Differential Involvement of Ca2+ Channels in Survival and Neurite Outgrowth of Cultured Embryonic Cockroach Brain Neurons

2002 ◽  
Vol 88 (3) ◽  
pp. 1475-1490 ◽  
Author(s):  
Pascal Benquet ◽  
Janine Le Guen ◽  
Yves Pichon ◽  
François Tiaho
Keyword(s):  
Cell Death ◽  
Calcium Channels ◽  
Neurite Outgrowth ◽  
Calcium Current ◽  
Culture Media ◽  
Calf Serum ◽  
Free Calcium ◽  
Extracellular Potassium ◽  
Patch Clamp Technique ◽  
Brain Neurons

The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents. VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil. The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about −30 and −20 mV in high-potassium culture media). As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM. The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network. In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I Ba. This effect could not be prevented by addition of fetal calf serum to the culture medium. A similar effect was obtained using intra- or extracellular calcium chelating agents (10 μM BAPTA-AM or 10 mM EGTA). Quite unexpectedly, blockade of the P/Q-like (ω-Aga WA-sensitive) component of the calcium current by 500 nM of ω-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism. As expected from their lack of effect on I Ba, isradipine, nifedipine, and ω-CgTx GVIA did not induce cell death. When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion. In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM. However, these agents, as well as ω-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons. This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites. These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons. The differential contribution of the P/Q-like and R-like (ω-Aga WA-sensitive) calcium channels in these processes is discussed.

scholarly journals Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells.

1988 ◽  
Vol 92 (4) ◽  
pp. 531-548 ◽  
Author(s):  
D Kalman ◽  
P H O'Lague ◽  
C Erxleben ◽  
D L Armstrong
Keyword(s):  
Calcium Channels ◽  
Calcium Current ◽  
State Level ◽  
Gh3 Cells ◽  
Free Calcium ◽  
Pipette Solution ◽  
Calcium Dependent ◽  
Dependent Inactivation ◽  
Exogenous Calcium ◽  
Free Membrane

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.

Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons

1995 ◽  
Vol 74 (6) ◽  
pp. 2251-2257 ◽  
Author(s):  
I. Ehrlich ◽  
K. S. Elmslie
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
G Proteins ◽  
Calcium Current ◽  
Voltage Dependence ◽  
Sympathetic Neurons ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Kinetics Of

1. We studied the mechanism of voltage-dependent inhibition of N-type calcium current by norepinephrine (NE) and vasoactive intestinal peptide (VIP) in adult rat superior cervical ganglion (SCG) neurons using the whole cell patch-clamp technique. 2. The voltage dependence of inhibition is manifest in the reversal of inhibition by strong depolarization. We tested the hypothesis that this voltage dependence results from disruption of G proteins binding to calcium channels. According to this hypothesis, the kinetics of calcium current reinhibition following a strong depolarization should become faster for higher concentrations of active G proteins. 3. Assuming that larger inhibitions result from higher concentrations of active G proteins, we used different concentrations of NE to alter the amplitude of inhibition and, thus, the active G protein concentration. We found that the kinetics of reinhibition at -80 mV following a depolarizing pulse to +80 mV were faster for larger inhibitions. 4. VIP induces voltage-dependent inhibition of N-current via a different G protein (Gs) than that of NE (Go). We found that the effect of VIP on reinhibition kinetics was identical to that produced by NE. 5. Combined application of NE and VIP did not greatly increase the amplitude of the inhibition but significantly increased the rate of reinhibition. Thus NE plus VIP appear to greatly increase the concentration of the molecule binding to the channel (G protein according to the hypothesis). 6. The kinetics of calcium current disinhibition during strong depolarization (step to +80 mV) did not change with the size of the inhibition induced by NE, VIP or application of NE and VIP together. 7. Both the concentration-dependent reinhibition kinetics and concentration-independent disinhibition kinetics are consistent with the hypothesis that active G proteins bind directly to N-type calcium channels to modulate their activity in rat sympathetic neurons.

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the α1-adrenoceptors

2003 ◽  
Vol 81 (3) ◽  
pp. 234-246 ◽  
Author(s):  
Ghassan Bkaily ◽  
Nesrine El-Bizri ◽  
Michel Bui ◽  
Rami Sukarieh ◽  
Danielle Jacques ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Calcium Channels ◽  
Adrenergic Receptor ◽  
Calcium Current ◽  
Single Cells ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Extracellular Loop ◽  
Cell Contraction ◽  
Patch Clamp Technique

The effects of methoxamine, a selective α1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of α1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of α1-adrenoreceptors significantly increased the L-type calcium current (ICa(L)) but had no effect on the T-type calcium current (ICa(T)), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective α1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in ICa(L), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional α1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of α1-receptor agonists like methoxamine.Key words: α1-adrenoceptor, autoantibody, heart cells, calcium, ion channels, confocal microscopy.

ω-AgaIVA–Sensitive (P/Q-type) and –Resistant (R-type) High-Voltage–Activated Ba2+ Currents in Embryonic Cockroach Brain Neurons

1999 ◽  
Vol 82 (5) ◽  
pp. 2284-2293 ◽  
Author(s):  
Pascal Benquet ◽  
Janine Le Guen ◽  
Govindan Dayanithi ◽  
Yves Pichon ◽  
François Tiaho
Keyword(s):  
Steady State ◽  
Calcium Channels ◽  
Primary Culture ◽  
High Voltage ◽  
Reversal Potential ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Brain Neurons ◽  
Voltage Dependent

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba2+ currents ( I Ba) were characterized in embryonic cockroach brain neurons in primary culture. I Ba was characterized by a threshold of approximately −30 mV, a maximum at ∼0 mV, and a reversal potential near +40 mV. Varying the holding potential from −100 to −40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V 0.5 of −10 ± 2 (SE) mV and −30 ± 1 mV, respectively. I Ba was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 μM) and antagonist isradipine (10 μM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner ( IC 50 = 170 μM). The toxin ω-CgTxGVIA (1 μM) had no significant effect on IBa. Micromolar doses of ω-CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 μM, 38% of the peak current was blocked after 1 h. In contrast, IBa was potently and irreversibly blocked by nanomolar concentrations of ω-AgaTxIVA in ∼81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0.4–1 μM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC 50 of 17 nM and a Hill coefficient ( n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific ( IC 50 = 9 nM; n = 1) and nonspecific ( IC 50 = 900 nM; n = 1) ω-AgaTxIVA–sensitive components. In the remaining 19% of the neurons, concentrations ≥100 nM ω-AgaTxIVA had no visible effect on IBa. On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage–activated (HVA) calcium channels: a specific ω-AgaTxIVA–sensitive component and DHP-, ω-CgTxGVIA–, and ω-AgaTxIVA–resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.

scholarly journals Long-QT founder variant T309I-Kv7.1 with dominant negative pattern may predispose delayed afterdepolarizations under β-adrenergic stimulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Iva Synková ◽  
Markéta Bébarová ◽  
Irena Andršová ◽  
Larisa Chmelikova ◽  
Olga Švecová ◽  
...  
Keyword(s):  
Calcium Current ◽  
Cell Model ◽  
Dominant Negative ◽  
Qtc Interval ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Long Qt ◽  
Channel Trafficking ◽  
Whole Cell Patch Clamp ◽  
Delayed Afterdepolarizations

AbstractThe variant c.926C > T (p.T309I) in KCNQ1 gene was identified in 10 putatively unrelated Czech families with long QT syndrome (LQTS). Mutation carriers (24 heterozygous individuals) were more symptomatic compared to their non-affected relatives (17 individuals). The carriers showed a mild LQTS phenotype including a longer QTc interval at rest (466 ± 24 ms vs. 418 ± 20 ms) and after exercise (508 ± 32 ms vs. 417 ± 24 ms), 4 syncopes and 2 aborted cardiac arrests. The same haplotype associated with the c.926C > T variant was identified in all probands. Using the whole cell patch clamp technique and confocal microscopy, a complete loss of channel function was revealed in the homozygous setting, caused by an impaired channel trafficking. Dominant negativity with preserved reactivity to β-adrenergic stimulation was apparent in the heterozygous setting. In simulations on a human ventricular cell model, the dysfunction resulted in delayed afterdepolarizations (DADs) and premature action potentials under β-adrenergic stimulation that could be prevented by a slight inhibition of calcium current. We conclude that the KCNQ1 variant c.926C > T is the first identified LQTS-related founder mutation in Central Europe. The dominant negative channel dysfunction may lead to DADs under β-adrenergic stimulation. Inhibition of calcium current could be possible therapeutic strategy in LQTS1 patients refractory to β-blocker therapy.

scholarly journals Oxytocin Inhibits T-Type Calcium Current of Human Decidual Stromal Cells

2005 ◽  
Vol 90 (7) ◽  
pp. 4191-4197 ◽  
Author(s):  
Bo Liu ◽  
Stephen J. Hill ◽  
Raheela N. Khan
Keyword(s):  
Stromal Cells ◽  
Calcium Current ◽  
Elective Cesarean Section ◽  
Dependent Manner ◽  
Uterine Contractility ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Decidual Cells ◽  
Elective Cesarean

Abstract Context: Little is known about the crosstalk between the decidua and myometrium in relation to human labor. The hormone oxytocin (OT) is considered to be a key mediator of uterine contractility during parturition, exerting some of its effects through calcium channels. Objective: The objective was to characterize the effect of OT on the T-type calcium channel in human decidual stromal cells before and after the onset of labor. Design: The nystatin-perforated patch-clamp technique was used to record inward T-type calcium current (ICa(T)) from acutely dispersed decidual stromal cells obtained from women at either elective cesarean section [CS (nonlabor)] or after normal spontaneous vaginal delivery [SVD (labor)]. Setting: These studies took place at the University of Nottingham Medical School. Results: I Ca(T) of both SVD and CS cells were blocked by nickel (IC50 of 5.6 μm) and cobalt chloride (1 mm) but unaffected by nifedipine (10 μm). OT (1 nm to 3.5 μm) inhibited ICa(T) of SVD cells in a concentration-dependent manner, with a maximal inhibition of 79.0% compared with 26.2% in decidual cells of the CS group. OT-evoked reduction of ICa(T) was prevented by preincubation with the OT antagonist L371,257 in the SVD but not CS group. OT, in a concentration-dependent manner, displaced the steady-state inactivation curve for ICa(T) to the left in the SVD group with no significant effect on curves of the CS group. Conclusion: Inhibition of ICa(T) by OT in decidual cells obtained during labor may signify important functional remodeling of uterine signaling during this period.

scholarly journals Cell death delineates axon pathways in the hindlimb and does so independently of neurite outgrowth

1988 ◽  
Vol 130 (2) ◽  
pp. 558-572 ◽  
Author(s):  
Kathryn W. Tosney ◽  
Sally Schroeter ◽  
Jennifer A. Pokrzywinski
Keyword(s):  
Cell Death ◽  
Neurite Outgrowth

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

scholarly journals L-type calcium channels refine the neural population code of sound level

2016 ◽  
Vol 116 (6) ◽  
pp. 2550-2563 ◽  
Author(s):  
Calum Alex Grimsley ◽  
David Brian Green ◽  
Shobhana Sivaramakrishnan
Keyword(s):  
Calcium Channels ◽  
Calcium Current ◽  
Dynamic Range ◽  
Sound Level ◽  
Neural Population ◽  
Total Calcium ◽  
Local Networks ◽  
Sound Levels ◽  
Local Circuits

The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1–1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level.

scholarly journals Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hongfei Chen ◽  
Zhousheng Jin ◽  
Fangfang Xia ◽  
Zhijian Fu
Keyword(s):  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

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