Flufenamic Acid Affects Multiple Currents and Causes Intracellular Ca2+ Release in Aplysia Bag Cell Neurons

2008 ◽  
Vol 100 (1) ◽  
pp. 38-49 ◽  
Author(s):  
Kate E. Gardam ◽  
Julia E. Geiger ◽  
Charlene M. Hickey ◽  
Anne Y. Hung ◽  
Neil S. Magoski
Keyword(s):  
Channel Blocker ◽  
Reversal Potential ◽  
Membrane Conductance ◽  
Cell Voltage ◽  
Cation Channel ◽  
Flufenamic Acid ◽  
Whole Cell ◽  
Positive Shift ◽  
Intracellular Ca ◽  
Bag Cell Neurons

Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward ( Iout) or inward ( Iin) currents. Iout had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. Iin developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of Iin, as strong intracellular Ca2+ buffering effectively eliminated Iin but did not alter Iout. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.

GABA-Activated Conductance in Cultured Rat Inferior Colliculus Neurons

1997 ◽  
Vol 77 (2) ◽  
pp. 994-1002 ◽  
Author(s):  
Hidenobu Hosomi ◽  
Masahiro Mori ◽  
Mutsuo Amatsu ◽  
Yasuhiro Okada
Keyword(s):  
Inferior Colliculus ◽  
Voltage Clamp ◽  
Reversal Potential ◽  
Membrane Conductance ◽  
External Solution ◽  
Cell Voltage ◽  
Equilibrium Potential ◽  
Fluorescence Measurements ◽  
Intracellular Ca ◽  
Induced Currents

Hosomi, Hidenobu, Masahiro Mori, Mutsuo Amatsu, and Yasuhiro Okada. GABA-activated conductance in cultured rat inferior colliculus neurons. J. Neurophysiol. 77: 994–1002, 1997. With the use of a whole cell voltage-clamp technique and fura-2 fluorescence measurements, the actions of γ-aminobutyric acid (GABA) on cultured neurons from rat inferior colliculus were investigated. GABA (10–1,000 μM) induced currents in neurons held under voltage clamp that were inhibited by bicuculline (20 μM). Muscimol (100 μM) also evoked the currents, whereas baclofen (100 μM) affected neither the holding currents nor K+ conductance due to depolarizing pulses. The current density–voltage relation of GABA-induced currents, with equal concentrations of Cl− in the internal and external solutions, reversed near 0 mV. Reduction of the internal Cl− concentration shifted the reversal potential in the negative direction as predicted from the Cl− equilibrium potential. Baclofen did not affect Ca2+ conductance due to depolarizing pulses. The extracellular application of 150 mM KCl or 1.0 mM glutamate increased the intracellular Ca2+ concentration ([Ca2+]i) of cultured inferior colliculus neurons only when neurons were bathed in a Ca2+-containing external solution. However, GABA (1.0 mM) failed to increase [Ca2+]i at all concentrations of external Ca2+ used, indicating that GABA neither depolarized the cultured inferior colliculus neurons sufficiently to activate the voltage-dependent Ca2+ conductances nor evoked Ca2+ release from intracellular stores. These results suggest that in cultured rat inferior colliculus neurons, GABAA receptor channels may be predominantly responsible for the membrane conductance evoked by GABA and subsequent hyperpolarization of the neurons.

Activity-Dependent Initiation of a Prolonged Depolarization in Aplysia Bag Cell Neurons: Role for a Cation Channel

2007 ◽  
Vol 97 (3) ◽  
pp. 2465-2479 ◽  
Author(s):  
Anne Y. Hung ◽  
Neil S. Magoski
Keyword(s):  
Pulse Duration ◽  
Action Potentials ◽  
Reversal Potential ◽  
Egg Laying ◽  
Cation Channel ◽  
Flufenamic Acid ◽  
Depolarization Current ◽  
Calmodulin Kinase ◽  
Current Voltage ◽  
Bag Cell Neurons

The translation of prior activity into changes in excitability is essential for memory and the initiation of behavior. After brief synaptic input, the bag cell neurons of Aplysia californica undergo a nearly 30-min afterdischarge to release egg-laying hormone. The present study examines a prolonged depolarization in cultured bag cell neurons. A 5-Hz, 10-s action potential train elicited a depolarization of about 10 mV, which lasted ≤30 min and was reduced by calmodulin kinase inhibition. Very broad action potentials (resulting from TEA application) decreased prolonged depolarization amplitude, indicating that strong Ca2+ influx did not necessarily promote the response. The prolonged depolarization current ( IPD) was recorded after 5-Hz, 10-s trains of square voltage pulses of varying duration (10–150 ms). Despite Ca2+ influx increasing steadily with pulse duration, IPD was most reliably initiated at 100 ms, suggesting a Ca2+ window or limit exists for triggering IPD. Consistent with this, modestly broader action potentials, evoked by lengthening the train current-pulse duration, resulted in smaller prolonged depolarizations. With respect to the properties of IPD, it displayed a linear current–voltage relationship with a reversal potential of about −45 mV that was shifted to approximately −25 mV by lowering internal K+ or about −56 mV by lowering external Na+ and Ca2+. IPD was blocked by Gd3+, but was not antagonized by MDL-123302A, SKF-96365, 2-APB, tetrodotoxin, or flufenamic acid. Optimal Ca2+ influx may activate calmodulin kinase and a voltage-independent, nonselective cation channel to initiate the prolonged depolarization, thereby contributing to the afterdischarge and reproduction.

Acute reduction in whole cell conductance in anoxic turtle brain

1999 ◽  
Vol 277 (3) ◽  
pp. R887-R893 ◽  
Author(s):  
H. S. Ghai ◽  
L. T. Buck
Keyword(s):  
Brain Slices ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Recording Electrode ◽  
Whole Cell ◽  
Artificial Cerebrospinal Fluid ◽  
Intracellular Ca ◽  
Specific Antagonist ◽  
Acute Changes ◽  
Dose Dependent

We tested the effect of anoxia, a “mimic” turtle artificial cerebrospinal fluid (aCSF) consisting of high Ca2+ and Mg2+ concentrations and low pH and adenosine perfusions, on whole cell conductance ( G w) in turtle brain slices using a whole cell voltage-clamp technique. With EGTA in the recording electrode, anoxic or adenosine perfusions did not change G w significantly (values range between 2.15 ± 0.24 and 3.24 ± 0.56 nS). However, perfusion with normoxic or anoxic mimic aCSF significantly decreased G w. High [Ca2+] (4.0 or 7.8 mM) perfusions alone could reproduce the changes in G w found with the mimic perfusions. With the removal of EGTA from the recording electrode, G wdecreased significantly during both anoxic and adenosine perfusions. The A1-receptor agonist N 6-cyclopentyladenosine reduced G w in a dose-dependent manner, whereas the A1-receptor specific antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked both the adenosine- and anoxic-mediated changes in G w. These data suggest a mechanism involving A1-receptor-mediated changes in intracellular [Ca2+] that result in acute changes in G w with the onset of anoxia.

Altered contractile and ion channel function in rabbit portal vein with dietary atherosclerosis

1995 ◽  
Vol 268 (6) ◽  
pp. H2522-H2530 ◽  
Author(s):  
R. H. Cox ◽  
T. N. Tulenko
Keyword(s):  
Portal Vein ◽  
Ion Channel ◽  
Plasma Cholesterol ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Molar Ratio ◽  
Whole Cell ◽  
Ion Channel Properties ◽  
Rabbit Portal Vein ◽  
K Currents

This study was performed to determine the effects of dietary atherosclerosis on the pharmacology and ion channel properties of rabbit portal vein (PV). New Zealand White rabbits were fed normal rabbit chow +/- 2% cholesterol for 10 wk. Contractions to norepinephrine (NE) and serotonin were studied under isometric conditions with longitudinal strips. Ca2+ and K+ currents (ICa and IK, respectively) were recorded in freshly dispersed myocytes by whole cell voltage clamp methods. Cholesterol feeding increased total plasma cholesterol levels from 28.4 +/- 5.2 to 1,387 +/- 172 mg/dl as well as the cholesterol-to-phospholipid molar ratio of the PV from 0.34 +/- 0.02 to 0.66 +/- 0.08. Only maximum contractile responses to serotonin were larger in atherosclerotic PV when normalized to the maximum KCl response. Concentration-active stress curves of the atherosclerotic PV to NE and serotonin were shifted to the left. Maximum values of ICa were larger in myocytes from atherosclerotic compared with control animals (4.4 +/- 0.4 vs. 3.1 +/- 0.2 pA/pF, P < 0.05). The voltage dependence of activation and availability of ICa was shifted toward more negative potentials by approximately 10 mV. Whole cell K+ currents were smaller in atherosclerotic myocytes. At a test voltage of +20 mV, IK averaged 14.9 +/- 2.8 pA/pF in control compared with 7.7 +/- 0.8 pA/pF in atherosclerotic myocytes from a holding potential of -80 mV with external Ca2+ concentration of 5 mM. The reversal potential for IK tail currents was significantly less negative in atherosclerotic myocytes (-70 +/- 1 vs. -64 +/- 1 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stretch-activated whole cell currents in adult rat cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H548-H557 ◽  
Author(s):  
Tao Zeng ◽  
Glenna C. L. Bett ◽  
Frederick Sachs
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Cellular Level ◽  
Whole Cell ◽  
Adult Rat ◽  
Longitudinal Stretch ◽  
Single Channel Currents ◽  
Channel Currents

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 μM Gd3+ but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately −6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 − 5.0SL (μm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

Mitochondrial Ca2+ Activates a Cation Current in Aplysia Bag Cell Neurons

2010 ◽  
Vol 103 (3) ◽  
pp. 1543-1556 ◽  
Author(s):  
Charlene M. Hickey ◽  
Julia E. Geiger ◽  
Chris J. Groten ◽  
Neil S. Magoski
Keyword(s):  
Endoplasmic Reticulum ◽  
Ion Channels ◽  
Time Course ◽  
Reversal Potential ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Inward Current ◽  
Cation Current ◽  
Intracellular Ca ◽  
Bag Cell Neurons

Ion channels may be gated by Ca2+ entering from the extracellular space or released from intracellular stores—typically the endoplasmic reticulum. The present study examines how Ca2+ impacts ion channels in the bag cell neurons of Aplysia californica. These neuroendocrine cells trigger ovulation through an afterdischarge involving Ca2+ influx from Ca2+ channels and Ca2+ release from both the mitochondria and endoplasmic reticulum. Liberating mitochondrial Ca2+ with the protonophore, carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone (FCCP), depolarized bag cell neurons, whereas depleting endoplasmic reticulum Ca2+ with the Ca2+-ATPase inhibitor, cyclopiazonic acid, did not. In a concentration-dependent manner, FCCP elicited an inward current associated with an increase in conductance and a linear current/voltage relationship that reversed near −40 mV. The reversal potential was unaffected by changing intracellular Cl−, but left-shifted when extracellular Ca2+ was removed and right-shifted when intracellular K+ was decreased. Strong buffering of intracellular Ca2+ decreased the current, although the response was not altered by blocking Ca2+-dependent proteases. Furthermore, fura imaging demonstrated that FCCP elevated intracellular Ca2+ with a time course similar to the current itself. Inhibiting either the V-type H+-ATPase or the ATP synthetase failed to produce a current, ruling out acidic Ca2+ stores or disruption of ATP production as mechanisms for the FCCP response. Similarly, any involvement of reactive oxygen species potentially produced by mitochondrial depolarization was mitigated by the fact that dialysis with xanthine/xanthine oxidase did not evoke an inward current. However, both the FCCP-induced current and Ca2+ elevation were diminished by disabling the mitochondrial permeability transition pore with the alkylating agent, N-ethylmaleimide. The data suggest that mitochondrial Ca2+ gates a voltage-independent, nonselective cation current with the potential to drive the afterdischarge and contribute to reproduction. Employing Ca2+ from mitochondria, rather than the more common endoplasmic reticulum, represents a diversification of the mechanisms that influence neuronal activity.

Protein Binding-dependent Decreases in hERG Channel Blocker Potency Assessed by Whole-Cell Voltage Clamp in Serum

2010 ◽  
Vol 55 (4) ◽  
pp. 368-376 ◽  
Author(s):  
Michael Margulis ◽  
Steve Sorota ◽  
Inhou Chu ◽  
Anthony Soares ◽  
Tony Priestley ◽  
...  
Keyword(s):  
Protein Binding ◽  
Voltage Clamp ◽  
Channel Blocker ◽  
Cell Voltage ◽  
Herg Channel ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp

Sour Transduction Involves Activation of NPPB-Sensitive Conductance in Mouse Taste Cells

1998 ◽  
Vol 80 (4) ◽  
pp. 1852-1859 ◽  
Author(s):  
Takenori Miyamoto ◽  
Rie Fujiyama ◽  
Yukio Okada ◽  
Toshihide Sato
Keyword(s):  
Citric Acid ◽  
Channel Blocker ◽  
Reversal Potential ◽  
Cation Channel ◽  
Induced Responses ◽  
Induced Current ◽  
Sour Taste ◽  
Taste Cells ◽  
Mouse Taste ◽  
Reversal Potentials

Miyamoto, Takenori, Rie Fujiyama, Yukio Okada, and Toshihide Sato. Sour transduction involves activation of NPPB-sensitive conductance in mouse taste cells. J. Neurophysiol. 80: 1852–1859, 1998. We examined the sour taste transduction mechanism in the mouse by applying whole cell patch-clamp technique to nondissociated taste cells from the fungiform papillae. Localized stimulation with 0.5 M NaCl and 25 mM citric acid (pH 3.0) of the apical membrane enabled us to obtain responses from single taste cells under a quasi-natural condition. Of 28 taste cells examined, 11 cells (39%) responded to 0.5 M NaCl alone and 2 cells (7%) responded to 25 mM citric acid alone, indicating the presence of salty- and sour-specific taste cells. Ten cells (36%) responded to both NaCl and citric acid and 5 cells (18%) responded to neither salt nor citric acid. Amiloride reversibly suppressed NaCl-induced responses in mouse taste cells but not citric acid-induced responses. On the other hand, a Cl− channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), reversibly suppressed all the citric-acid-induced responses. Most of the NaCl-induced current responses displayed an inwardly rectifying property, whereas all the citric-acid-induced responses displayed an outwardly rectifying property. The reversal potential for NPPB-sensitive component in citric-acid-induced current responses was −2 ± 7 mV (mean ± SE, n = 4), which was close to the equilibrium potential of Cl− ( E Cl), whereas the reversal potential for NPPB-insensitive component was 34 ± 8 mV ( n = 4). The reversal potential of citric-acid-induced current responses (19 ± 8 mV, n = 4) was mostly present at the middle point between reversal potentials of NPPB-sensitive and -insensitive current components. In some taste cells, an inorganic cation channel blocker, Cd2+, suppressed citric-acid-induced responses, but an inorganic stretch-activated cation channel blocker, Gd3+, did not affect these responses. These results suggest that salt- and acid-induced responses were mediated by differential transduction mechanisms in mouse taste cells and that NPPB-sensitive Cl− channels play a more important role to sour taste transduction rather than amiloride-sensitive Na+ channels. However, the fact that the reversal potentials of citric-acid-induced responses had more positive than E Cl suggests that Ca2+ or H+ permeable and poorly selective cation channels, which should be amiloride insensitive, may be activated by citric acid.

Activation of a Calcium-Activated Cation Current During Epileptiform Discharges and Its Possible Role in Sustaining Seizure-Like Events in Neocortical Slices

2004 ◽  
Vol 92 (2) ◽  
pp. 862-872 ◽  
Author(s):  
Yitzhak Schiller
Keyword(s):  
Brain Slices ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Epileptic Seizures ◽  
Flufenamic Acid ◽  
Calcium Stores ◽  
Epileptiform Discharges ◽  
Cation Current ◽  
Paroxysmal Depolarization Shift ◽  
Internal Calcium

Epileptic seizures are composed of recurrent bursts of intense firing separated by periods of electrical quiescence. The mechanisms responsible for sustaining seizures and generating recurrent bursts are yet unclear. Using whole cell voltage recordings combined with intracellular calcium fluorescence imaging from bicuculline (BCC)-treated neocortical brain slices, I showed isolated paroxysmal depolarization shift (PDS) discharges were followed by a sustained afterdepolarization waveform (SADW) with an average peak amplitude of 3.3 ± 0.9 mV and average half-width of 6.2 ± 0.6 s. The SADW was mediated by the calcium-activated nonspecific cation current ( Ican) as it had a reversal potential of –33.1 ± 6.8 mV, was unaffected by changing the intracellular chloride concentrations, was markedly diminished by buffering [Ca2+]i with intracellular bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid (BAPTA), and was reversibly abolished by the Ican blocker flufenamic acid (FFA). The Ca2+ influx responsible for activation of Ican was mediated by both N-methyl-d-aspartate-receptor channels, voltage-gated calcium channels and, to a lesser extent, internal calcium stores. In addition to isolated PDS discharges, BCC-treated brain slices also produced seizure-like events, which were accompanied by a prolonged depolarizing waveform underlying individual ictal bursts. The similarities between the initial part of this waveform and the SADW and the fact it was markedly reduced by buffering [Ca2+]i with BAPTA strongly suggested it was mediated, at least in part, by Ican. Addition of FFA reversibly eliminated recurrent bursting, and transformed seizure-like events into isolated PDS responses. These results indicated Ican was activated during epileptiform discharges and probably participated in sustaining seizure-like events.

Properties of GABA-activated whole-cell currents in bipolar cells of the rat retina

1990 ◽  
Vol 4 (4) ◽  
pp. 349-357 ◽  
Author(s):  
Hermes H. Yeh ◽  
Maria B. Lee ◽  
Jane E. Cheun
Keyword(s):  
Reversal Potential ◽  
Cell Voltage ◽  
Bipolar Cells ◽  
Whole Cell ◽  
Rat Retina ◽  
Enzymatic Dissociation ◽  
Adult Rat ◽  
Cell Responses ◽  
Conductance Increase ◽  
Distinct Morphology

AbstractThis paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1–100 μM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet &lsqb;CI] paralleled that predicted based on the Nernst equation for Cl−. IGABA was mimicked by muscimol (5–20 μM) and antagonized by bicuculline (20–100 μM). Baclofen (0.1–1.0 mM) produced no apparent conductance change. “Hot spots” of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine.In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.

Sign in / Sign up

Export Citation Format

Share Document