Biomedical subjects

J J Pancrazio

Publications and source records attributed to J J Pancrazio.

At least 37 records · Page 2Linked to original sources

PubMed1999

Characterization of rat spinal cord neurons cultured in defined media on microelectrode arrays.

Previous efforts to utilize mammalian spinal cord neurons as biosensor elements have relied on neuronal: glial co-cultures maintained in serum-containing media. We have examined the feasibility of culturing primary spinal cord neurons in serum-free medium, modified for neuronal longevity, on fabricated microelectrode arrays. Embryonic day 15 rat spinal cord cells were plated on trimethoxysilyl-propyldiethylenetriamine coated microelectrode arrays comprised of gold recording sites passivated with silicon nitride. Immunocytochemistry was performed to verify the presence of neurons and quantitatively assess astrocytes using antibodies against glial fibrillary acidic protein on the silicon nitride substrates. Modifications to culture media enabled viable neuronal culture to extend from approximately 14 days in vitro (DIV) to 40 DIV on the arrays containing only 1.1 +/- 0.5% (mean +/- SEM) astrocytes. Extracellular recording revealed tetrodotoxin-sensitive spontaneous electrical activity from the enriched neuronal culture. Threshold detection of extracellular potentials showed an increase in spike rate as a function of glutamate concentration with neurotoxicity at elevated levels. This approach suggests that functional measures related to biosensor applications, pharmacological screening, or the evaluation of neurological disease models can be implemented in a defined culture system.

Action Potentials↗

PubMed1999

Kir 4.1 channel expression in neuroblastomaxglioma hybrid NG108-15 cell line.

To study a possible involvement of inwardly rectifying K+ 4.1 (Kir 4. 1) channels in neural cell development, RT-PCR, immunocytochemistry and whole-cell patch-clamp techniques were used to assess expression of Kir 4.1 channels in proliferating and differentiated NG108-15 cells. RT-PCR revealed co-expression of Kir 4.1 and rat ether-a-go-go-related gene (R-ERG) mRNAs in both proliferating and differentiated cells. The relative Kir 4.1 mRNA concentration increased markedly as cells progressed from undifferentiated to differentiated cells. Kir 4.1-immunoreactivity was barely detectable in undifferentiated cells, but clearly detected in differentiated cells, indicating that Kir 4.1 gene and protein expressions are developmentally regulated. However, corresponding Kir 4.1 current could not be detected in differentiated cells using whole-cell patch-clamp recording. The 'silent' channel/receptor, often found in tumor cells, may carry genetic defects, which prevent functional expression of the channel. NG108-15 may serve as unique model for studying the relationship between the expression of an ion channel gene and the electrophysiological phenotype it encodes.

Animals↗

PubMed1999

Volatile anesthetic sensitivity of T-type calcium currents in various cell types.

UNLABELLED: We evaluated the effects of volatile anesthetics on T-type calcium current (ICa,T) present in four different cell types using the whole cell version of the patch clamp technique. In dorsal root ganglion neurons and in two neuroendocrine cells--adrenal glomerulosa cells (AG) and thyroid C-cells--ICa,T was reversibly decreased by volatile anesthetics at clinically relevant concentrations, with isoflurane and enflurane being more potent that halothane. In AG cells, the most sensitive cell type tested, ICa,T was reduced 47%+/-4% (n = 6) by isoflurane (0.7 mM) and 56%+/-2% (n = 5) by enflurane (1.2 mM), but by only 24%+/-1% (n = 5; P < 0.05) by halothane (0.7 mM). Isoflurane caused a significant increase in the rate of deactivation of ICa,T in AG cells. In ventricular myocytes, however, ICa,T was much less sensitive to both isoflurane and halothane. The differential sensitivity of ICa,T in various cell types to the anesthetics may reflect differences in the channels expressed in these tissues or differences in the cellular intermediates involved in anesthetic action. Depression of ICa,T in neuronal cells may contribute to anesthetic action through decreases in cellular excitability. IMPLICATIONS: Using the patch clamp technique, we showed that T-type calcium channels, which promote cellular excitability, are inhibited by volatile anesthetics in neuronal and neuroendocrine cells, but not in ventricular myocytes. Inhibition of neuronal T-type channels may contribute to the mechanism of action of volatile anesthetics.

Anesthetics, Inhalation↗

PubMed1999

A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.

The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Animals↗

PubMed1999

Inhibition of spontaneous GABAergic transmission by trimethylolpropane phosphate.

Trimethylolpropane phosphate (TMPP) is a neuroactive organophosphate generated during partial pyrolysis of a synthetic ester turbine engine lubricant. While TMPP had been shown to have little affinity for acetylcholinesterase, previous binding studies and 6Cl- flux measurements have implicated TMPP as an antagonist of GABA, receptor/Cl- channels. Using the whole-cell patch clamp method, spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by bicuculline-sensitive GABA(A) receptors were measured in neurons cultured from the rat embryonic hippocampus for 13-21 days. Experiments were conducted in the presence of tetrodotoxin and 6-cyano-7-nitroquinoxaline to inhibit spontaneous presynaptic action potentials and glutamate transmission, respectively, thus isolating GABAergic sIPSCs for study. TMPP induced a concentration-dependent inhibition of sIPSC amplitude and frequency suggesting both postsynaptic and presynaptic actions. Administration of 5 microM TMPP reversibly diminished sIPSC amplitude by 23 +/- 8% (mean SEM, n=5 cells) while markedly decreasing the mean sIPSC frequency by 40 +/- 2% (n=5). The mean time constant of sIPSC decay was reversibly decreased by 20 +/- 4% (n=3) in the presence of 20 microM TMPP, suggesting an increase in the rate of inactivation. To directly verify the blockade of ionotropic GABA receptors by TMPP, the effects of TMPP were examined on whole-cell Cl- current responses activated by exogenous GABA. Administration of TMPP (5 microM) depressed peak whole-cell GABA-induced currents to 73 1% (n=4) of control levels, consistent with the results on sIPSC amplitude. Our data directly demonstrate that TMPP directly inhibits GABA(A) receptor function, as indicated by the blockade of whole-cell GABA-mediated Cl- current and the reduction in sIPSC amplitude. Furthermore, TMPP exerts a presynaptic effect on GABAergic transmission, as evidenced by the reduction in sIPSC frequency, which may be independent of a GABA(A) receptor. The molecular basis for the presynaptic action of TMPP remains to be elucidated.

Animals↗

PubMed1998

Central neuronal synapse formation on micropatterned surfaces.

Controlling synapse formation is a key to patterning of neurons into functional circuits and networks in vitro. However, the process of synapse formation among neurons grown on artificial surfaces is relatively unstudied. We cultured embryonic hippocampal cells on trimethoxysilylpropyl-diethylenetriamine (DETA) and tridecafluoro-1, 1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F), and on patterns composed of DETA lines separated by 13F spaces. For comparison, neurons were concurrently plated on surfaces coated with uniform poly-d-lysine (PDL). Pre- and postsynaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Spontaneous (SPCs) and evoked (EPCs) postsynaptic currents were recorded using dual patch-clamp techniques. We found that DETA promoted synapse formation, whereas evidence for synapse formation on 13F was barely detected. MAP-2+ neuronal soma and rapidly growing dendrites were co-localized with synapsin I puncta faithfully along DETA lines. The expression of synapsin I puncta, and MAP-2+ soma and dendrites correlated well with the appearance of SPCs. Synapsin I, MAP-2 and SPCs emerged together at days 3-4 and increased at day 7, when EPCs appeared. Synaptic signals occurring during 4-7 days in culture were all GABAergic. These results indicate that fully functional synapses are formed on silane surfaces, demonstrating the suitability of patterned silane surfaces for organizing synapse formation in vitro.

Animals↗

PubMed1998

Multiple ionic mechanisms mediate inhibition of rat motoneurones by inhalation anaesthetics.

1. We studied the effects of inhalation anaesthetics on the membrane properties of hypoglossal motoneurones in a neonatal rat brainstem slice preparation. 2. In current clamp, halothane caused a membrane hyperpolarization that was invariably associated with decreased input resistance; in voltage clamp, halothane induced an outward current and increased input conductance. Qualitatively similar results were obtained with isoflurane and sevoflurane. 3. The halothane current reversed near the predicted K+ equilibrium potential (EK) and was reduced in elevated extracellular K+ and in the presence of Ba2+ (2 mM). Moreover, the Ba2+-sensitive component of halothane current was linear and reversed near EK. The halothane current was not sensitive to glibenclamide or thyrotropin-releasing hormone (TRH). Therefore, the halothane current was mediated, in part, by activation of a Ba2+-sensitive K+ current distinct from the ATP- and neurotransmitter-sensitive K+ currents in hypoglossal motoneurones. 4. Halothane also inhibited Ih, a hyperpolarization-activated cationic current; this was primarily due to a decrease in the absolute amount of current, although halothane also caused a small, but statistically significant, shift in the voltage dependence of Ih activation. Extracellular Cs+ (3 mM) blocked Ih and a component of halothane-sensitive current with properties reminiscent of Ih. 5. A small component of halothane current, resistant to Ba2+ and Cs+, was observed in TTX-containing solutions at potentials depolarized to approximately -70 mV. Partial Na+ substitution by N-methyl-D-glucamine completely abolished this residual current, indicating that halothane also inhibited a TTX-resistant Na+ current active near rest potentials. 6. Thus, halothane activates a Ba2+-sensitive, relatively voltage-independent K+ current and inhibits both Ih and a TTX-insensitive persistent Na+ current in hypoglossal motoneurones. These effects of halothane decrease motoneuronal excitability and may contribute to the immobilization that accompanies inhalation anaesthesia.

Anesthetics, Inhalation↗

PubMed1998

Description and demonstration of a CMOS amplifier-based-system with measurement and stimulation capability for bioelectrical signal transduction.

An extracellular recording system incorporating an electrode array and an amplifier/stimulator CMOS chip is described and characterized. Important features of this custom VLSI chip include 16 instrumentation amplifiers with a gain of 50 and the incorporation of a cross-point array allowing designation of an extracellular microelectrode as either a stimulator or sensor. The planar array consisted of 32 microelectrodes, 14 microns in diameter, and four larger reference electrodes. Microelectrodes, interconnecting traces, and bond pads were patterned with a 500-nm layer of gold. The interconnecting traces were passivated with a 1-micron thick layer of silicon nitride to provide chemical and electrical insulation and microelectrode impedance was lowered utilizing electrode position of platinum black. The amplifier exhibited a nearly flat frequency response with high pass and low pass corner frequencies of 0.7 Hz and 50 kHz, respectively. The input referred noise over the 50 kHz bandwidth was 12-16 microVRMS, well below the magnitude of previously reported extracellular potentials. Crosstalk between neighboring channels resulted in an output signal below the amplifier noise level, even for relatively large extracellular potentials. Using this system, extracellular recording were demonstrated yielding typical peak-to-peak biopotentials of magnitude 0.9-2.1 mV and 100-400 microV for chick cardiac myocytes and rat spinal cord neurons, respectively. The key components of this extracellular recording system can be manufactured using industry standard thin film photolithographic techniques.

Amplifiers, Electronic↗

PubMed1998

Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons.

High resolution substrates, created using patterned self-assembled monolayers, are shown to direct axonal and dendritic process extension at the level of a single hippocampal neuron. Axons and dendrites were identified using morphological characteristics and immunocytochemical markers. Patterns were formed on glass coverslips from a co-planar monolayer of cell adhesive aminosilanes and non-adhesive fluorinated silanes. On patterned surfaces, the percentage of the total number of cells attached to the 0.71 mm2 substrate field with compliance to the 25-micron diameter 'somal adhesion site' reached 41 +/- 7% (mean +/- S.D., 428 cells counted). A total of 76 +/- 11% of cells that adhered to a somal attachment site developed a lone process > or = 100 microns oriented in the direction of the continuous aminosilane pathway which was shown to express axonal markers. Cells on either the fluorinated silane, which is non-permissive for neurite outgrowth, or localized on an aminosilane region only 5 microns wide failed to extend major processes. This approach is amenable to a variety of industry standard fabrication techniques and may be used to study the role of fine scale spatial cues in neuronal development and synapse formation.

Animals↗

PubMed1998

Influence of extracellular matrix proteins on membrane potentials and excitability in NG108-15 cells.

Previous studies have demonstrated that components of the extracellular matrix can induce neurite extension and cell adhesion in the neuroblastoma x glioma hybrid cell line, NG108-15. Using standard intracellular recording techniques, we examined the resting membrane potential (RMP) and membrane excitability of NG108-15 cells differentiated under serum-free media with representative extracellular matrix (ECM) protein components as the substrate. Surfaces coated with collagen IV and a laminin-1 synthetic peptide induced a significantly (P < 0.05) more hyperpolarized RMP than control polystyrene surfaces. For example, after > or =8 days in culture NG108-15 cells plated on polystyrene exhibited a RMP of -33.2+/-0.8 mV (mean+/-SEM, n=158 cells) whereas cells cultured on the laminin-1 peptide C16 and collagen IV showed a RMP of -37.6+/-0.7 mV (n=157) and -37.5+/-1.5 mV (n=68), respectively. Furthermore, the proportions of cells on ECM substrates showing membrane excitability, i.e. evoked action potentials (APs) and the capability for regular firing, were significantly greater compared to those cells cultured on polystyrene. Among excitable cells cultured on the different substrates, characteristics of the action potentials, such as AP duration, amplitude, and the maximum rate of rise, dV/dtMAX, were examined in detail. While little or no differences were observed between polystyrene and the laminin-1 peptide groups, significant differences in the AP parameters were apparent for collagen IV. For example, dV/dtMAX for polystyrene and the laminin-1 peptide C16 were only 71.7+/-24.5 V/s (n=11) and 59.0+/-8.9 V/s (n=9), respectively, whereas cells cultured on collagen IV surfaces exhibited a dV/dtMAX reaching 156.1+/-22.0 V/s (n=7). These data support a role for ECM components in the maintenance of the RMP and membrane excitability in NG108-15 cells.

Action Potentials↗

PubMed1998

Neuronal and glial epitopes and transmitter-synthesizing enzymes appear in parallel with membrane excitability during neuroblastoma x glioma hybrid differentiation.

The membrane excitability and the presence of neural proteins, including neuronal and glial markers and neurotransmitter-synthesizing enzymes, were examined in parallel while the NG108-15 cell line was maintained in a serum-free medium. Whole-cell recordings in voltage-clamp or current-clamp configurations were used to evaluate the membrane excitability, and immunostaining was done with a panel of well-characterized antibodies against NSE, NF150, S-100 beta, GFAP, ChAT and TH. Culture for 4 to 10 days led to a striking rise in neurite outgrowth, electrical excitability and expression of neural proteins in type I neuron-like cells, which were of both neuronal and glial character, and expressed both cholinergic and adrenergic traits. After about 2 weeks, type II cells which lack neurite processes began to emerge. The type II cells proliferated, as revealed by BrdU uptake, and gradually overgrew differentiated cell types. They exhibited little or no membrane excitability and absence of immunoreactivity for the neuronal and glial specific proteins tested. These measurements indicate that the presence of these neural proteins at crucial stages of membrane excitability development is an important characteristics of NG108-15 cell differentiation, providing insights into the neural development and the reversible nature of neoplasia in the nervous system.

Animals↗

PubMed1998

Inhibition of neuronal Na+ channels by antidepressant drugs.

Although tricyclic antidepressant (TCA) blockade of cardiac Na+ channels is appreciated, actions on neuronal Na+ channels are less clear. Therefore, the effects of TCAs (amitriptyline, doxepin and desipramine) as well as trazdone and fluoxetine on voltage-gated Na+ current (INa) were examined in bovine adrenal chromaffin cells using the whole-cell patch-clamp method. Amitriptyline produced concentration-dependent depression of peak INa evoked from a holding potential of -80 mV with KD value of 20.2 microM and a Hill coefficient of 1.2. Although 20 microM amitriptyline induced no change in the rate or voltage dependence of INa activation, steady-state inactivation demonstrated a 15-mV hyperpolarizing shift. Similar results were observed for doxepin and desipramine. This shift in steady-state inactivation was associated with a slowed rate of recovery from the inactivated state. Contrasting results were observed with the atypical antidepressants: while 20 microM fluoxetine depressed peak INa by 61% and caused a 7-mV hyperpolarizing shift in steady-state inactivation, 100 microM trazodone decreased peak INa by only 19% and caused only a 3-mV shift. Although the magnitude of fluoxetine effects was similar to those of the TCAs, the onset of fluoxetine effects was substantially slower than for amitriptyline. In voltage-clamp and current-clamp measurements from neonatal rat dorsal root ganglion neurons, 20 microM amitriptyline decreased INa by 52% and depressed action potential dynamics consistent with enhanced Na+ channel inactivation. The effects of the TCAs on INa are similar to local anesthetic behavior and could contribute to certain analgesic actions.

Amitriptyline↗

PubMed1996

Halothane and isoflurane preferentially depress a slowly inactivating component of Ca2+ channel current in guinea-pig myocytes.

1. The effects of the inhalational anaesthetics halothane and isoflurane on the high-voltage-activated Ca2+ channels were determined in isolated guinea-pig ventricular myocytes using the patch-clamp technique. 2. Recording solutions were equilibrated with inhalational anaesthetic vapour delivered from a calibrated vaporizer set at clinically relevant ranges of partial pressure. Anaesthetic concentrations in solution were determined using gas chromatography. 3. Halothane (0.9 mM in solution) and isoflurane (0.8 mM in solution) decreased peak whole-cell CA2+ current (ICa) by approximately 40 and approximately 20%, respectively, while increasing the apparent rate of inactivation. 4. The sum of fast and slow exponential decay functions was required to fit the inactivation phase of ICa. The anaesthetics preferentially affected the slow component of inactivation while also increasing the rate of slow inactivation. The physiological significance of these effects was addressed by examining ICa evoked by a ventricular action potential waveform. 5. Measurement of the current carried by Ba2+ through Ca2+ channels (IBa) permitted the isolation of the slow component of inactivation. Halothane and isoflurane diminished peak IBa at 0 mV by approximately 45 and approximately 20% respectively, with similar changes in rate and magnitude of the slowly inactivating component as with ICa. 6. Cell-attached patch-clamp measurements of Ca2+ channel activity revealed that halothane did not alter single-channel conductance. Instead, the anaesthetic reduced channel open probability to the same extent as observed during the whole-cell recording, an effect partially due to an increase in null sweeps. In patches with a single channel present, the open-time distribution, fitted by a single exponential, showed a decrease in mean open time. The closed-time distribution, fitted by the sum of slow and fast exponential components, revealed an anaesthetic-induced increase in the duration of the slow component with no effect on the fast component. Results are presented in terms of a channel-gating model, and model predictions are examined with a computer simulation.

Animals↗

PubMed1996

Myocardial depressant effects of sevoflurane. Mechanical and electrophysiologic actions in vitro.

BACKGROUND: The effects of anesthetic concentrations of sevoflurane were studied in isolated myocardial tissue to delineate the mechanisms by which cardiac function is altered. METHODS: Isometric force of isolated guinea pig ventricular papillary muscle was studied at 37 degrees C in normal and 26 mM K+ Tyrode's solution at various stimulation rates. Normal and slow action potentials were evaluated using conventional microelectrodes. Effects of sevoflurane on sarcoplasmic reticulum function in situ were also evaluated by its effect on rapid cooling contractures, which are known to activate Ca2+ release from the sarcoplasmic reticulum, and on concentrations of rat papillary muscle. Finally, Ca2+ and K+ currents of isolated guinea pig ventricular myocytes were examined using the whole-cell patch clamp technique. RESULTS: Sevoflurane equivalent to 1.4% and 2.8% depressed guinea pig myocardial contractions to approximately 85 and approximately 65% of control, respectively, although the maximum rate of force development at 2 or 3 Hz and force in rat myocardium after rest showed less depression. In the partially depolarized, beta-adrenergically stimulated myocardium, sevoflurane selectively depressed late peak force without changing early peak force, whereas it virtually abolished rapid cooling contractures. Sevoflurane did not alter the peak amplitude or maximum depolarization rate of normal and slow action potentials, but action potential duration was significantly prolonged. In isolated guinea pig myocytes at room temperature, 0.7 mM sevoflurane (equivalent to 3.4%) depressed peak Ca2+ current by approximately 25% and increased the apparent rate of inactivation. The delayed outward K+ current was markedly depressed, but the inwardly rectifying K+ current was only slightly affected by 0.35 mM sevoflurane. CONCLUSIONS: These results suggest that the direct myocardial depressant effects of sevoflurane are similar to those previously described for isoflurane. The rapid initial release of Ca2+ from the sarcoplasmic reticulum is not markedly decreased, although certain release pathway, specifically those induced by rapid cooling, appear to be depressed. Contractile depression may be partly related to the depression of Ca2+ influx through the cardiac membrane. The major electrophysiologic effect of sevoflurane seems to be a depression of the delayed outward K+ current, which appears to underlie the increased action potential duration.

Anesthetics, Inhalation↗

PubMed1996

Volatile anesthetics reduce low-voltage-activated calcium currents in a thyroid C-cell line.

BACKGROUND: Volatile anesthetics may act in part by inhibiting voltage-dependent calcium channels. The effects of several volatile agents on three types of calcium channels in a thyroid C-cell line were examined. METHODS: Whole-cell calcium currents were recorded using standard patch clamp techniques. Current-voltage relationships were derived before, during, and after application of isoflurane, enflurane, or halothane. Low-voltage-activated (LVA; T type) calcium currents were isolated based on the voltage range of activation. High-voltage-activated (HVA) calcium currents were separated into L and N types using omega-conotoxin GVIA (omega-CTX) and nicardipine. RESULTS: All three agents reversibly decreased both LVA and HVA currents at clinically relevant concentrations. Isoflurane and enflurane both reduced peak LVA current more than peak HVA current: -33 +/- 6% (mean +/- SE) versus -22 +/- 4% for 0.71 mM isoflurane (n = 6), and -46 +/- 6% versus -35 +/- 5% for 1.21 mM enflurane (n = 6). In contrast, halothane depressed LVA and HVA currents to a similar extent: -22 +/- 4% versus -29 +/- 3% for 0.65 mM halothane (n = 6). Isoflurane had no effect on LVA whole-cell current kinetics. Pretreatment with either omega-CTX (400 nM) or nicardipine (1 microM) did not change the sensitivity of HVA current to isoflurane. CONCLUSIONS: Isoflurane and enflurane block LVA calcium channels more potently than either L-type or N-type calcium channels, but halothane shows no such preferential effect. These results may have implications for the mechanism action of volatile anesthetics.

Anesthetics, Inhalation↗

PubMed1995

Ion channel events simulated with the program SIMSTATE.

Ion channel transitions between conducting (open) and non-conducting (closed) states are often described in terms of a chemical kinetic model, where the rate constants describing the transitions between states can be derived by analysing a data record and measuring channel dwell times. In this paper, a menu-driven program for IBM-compatible microcomputers, SIMSTATE, is described which permits simulations of one or more channels according to a user-specified set of transition rates. Rates can be constant, voltage- or ligand-activated, exhibit co-operativity, or dependent on the calcium concentration resulting from the opening of one or more nearby Ca channels. To illustrate the functionality of SIMSTATE, open and closed events are simulated for a well-known Ca channel model and an example of co-operative gating is examined. Furthermore, the control of a Ca-dependent K channel by a nearby Ca channel, which also opens and closes according to a user-specified transition rates, is described. Potential uses for SIMSTATE as a tool for theoretical analysis, education and experimental design are discussed.

Algorithms↗

PubMed1994

A major role for calcium-dependent potassium current in action potential repolarization in adrenal chromaffin cells.

To determine the extent which Ca dependent K current (IKCa) contributes during an action potential (AP), bovine chromaffin cells were voltage-clamped using a pre-recorded AP as the command voltage waveform. Based on (1) differential sensitivity of IKCa and Ca-independent K current (IK) to tetraethylammonium; (2) measurements of AP currents under conditions where Ca activation of IKCa had been abolished; and (3) blockade by charybdotoxin, IKCa comprised 70-90% of the outward K current during AP repolarization. In addition, observations are made concerning the form of AP-evoked Ca current.

Action Potentials↗

PubMed1994

Differential anesthetic-induced opening of calcium-dependent large conductance channels in isolated ventricular myocytes.

Under conditions of low Ca buffering of the pipette intracellular dialyzing solution, the opening of Ca dependent large conductance (approximately 310 pS) channels (LCCs) was observed in isolated ventricular myocytes using the whole-cell patch clamp technique. With Na-Ca exchange current (INaCa) suppressed by elimination of intracellular and extracellular Na, sustained LCC activity, which is markedly enhanced by caffeine-stimulated Ca release from the sarcoplasmic reticulum (SR), was increased by application of the inhalational anesthetic halothane, but not isoflurane. Halothane (0.90 mM in solution) reversibly increased the frequency of LCC openings, fo, by a factor of approximately 30 with a concurrent rise in the observed probability of opening, NPo, by a factor of approximately 50. The effect of halothane on LCC activation was suppressed by either strong Ca buffering in the whole-cell pipette solution or pretreatment of the myocytes with ryanodine (10 microM) to decrease SR Ca release. In the presence of intracellular and extracellular Na, a transient inward current was evoked by application of caffeine (5 mM) or halothane (1.80 mM) suggesting that Ca release from the SR by either agent can activate INaCa. Our findings are consistent with the notion that halothane, in contrast to isoflurane, causes SR release of Ca. The eventual depletion of SR activator Ca may account, at least in part, for the differential effects of these anesthetics on myocardial tension development.

Anesthetics↗