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At least 1,351 records · Page 75Linked to original sources
PubMed2005

Investigation of the enhancing mechanism of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate effect on the intestinal permeability of polar molecules utilizing a voltage clamp method.

Oral administration of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) has been reported to increase the bioavailability of various macromolecules. The present study was aimed to study the effect of SNAC on the intestinal tissue permeability of polar charged molecules, using 6-carboxy-fluorescein (6-CF) as a model. The effects of SNAC on rat intestinal permeability was investigated ex vivo by utilizing voltage clamp experiments in a side-by-side diffusion chamber model in comparison with the effect of EDTA (10mM). The intestinal permeability of 6-CF was increased two-fold in the presence of 33-66 mM SNAC, and by 6.5-fold in the presence of 10mM EDTA. The voltage clamp experiments show that the effect of SNAC was particularly on the transcellular 7-folds increase (that was five times larger than the paracellular transport of the model agent). While EDTA affected predominantly paracellular pathway transport, SNAC 33-66 mM had no effect on [(3)H]-mannitol transport or any toxic effect on tissue integrity measured by TEER values. In conclusion, this study demonstrates that SNAC can facilitate passive transport of polar charged molecules through the membrane of epithelial enterocytes. This is noteworthy in view of the very low tendency of a charged molecule to permeate across the lipophilic inter-phase of the enterocytes membrane.

Animals↗
PubMed2004

MRCI: a flexible real-time dynamic clamp system for electrophysiology experiments.

We present a real-time simulation system that enables modeled dynamical systems to interact with physical experimental systems, and is specifically aimed towards execution of the dynamic clamp protocol. Model reference current injection (MRCI) operates under Real-Time Linux (RT-Linux or RTL) and provides a simple equation-oriented language for describing dynamical system models. Features include scripting of commands to implement repeatable protocols, the ability to output pre-computed waveforms through any variable or parameter of the model, the means to conduct time measurements and assess the computational performance of the real-time system, and an installation program that installs the software and accompanying device drivers with minimal input from the user. Tested models operate as fast as 30 kHz, with actual maximum rates dependent on model complexity. We present sample models that exhibit the main features of the modeling language. Experiments demonstrate the abilities of the system by creating a hybrid network of real and simulated neurons, and playing a pre-defined synaptic waveform into a synaptic conductance variable. We conclude by introducing a waveform reconstruction technique that is useful for establishing the presence of significant experimental error in implementations of the dynamic clamp protocol.

Action Potentials↗
PubMed2005

Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions.

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

Action Potentials↗
PubMed2005

Fluctuations in Xenopus oocytes protein phosphorylation levels during two-electrode voltage clamp measurements.

The biophysical and pharmacological properties of ion channels and transporters are often studied in exogenous expression systems using either the two-electrode voltage clamp (TEVC) in Xenopus oocytes or the patch clamp techniques. Cells machinery is trusted to produce active proteins that are correctly phosphorylated and glycosylated. However, native physiological cellular processes that might be altered during the course of the experiment are often ignored. Here, we detected and quantified the effects of various electrophysiological recording conditions on the phosphorylation levels of Xenopus oocytes proteins, including membrane proteins, as phosphorylation/dephosphorylation events modulate ion channels gating and cell surface expression. Two strategies were chosen to determine relative protein phosphorylation levels: a direct detection with a phospho-Ser/Thr PKA substrate antibody, and a functional method employing two different leak potassium channels as indicators, chosen based on their opposite responses to protein kinase phosphorylation. We report that holding potential, and bath solution properties such as pH, osmolarity, temperature and ion composition, dramatically affect protein phosphorylation levels in Xenopus oocytes. Our results might explain some of the fluctuations in the biophysical properties of expressed channels, often observed during electrophysiological measurements. Minimizing possible misinterpretations could be achieved using either mutated, kinase insensitive, channels or kinases/phosphatases modulators.

Animals↗
PubMed2006

Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.

Viable neurons in brain slices are crucial for electrophysiological studies. The present study describes a new method for obtaining viable cells in several regions of the central nervous system including the ventral tegmental area, the hypothalamus, the periaqueductal grey matter and the spinal cord. The essence of the method was to use a modified artificial cerebrospinal fluid (ACSF) in which all NaCl was replaced initially by equi-osmotic glycerol. This modified glycerol-based ACSF was used during slice preparation. The underlying principle for the modification is to prevent the possible acute neurotoxic effects of passive chloride entry, subsequent cell swelling and lysis. This method significantly increased the live/dead ratio in morphology compared to the normal ACSF or sucrose-base ACSF, in which NaCl was replaced by sucrose. An examination of some electrophysiological and pharmacological properties of the neurons in these preparations, by means of current-clamp and voltage-clamp recordings, revealed similar properties of those neurons obtained with the traditional ACSF method. Due to the increase in the number of viable neurons, the new ACSF increases the productivity of experiments. Based on our data, we propose that this glycerol-based solution may protect CNS neurons.

Animals↗
PubMed2006

Hodgkin-Huxley type ion channel characterization: an improved method of voltage clamp experiment parameter estimation.

The Hodgkin-Huxley formalism for quantitative characterization of ionic channels is widely used in cellular electrophysiological models. Model parameters for these individual channels are determined from voltage clamp experiments and usually involve the assumption that inactivation process occurs on a time scale which is infinitely slow compared to the activation process. This work shows that such an assumption may lead to appreciable errors under certain physiological conditions and proposes a new numerical approach to interpret voltage clamp experiment results. In simulated experimental protocols the new method was shown to exhibit superior accuracy compared to the traditional least squares fitting methods. With noiseless input data the error in gating variables and time constants was less than 1%, whereas the traditional methods generated upwards of 10% error and predicted incorrect gating kinetics. A sensitivity analysis showed that the new method could tolerate up to approximately 15% perturbation in the input data without unstably amplifying error in the solution. This method could also assist in designing more efficient experimental protocols, since all channel parameters (gating variables, time constants and maximum conductance) could be determined from a single voltage step.

Animals↗
PubMed2005

Effects of N-methyl-D-aspartate glutamate receptor antagonists on oscillatory signal propagation in the guinea-pig accessory olfactory bulb slice: characterization by optical, field potential and patch clamp recordings.

To characterize the role of N-methyl-d-aspartate glutamate receptors in oscillations induced by a single electrical stimulation of the vomeronasal nerve layer, optical, field potential and patch clamp recordings were carried out in guinea-pig accessory olfactory bulb slice preparations. Bath application of the N-methyl-D-aspartate receptor antagonists, 2-amino-5-phosphonovaleric acid or MK-801, produced an increase in frequency of oscillating waves (oscillation) in external plexiform and mitral cell layers. The removal of Mg2+ from perfusate abolished oscillations, while subsequent application of 2-amino-5-phosphonovaleric acid or MK-801 restored oscillations. Vomeronasal nerve layer-evoked postsynaptic currents were analyzed by whole-cell clamp recordings from mitral and granule cells. A long-lasting excitatory postsynaptic current and periodic inhibitory postsynaptic currents, which were superimposed on the long excitatory postsynaptic current, were observed in mitral cells. The frequency of the periodic inhibitory postsynaptic currents correlated with the frequency of oscillations observed in the optical and field potential recordings. Furthermore, periodic inhibitory postsynaptic currents were blocked by puff application of bicuculline to the external plexiform layer/mitral cell layer, where mitral cells make dendrodendritic synapses with granule cells. In addition, puff application of the non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, to the external plexiform layer/mitral cell layer suppressed an early phase of periodic inhibitory postsynaptic currents (membrane oscillation), whereas 2-amino-5-phosphonovaleric acid suppressed the late phase of periodic inhibitory postsynaptic currents. These data indicate that periodic excitatory postsynaptic currents of granule cells induce relevantly periodic inhibitory postsynaptic currents in mitral cells via dendrodendritic synapses and suggest that feedback inhibition regulates generation of oscillation via activation of non-N-methyl-d-aspartate glutamate receptors and gradual attenuation of oscillation via activation of N-methyl-D-aspartate receptors on granule cells.

Animals↗
PubMed1998

Voltage clamp studies on S-layer-supported tetraether lipid membranes.

Isolated subunits from the cell surface proteins (S-layer) of Bacillus coagulans E38-66 have been recrystallized on a glycerol dialkyl nonitol tetraether lipid (GDNT)-monolayer and the electrophysical features of this biomimetic membrane have been investigated in comparison to unsupported GDNT-monolayers. The GDNT-monolayer, spread on a Langmuir-Blodgett trough, was clamped with the tip of a glass patch pipette. In order to investigate the barrier function and potential to incorporate functional molecules, voltage-clamp examinations on plain and S-layer-supported GDNT-monolayers were per-formed. Our results indicate the formation of a tight GDNT-monolayer sealing the tip of the glass pipette, and a decrease in conductance of the GDNT-monolayer upon recrystallization of the S-layer protein. Thus, the S-layer protein, apparently, did not penetrate or rupture the lipid monolayer. The valinomycin-mediated increase in conductance was less pronounced for the S-layer-supported than for the plain GDNT-monolayer, indicating differences in the accessibility and/or in the fluidity of the lipid membranes. Furthermore. in contrast to plain GDNT-monolayers. S-layer supported GDNT-monolayers with high valinomycin-mediated conductance persisted over long, periods of time, indicating enhanced stability. These composite S-layer/lipid films may constitute a new tool for electrophysical and electrophysiological studies on membrane-associated and membrane-integrated biomolecules.

Anti-Bacterial Agents↗
PubMed2001

Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements.

Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the half-inactivation voltage and net gating charge movement.

Animals↗
PubMed1997

Noise analysis of ion channels in non-space-clamped cables: estimates of channel parameters in olfactory cilia.

Ion channels in the cilia of olfactory neurons are part of the transduction machinery of olfaction. Odorant stimuli have been shown to induce a biphasic current response, consisting of a cAMP-activated current and a Ca(2+)-activated Cl- current. We have developed a noise analysis method to study ion channels in leaky cables, such as the olfactory cilium, under non-space-clamp conditions. We performed steady-state noise analysis on ligand-induced currents in excised cilia, voltage-clamped at input and internally perfused with cAMP or Ca2+. The cAMP-activated channels analyzed by this method gave results similar to those of single-channel recordings (gamma = 8.3 pS). Single-channel currents have not yet been recorded for the Ca(2+)-activated Cl- channels. Using our noise analysis method, we estimate a unit conductance, gamma = 0.8 pS, for these channels. The density of channels was found to be approximately 70 channels/micron2 for both channel species.

Analysis of Variance↗
PubMed1998

Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study.

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.

Action Potentials↗
PubMed1998

Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.

An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17 mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.

Animals↗
PubMed1998

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.

Animals↗
PubMed1999

Two-microelectrode voltage clamp of Xenopus oocytes: voltage errors and compensation for local current flow.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The most common method for their electrophysiological investigation is the two-microelectrode voltage clamp technique. The quality of voltage clamp recordings obtained with this technique is poor when membrane currents are large and when rapid charging of the membrane is desired. Detailed mathematical modeling of the experimental setup shows that the reasons for this weak performance are the electrical properties of the oocytes and the geometry of the setup. We measured the cytosolic conductivity to be approximately 5 times lower than that of the typical bath solution, and the specific membrane capacitance to be approximately 6 times higher than that of a simple lipid bilayer. The diameter of oocytes is typically approximately 1 mm, whereas the penetration depth of the microelectrodes is limited to approximately 100 microm. This eccentric current injection, in combination with the large time constants caused by the low conductivity and the high capacitance, yields large deviations from isopotentiality that decay slowly with time constants of up to 150 micros. The inhomogeneity of the membrane potential can be greatly reduced by introducing an additional, extracellular current-passing electrode. The geometrical and electrical parameters of the setup are optimized and initial experiments show that this method should allow for faster and more uniform control of membrane potential.

Animals↗
PubMed1999

Patch-clamp recording of charge movement, Ca2+ current, and Ca2+ transients in adult skeletal muscle fibers.

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/microF, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.

Animals↗
PubMed1999

Common components of patch-clamp internal recording solutions can significantly affect protein kinase A activity.

Common components of whole-cell internal recording solutions were tested both in vitro and in patch-clamp experiments for their effects on the activity of cAMP-dependent protein kinase. Potassium fluoride (KF), 440 mM trimethylamine chloride and exclusion of bovine serum albumin (BSA) decreased the activity of the enzyme, while ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and the potassium salts of aspartate, gluconate, methylsulfate and monobasic phosphate increased its activity. Addition of KF to the internal solution produced a hyperpolarizing shift in the V1/2 of Ih channel activation, consistent with the KF-induced reduction of protein kinase A activity. Therefore, consideration of the composition of internal solutions is warranted when studying channel physiology by patch-clamp techniques.

Animals↗
PubMed2003

Five weeks of insulin-like growth factor-I treatment does not alter glucose kinetics or insulin sensitivity during a hyperglycemic clamp in older women.

Insulin sensitivity and the activity of the hypothalamic-growth hormone (GH)- insulin-like growth factor-I (IGF-I) axis both decline with age. Treatment with IGF-I increases insulin sensitivity in healthy young subjects. We hypothesized that increasing plasma IGF-I in postmenopausal women to levels characteristic of young women would enhance insulin sensitivity. To test the hypothesis, fasting glucose kinetics and insulin sensitivity were measured in 24 healthy, normoglycemic, postmenopausal women before and after 5 weeks of treatment with either recombinant human (rh)IGF-I (15 microg/kg body weight/d twice daily) or placebo in a double-blind study. Diet energy content and composition were rigidly controlled to maintain energy balance. A hyperglycemic clamp (8 mmol/L) coupled with stable isotope infusion ([6,6(2)H]glucose) was performed before and after treatment to assess whole-body insulin sensitivity; defined as the glucose rate of disappearance (Rd) or rate of infusion (GRIF) scaled to the steady-state insulin concentration (I). There were no differences in fasting glucose or insulin concentrations, glucose kinetics, or glucose oxidation after either treatment. During the clamps, steady-state insulin concentrations with placebo (pre = 151 +/- 28 pmol/L, post = 173 +/- 31 pmol/L) were slightly different than with IGF-I (pre = 182 +/- 37 pmol/L, post = 163 +/- 33 pmol/L), but the variations were not significant. No significant changes in whole-body insulin sensitivity were observed after treatment with IGF-I, calculated as Rd/I (pre = 17.7 +/- 2.6 microg/kg/min/pmol/L, post = 19.3 +/- 2.0 microg/kg/min/pmol/L for IGF-I v pre = 24.2 +/- 2.5 microg/kg/min/pmol/L, post = 22.8 +/- 3.4 microg/kg/min/pmol/L for placebo) or as GRIF/I (pre = 18.0 +/- 3.9 microg/kg/min/pmol/L, post = 22.3 +/- 3.5 microg/kg/min/pmol/L for IGF-I v pre = 26.4 +/- 6.2 microg/kg/min/pmol/L, post = 26.9 +/- 4.8 microg/kg/min/pmol/L for placebo). Baseline insulin sensitivity in women using hormone replacement therapy (HRT, n = 15) was similar to nonusers (n = 9), but HRT users derived a greater portion of energy expenditure from carbohydrate oxidation compared with nonusers. HRT use had no impact on the response to IGF-I. Overall, we observed subtle, but physiologically insignificant, variations after IGF-I treatment in the direction of enhanced insulin sensitivity. The data suggest that 5 weeks of low-dose rhIGF-I treatment has no material influence on whole-body insulin sensitivity in normoglycemic postmenopausal women.

Aging↗
PubMed1996

Interstitial insulin during euglycemic-hyperinsulinemic clamp in obese and lean individuals.

Transcapillary insulin transport has been considered a rate-limiting step of insulin action. However, direct measurement of interstitial insulin levels during physiologic levels of insulinemia have not been performed. We determined changes in interstitial insulin in eight healthy non-obese men and seven healthy obese men by microdialysis during a euglycemic-hyperinsulinemic clamp. Interstitial insulin was determined in the subcutaneous tissue of the abdomen and thigh. Steady-state insulin concentrations were reached approximately 10 minutes after the start of insulin infusion in the subcutaneous tissue of the abdomen and thigh and returned to basal levels approximately 10 minutes after the infusion was discontinued. There was no difference in the rapidity of change in interstitial insulin between obese and lean individuals at either site studied, irrespective of the pattern of fat distribution. The relative change in dialysate insulin concentration during the euglycemic clamp did not differ between obese and lean individuals at either site studied. It was also unaffected by the waist to hip ratio. The rapid change in interstitial insulin concentration could be of physiologic significance in determining the effects of changes in circulating insulin concentration. We conclude that transcapillary insulin transport in adipose tissue is unaffected by obesity and the pattern of fat distribution in healthy men. It is also concluded that when interstitial insulin is determined directly, transcapillary insulin transport is rapid and does not demonstrate a significant lag phase.

Adult↗