and striatal synaptosomes, but not in cerebellar synaptosomes. Synaptosomes appear to be an adequate model to study the regional differences in the mechanism of toxic effect of excitatory amino acids.     

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.     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K⁺ and Cl⁻ currents (I _K and I _Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺] _i ), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn²⁺ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca²⁺] _i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of I _K and I _Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca²⁺ influx and [Ca²⁺] _i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca²⁺] _i increase. Received: 6 October 1997/Revised: 16 April 1998     

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K+ channel as studied by a kinetic analysis in Chara corallina

The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca²⁺]_c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca²⁺]_c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K⁺ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K⁺ transport of the plasma membrane: (i) light causes an uptake of Ca²⁺ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca²⁺]_c, (iii) this leads to a decrease in the activity of a K⁺ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca²⁺ fluxes coming out of the chloroplasts upon darkening cause a Ca²⁺-induced phosphorylation of myosin, which slows down cytoplasmic streaming. Received: 3 May 1997 / Accepted: 19 May 1998     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

Mechanisms underlying overdrive suppression and overdrive excitation in guinea pig sino-atrial node

Summary The hypothesis that the pause that follows overdrive of the sino-atrial node (SAN) might be the net result of overdrive excitation and overdrive suppression was tested by studying rate and force patterns induced by overdrive in isolated guinea pig SAN superfused in vitro. In Tyrode solution, the pause is short and changes but little with longer or faster drives. In high [K⁺]_o solution, longer overdrives increase force percent-wise more than in Tyrode solution, shorten the pause and are followed by greater rate and force. When the SAN (quiescent in high [K⁺]_o) is driven at 6/min, faster overdrives are followed by stronger, slowly decreasing contractions. Alternating 10 s drives with 10 s pauses have little effect on force and rate in Tyrode solution, but progressively increase force and rate in high [K⁺]_o. Cesium has effects similar to high [K⁺]_o. High [Ca²⁺]_o increases force and in high [K⁺]_o increases the rate as well as it shortens the pause, whereas Ni²⁺ decreases force as well as rate and lengthens the pause. Barium dissociates the effects on force and rate.␣Lidocaine and tetrodotoxin decrease rate and force, and increase the pause duration. In overdrive excitation, the increase in rate is associated with an enhancement of diastolic voltage oscillations. It is concluded that in SAN the prevalence of Ca²⁺ load leads to overdrive excitation whereas the prevalence of␣Na⁺ load leads to overdrive suppression. In Tyrode solution, the pause after drive appears to be the net␣result of these two different mechanisms.     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

Ca2+/Calmodulin Kinase II and Decreases in Intracellular pH are Required to Activate K+ Channels After Substantial Swelling in Villus Epithelial Cells

To assess the activation of the charybdotoxin-insensitive K⁺ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH _i ), intracellular calcium ([Ca²⁺] _i ) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH _i and [Ca²⁺] _i of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca²⁺] _i to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca²⁺/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K⁺ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K⁺ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K⁺ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by Ca²⁺/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca²⁺] _i are a critical determinant of this activation. Received: 30 March 1999/Revised: 6 July 1999     

The role of cytosolic free calcium in the regulation of pyruvate dehydrogenase in synaptosomes

Calcium homeostasis and mitochondrial oxidative metabolism interact closely in brain and both processes are impaired during hypoxia. Since the regulation of the pyruvate dehydrogenase complex (PDHC) may link these two processes, the relation of cytosolic free calcium ([Ca²⁺]_i) to the activation state of PDHC (PDH_a) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca²⁺]_i. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca²⁺]_i and PDH_a. Furthermore, in the presence of KCN, PDH_a became more sensitive to K⁺ depolarization as indicated by larger changes in PDH_a than in [Ca²⁺]_i. The calcium ionophore Br-A23187 elevated [Ca²⁺]_i, but did not affect PDH_a. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a even if [Ca²⁺]_i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDH_a is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDH_a. These data also further support the notion that PDH_a is regulated by [Ca²⁺]_i as well as by other factors such as ATP. Our results are consistent with the concept that PDH_a in nerve endings may be affected by [Ca²⁺]_i and that these two processes are clearly linked.Abbreviations (PDH_a) Activation state of the pyruvate dehydrogenase complex - ([Ca²⁺]_i) cytosolic free calcium concentrations - (MOPS) 3(N-morpholino)propanesulfonic acid - (fura-2AM) fura-2 acetoxymethyl ester - (AABS) p-(p-aminophenylazo)benzene sulfonic acid - (PDHC) pyruvate dehydrogenase complex - (TES) N-tris{[hydroxymethyl]methyl}-2-amino-ethanesulfonic acid - (SNK-test) Student-Newman-Keuls test     

Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor-Differentiated PC12 Cells Induced by Chemical Hypoxia

Abstract: Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca²⁺ in growth cones and cell bodies. Hypoxia increased [Ca²⁺]_i and exaggerated its response to K⁺ depolarization in both parts of the cells. [Ca²⁺]_i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K⁺ or hypoxia. Ca²⁺-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca²⁺]_i following K⁺ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker ω-conotoxin GVIA (ω-CgTX) diminished K⁺-induced changes in [Ca²⁺]_i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω-CgTX diminished K⁺-induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω-CgTX diminished the [Ca²⁺]_i response to K⁺ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca²⁺ entry with K⁺ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca²⁺ regulation in the growth cone and cell body of the same cell.     

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.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Enhanced Depolarization-Evoked Calcium Signal and Reduced [ATP]/[ADP] Ratio Are Unrelated Events Induced by Oxidative Stress in Synaptosomes

Abstract: Oxidative insult elicited by hydrogen peroxide (H₂O₂) was previously shown to increase the basal intracellular Ca²⁺ concentration in synaptosomes. In the present study, the effect of H₂O₂ on the depolarization-evoked [Ca²⁺] signal was investigated. Pretreatment of synaptosomes with H₂O₂ (0.1–1 mM) augmented the [Ca²⁺] rise elicited by high K⁺ depolarization with essentially two alterations, the sudden sharp rise of [Ca²⁺]_i due to K⁺ depolarization is enhanced and, instead of a decrease to a stable plateau, a slow, steady rise of [Ca²⁺]_i follows the peak [Ca²⁺]_i. H₂O₂ in the same concentration range lowered the ATP level and the [ATP]/[ADP] ratio. When carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (1 µM) or rotenone (2 µM)/oligomycin (10 µM) was applied initially to block mitochondrial ATP production, the lowered [ATP]/[ADP] ratio was further reduced by subsequent addition of 0.5 mM H₂O₂. The decline of the [ATP]/[ADP] ratio was parallel with but could not explain the enhanced K⁺-evoked [Ca²⁺]_i signal, indicated by experiments in which the [ATP]/[ADP] ratio was decreased by FCCP (0.1 µM) or rotenone (2 µM) to a similar value as by H₂O₂ without causing any alteration in the [Ca²⁺]_i signal. These results indicate that H₂O₂-evoked oxidative stress, in its early phase, gives rise to a complex dysfunction in the Ca²⁺ homeostasis and, parallel with it, to an impaired energy status.     

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca²⁺)-activated K⁺ (K_Ca) channels regulate membrane excitability and are activated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), leading to membrane hyperpolarization. Most patch clamp experiments that measure K_Ca currents use steady-state [Ca²⁺] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca²⁺]_i changes dynamically, for example during [Ca²⁺]_i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca²⁺]_i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K_Ca subtype in isolation were used to simultaneously measure agonist-evoked [Ca²⁺]_i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca²⁺]_i oscillations induced a corresponding K_Ca current that faithfully followed the [Ca²⁺]_i in 13–50% of cells, suggesting a good synchronization. However, [Ca²⁺]_i and K_Ca current was much less synchronized in 50–76% of cells that exhibited Ca²⁺-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca²⁺]_i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca²⁺]_i and K_Ca current was least synchronized, 36% of total [Ca²⁺]_i spikes occurred without activating a corresponding K_Ca current spike, suggesting that BK_Ca channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca²⁺]_i and K_Ca current suggests that this relationship is more complex than could be predicted from steady-state [Ca²⁺]_i and K_Ca current. These phenomena may be important for encoding stimulus–response coupling in various cell types.