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Alternative splicing and tissue specific expression of the 5' truncated bCCE 1 variant bCCE 1delta514.

In many non-excitable as well as electrically excitable cells, depletion of intracellular Ca2+ stores after stimulation of G protein coupled receptors or receptor tyrosine kinases is followed by Ca2+ entry across the plasma membrane, a mechanism referred to as capacitative calcium entry (CCE) [Putney, J.W., Cell Calcium 11 (1990) 611-624; Fasolato, C. et al., Trends Pharmacol Sci. 15 (1994) 77-83]. Recently, we reported that bCCE 1, a homologue of the Drosophila protein trp, exhibits the characteristics of CCE channels [Philipp, S. et al., EMBO J. 15 (1996) 6166-6171]. In this study, we report the cloning of a 5' truncated splice variant (bCCE 1delta514) of the full-length bCCE 1. The bCCE 1delta514 cDNA encodes a protein of 486 amino acids with the ATG triplet encoding M514 of bCCE 1 as translation initiation codon and, therefore, comprises two putative transmembrane segments corresponding to the predicted transmembrane segments 5 and 6 of bCCE 1. bCCE 1delta514 transcripts appear to be specifically expressed in the adrenal gland and genome analysis reveals an alternative splice site within an exon of the CCE 1 gene leading to the formation of bCCE 1delta514.

Adrenal Glands↗

Ion concentration and haematocrit as determinants of impedance in an erythrocyte suspension model of renal medullary tissue.

In order to analyse the respective roles of ion concentration and fractional volume of the interstitial compartment as determinants of the impedance, Z, of renal medullary tissue, a model was needed in which both these factors could be varied independently. An array of blood cell suspensions ions in saline (different haematocrit values and different NaCl concentrations) was used for this purpose. It was found that: (i) up to a measuring frequency of about 10 kHz, the complex consisting of needle electrodes and 'tissue' can be regarded as serially connected resistances, R, and capacitances; (ii) the frequency range 3-10 kHz can be regarded as optimal since it simultaneously assures low electrode polarization and a negligible role of tissue capacitance; (iii) increasing the haematocrit had two consequences--a reduced contribution of polarization impedance to the total impedance measured and a decreased sensitivity of ion concentration measurement from R-1 (conductance); (iv) passive electrical properties of renal medullary tissue were close to those of a 75% haematocrit cell suspension; (v) since in high haematocrit suspensions the resistive component of impedance predominates, within the frequency range 3-10 kHz either conductance or admittance, Z-1, can be used as an index of ion concentration; and (vi) impedance changes in kidney tissue are primarily determined by fluctuations of ion concentration with a less important contribution from interstitial volume changes.

Animals↗

Electrophysiologic characteristics of atrial myocytes in levo-thyroxine-treated rats.

To investigate whether thyroid hormone modulates electrical properties of atrial myocytes, electrocardiogram (ECG), action potentials (APs), and ionic currents were measured. Male Sprague-Dawley rats were randomly divided into control and levo-thyroxine (T4)-treated groups at 6 weeks of age. Levo-thyroxine (500 microg/kg of body weight) was injected daily into the peritoneal cavity for 14 days (T4-treated rats) and the same volume of saline was injected in control rats daily. ECG signals were recorded using apex-base leads. APs, voltage-dependent Na+ and L-type Ca2+ channel current (I(Na) and I(Ca(L))), inwardly rectifying K+ channel current (I(K1)), transient outward K+ channel current (I(to)), and delayed rectifier K+ channel current (I(K(delay))) were measured using patch-clamp techniques. T4 treatment significantly changed electrical properties in rat atrial myocytes, including (1) the increase in heart rate, (2) the increase in cell size, (3) the shortening of action potential duration (APD), (4) the increase in cell membrane capacitance (C(m)), and (5) the decrease in input resistance (R(in)). Although the current densities of I(Na) and I(K1) in T4-treated atrial myocytes did not differ from those in control cells, I(Ca(L)) was significantly decreased and I(K(delay)) was significantly increased in T4-treated rats. Thus, thyrotoxicosis could induce the shortening of APD by alterations in current density of both I(Ca(L)) and I(K(delay)) in rat atrial myocytes.

Action Potentials↗

The effects of the coronary capacitance on the interpretation of diastolic pressure-flow relationships.

The effects of coronary capacitance on instantaneous pressure-flow (P/F) relationships were analyzed using a theoretical model of coronary flow during diastole that included capacitance. The magnitude of the discrepancy between actual intramural and instantaneously derived P/F relationships was predicted to be dependent on the ratio of two natural decay constants (central aortic decay constant/intrinsic coronary decay constant). The effects of coronary capacitance are eliminated using constant pressure conditions. The instantaneous (dynamic) and constant pressure (static) P/F relationships were compared experimentally using a reservoir to provide constant pressure perfusion during prolonged diastoles in heart blocked dogs. In the presence of coronary tone, zero flow pressure intercepts (Pzf) of 27.1 +/- 6.6 and 11.0 +/- 3.0 mm Hg were obtained under dynamic and constant pressure conditions respectively, P less than 0.001. After maximal vasodilation, Pzf of 14.2 +/- 4.5 mmHg and 10.7 +/- 2.4 mmHg were obtained under dynamic and constant pressure conditions, respectively, P = NS. Pzf derived under constant pressure conditions were independent of the state of coronary vasomotor tone with a value about 11 mmHg. The slopes of the dynamic P/F relationships tended to be greater than those derived from constant pressure conditions. This may suggest an additional component of increasing coronary resistance during diastole that could not be readily assessed under dynamic conditions. We conclude that coronary capacitive effects and resistance changes during diastole severely limit the interpretation of instantaneous dynamic P/F relationships. Diastolic coronary perfusion ceases at about 11 mm Hg and is independent of coronary tone when capacitive effects are eliminated.

Animals↗

Carotid sinus baroreceptor reflex control and epinephrine. Influence on capacitive and resistive properties of the total pulmonary vascular bed of the dog.

To quantify the importance of the carotid sinus baroreceptor reflex and the interaction with epinephrine infusion on total pulmonary vascular capacity and resistance, I have simultaneously measured total pulmonary vascular compliance, changes in pulmonary blood volumes, and changes in resistances in seven sodium pentobarbital-anesthetized dogs. A preparation was used that bypasses the right and left hearts allowing for the simultaneous measurement of the pulmonary as well as the systemic vascular bed parameters. At intrasinus pressures of 50, 125, and 200 mm Hg, without epinephrine infusion, the pulmonary vascular resistance was 0.134, 0.121, and 0.109 mm Hg/(ml per min per kg) and the systemic vascular resistance was 1.21, 0.87, and 0.63 mm Hg/(ml per min per kg). Epinephrine infusion of 1 microgram/min per kg at each intrasinus pressure caused the resistances of both vascular beds to increase. Pulmonary vascular resistance increased to 0.157 mm Hg/(ml per min per kg) and showed no further changes with changes in ISP. However, systemic vascular resistance did decrease from 1.49 to 1.41 and 1.25 at intrasinus pressures of 50, 125, and 200 mm Hg. During control runs, pulmonary vascular capacity changed 1.06 ml/kg and systemic capacity changed 7.63 ml/kg for SP changes between 50 and 200 mm Hg. Both responses were greatly attenuated after epinephrine infusion and amounted to only 0.17 ml/kg for the pulmonary and vascular bed and 2.26 ml/kg for the systemic vascular bed. The differences in capacity changes between control and epinephrine runs for the two vascular beds were nearly equal to the change in capacities brought about by epinephrine when it was infused at a fixed intrasinus pressure of 125 mm Hg. The pulmonary vascular compliance during the control runs increased from 0.303 to 0.329 ml/mm Hg per kg when ISP was increased from 50 to 200 mm Hg. After epinephrine infusion, the pulmonary compliance was 0.273 ml/mm Hg per kg and showed no changes with intrasinus pressure. Similar results were obtained for the systemic vascular compliance which was 2.07 ml/mm Hg per kg at an ISP of 50 mm Hg and increased to 2.39 ml/mm Hg per kg to an ISP of 200 mm Hg during control runs. After epinephrine infusion, the systemic compliance was 1.89 ml/mm Hg per kg and again showed no changes with ISP. These data indicate that the baroreceptor reflex can exert control of pulmonary and systemic vascular resistance and capacitance and epinephrine can greatly attenuate the reflex control of both vascular beds.

Animals↗

Electrical impedance of cultured endothelium under fluid flow.

The morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro. Quantitative, real-time changes in the electrical impedance of endothelial monolayers were evaluated using a modified electric cell-substrate impedance sensing (ECIS) system. This ECIS/Flow system allows for a continuous evaluation of the cell monolayer impedance upon exposure to physiological fluid shear stress forces. Bovine aortic endothelial cells grown to confluence on thin film gold electrodes were exposed to fluid shear stress of 10 dynes/cm2 for a single uninterrupted 5 h time period or for two consecutive 30 min time periods separated by a 2 h no-flow interval. At the onset of flow, the monolayer electrical resistance sharply increased reaching 1.2 to 1.3 times the baseline in about 15 min followed by a sustained decrease in resistance to 1.1 and 0.85 times the baseline value after 30 min and 5 h of flow, respectively. The capacitance decreased at the onset of flow, started to recover after 15 min and after slightly overshooting the baseline values, decreased again with a prolonged exposure to flow. Measured changes in capacitance were in the order of 5% of the baseline values. The observed changes in endothelial impedance were reversible upon flow removal with a recovery rate that varied with the duration of the preceding flow exposure. These results demonstrate that the impedance of endothelial monolayers changes dynamically with flow indicating morphological and/or functional changes in the cell layer. This in vitro model system (ECIS/Flow) may be a very useful tool in the quantitative evaluation of flow-induced dynamic changes in cultured cells when used in conjunction with biological or biochemical assays able to determine the nature and mechanisms of the observed changes.

Animals↗

Modulation of motoneuron excitability by brain-derived neurotrophic factor.

The influence of neurotrophins on motoneuron survival and development has been well documented in cell cultures and neonates. In the present study, the role of brain-derived neurotrophic factor (BDNF) in the maintenance of motoneuron electrical properties was investigated. In adult male rats, BDNF- or saline-saturated gelfoam was inserted between the medial and lateral heads of the gastrocnemius muscles. After 5 days survival, in vivo intracellular recordings were obtained, and motoneuron biophysical properties were measured. In BDNF-treated rats, significant decreases in mean rheobase and in total cell capacitance of medial gastrocnemius motoneurons were observed. In addition, a concommitant increase in input resistance and decrease in membrane time constant were noted in BDNF-treated rats but were not statistically significant. No significant treatment effect was observed in motoneuron conduction velocity, action potential amplitude, equalizing time constant, electrotonic length, afterhyperpolarization amplitude and duration, and membrane potential sag during current injection. The observed changes in motoneuron rheobase and total cell capacitance suggest that application of BDNF produces an increase in motoneuron excitability coincident with a reduction in size. These data are discussed with respect to the possible role of BDNF as a muscle-derived trophic factor for the regulation of motoneuron excitability.

Animals↗

Some electrical circuit properties of the organ of Corti. II. Analysis including reactive elements.

The simplified network model of the organ of Corti-hair cell complex that was studied in previous publications (Dallos, P. (1983): In: Hearing - Physiological Bases and Biophysics, pp. 32-37, editors: R. Klinke and R. Hartmann; Dallos, P. (1983): Hearing Res. 12, 89-119) is extended to include the capacitive reactances of hair cell 12, 89-119) is extended to include the capacitive reactances of hair cell membranes. With the assumption that the circuit is parametrically excited by resistance changes of the apical hair cell membrane, intracellular responses for outer (eO) and inner (eI) hair cells and the organ of Corti potential (eOC) are computed for the linear case. In order that frequency-dependent effects be primarily determined by the network properties of the system, the eO/eOC and eI/eOC ratios are considered. The amplitude and phase dependence of these computed functions is compared to corresponding experimental results from the third turn of the guinea pig's cochlea. The eO/eOC function is predicted to be a simple low-pass filter whose cutoff frequency (1250 Hz) is determined by the parallel resistance-capacitance circuit of the cell's baso-lateral membrane. This circuit prediction is in excellent agreement with the data. The eI/eOC function is again derived as a low-pass filter. The cutoff frequency is obtained as 472 Hz, which corresponds to an RC membrane time constant of 0.34 ms. Experimental data are widely divergent from this predicted pattern. In order to bring the theoretical and experimental results in agreement, two steps are necessary. First, a differentiation (i.e., multiplication by j omega) needs to be included in the input to inner hair cells to account for their velocity dependence [4,10]. Second, an underdamped complex pole, located above the cell's best frequency must be introduced. The velocity dependence and the resonance represented by the underdamped pole-pair indicate that the electrical response pattern of inner hair cells is more complex than that of outer hair cells.

Cell Membrane↗

Parameter-extraction of a two-compartment model for whole-cell data analysis.

Neuronal modeling of patch-clamp data is based on approximations which are valid under specific assumptions regarding cell properties and morphology. Certain cells, which show a biexponential capacitance transient decay, can be modeled with a two-compartment model. However, for parameter-extraction in such a model, approximations are required regarding the relative sizes of the various model parameters. These approximations apply to certain cell types or experimental conditions and are not valid in the general case. In this paper, we present a general method for the extraction of the parameters in a two-compartment model without assumptions regarding the relative size of the parameters. All the passive electrical parameters of the two-compartment model are derived in terms of the available experimental data. The experimental data is obtained from a DC measurement (where the command potential is a hyperpolarizing DC voltage) and an AC measurement (where the command potential is a sinusoidal stimulus on a hyperpolarized DC potential) performed on the cell under test. Computer simulations are performed with a circuit simulator, XSPICE, to observe the effects of varying the two-compartment model parameters on the capacitive transients of the current response. Our general solution for the parameter-estimation of a two-compartment model may be used to model any neuron, which has a biexponential capacitive current decay. In addition, our model avoids the need for simplifying and perhaps erroneous approximations. Our equations may be easily implemented in hardware/software compensation schemes to correct the recorded currents for any series resistance or capacitive transient errors. Our general solution reduces to the results of previous researchers under their approximations.

Algorithms↗

Use of a spread sheet to calculate the current-density distribution produced in human and rat models by low-frequency electric fields.

The current-density distribution produced inside irregularly shaped, homogeneous human and rat models by low-frequency electric fields is obtained by a two-stage finite-difference procedure. In the first stage the model is assumed to be equipotential. Laplace's equation is solved by iteration in the external region to obtain the capacitive-current densities at the model's surface elements. These values then provide the boundary conditions for the second-stage relaxation solution, which yields the internal current-density distribution. Calculations were performed with the Excel spread-sheet program on a Macintosh-II microcomputer. A spread sheet is a two-dimensional array of cells. Each cell of the sheet can represent a square element of space. Equations relating the values of the cells can represent the relationships between the potentials in the corresponding spatial elements. Extension to three dimensions is readily made. Good agreement was obtained with current densities measured on human models with both, one, or no legs grounded and on rat models in four different grounding configurations. The results also compared well with predictions of more sophisticated numerical analyses. Spread sheets can provide an inexpensive and relatively simple means to perform good, approximate dosimetric calculations on irregularly shaped objects.

Animals↗

Effect of electrical current on temperature and pH in cerebellum and spinal cord.

Electrical currents similar to those used for relief of pain and spasticity were applied to the spinal cord and cerebellum of monkeys and cats. Direct coupled monopolar pulses increased pH at the tissue surface near a negative electrode and correspondingly decreased the pH near a positive electrode. Cerebrospinal fluid (CSF) circulation around the electrodes neutralized these changes. Capacitively coupled currents up to a 10 mA peak and 2.5 mucoul per peak failed to change tissue pH measurably. Current levels with power densities of 100 mW per cm2 did not cause any change in tissue temperature.

Animals↗

Neuron-transistor coupling: interpretation of individual extracellular recorded signals.

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.

Action Potentials↗

A new method for extracting cable parameters from input impedance data.

We establish conditions under which the input impedance and its first two derivatives, evaluated at the origin, uniquely determine the axial resistance, membrane capacitance, and membrane conductance of a finite uniform cable with one sealed end. We argue that these impedance data are readily available to the experimentalist and we demonstrate the recovery of a cable's electrical parameters from synthetic noisy measurements.

Algorithms↗

Pressure polishing: a method for re-shaping patch pipettes during fire polishing.

The resolution of patch-clamp recordings is limited by the geometrical and electrical properties of patch pipettes. The ideal whole-cell patch pipette has a blunt, cone-shaped tip and a low resistance. The best glasses for making patch pipettes are low noise, low capacitance glasses such as borosilicate and aluminasilicate glasses. Regrettably, nearly all borosilicate glasses form pipettes with sharp, cone-shaped tips and relatively high resistance. It is possible, however, to reshape the tip during fire polishing by pressurizing the pipette lumen during fire polishing, a technique we call 'pressure polishing.' We find that this technique works with pipettes made from virtually any type of glass, including thick-walled aluminasilicate glass. We routinely use this technique to make pipettes suitable for whole-cell patch-clamp recording of tiny neurons (1-3 microm in diameter). Our pipettes are made from thick-walled, borosilicate glass and have submicron tip openings and resistances <10 MOmega. Similar pipettes could be used to record from subcellular neuronal structures such as axons, dendrites and dendritic spines. Pressure polishing should also be useful in patch-clamp applications that benefit from using pipettes with blunt tips, such as perforated-patch whole-cell recordings, low-noise single channel recordings and experiments that require internal perfusion of the pipette.

Animals↗

Temperature-dependent quantized double layer charging of monolayer-protected gold clusters.

This paper describes low-temperature voltammetry of purified hexanethiolate-coated monolayer-protected Au140 clusters (C6 MPCs). Lowered temperatures enhance the resolution of quantized double layer (QDL) charging peaks in differential pulse voltammetry (DPV) observations. As many as 13 resolved peaks are seen in illustrative voltammetry at 263 K in CH2Cl2 solvent, and the concept of voltammetric peak capacity is introduced. For the one-electron MPC charge steps surrounding the E(PZC) of the MPC (small numbers of electrons added or removed from the core), the capacitance C(CLU) of the MPCs (measured from the voltage spacing between charging peaks) increases by approximately 15% as the solvent temperature is lowered from 273 to 210 K. The experimental C(CLU) temperature dependency (d[ln(C(CLU))]/dT approximately -0.0025, in 0.1 M electrolyte) is discussed in light of temperature dependencies of the compact and diffuse double layer capacitances. It is concluded that the observed temperature dependence is probably a mixed diffuse, compact dependence. The regular voltage spacing of MPC charging peaks near the potential of zero charge is generally consistent with electrical double layer properties, but the irregular pattern of charging of the nanoparticles seen at higher charge states suggests intervention of the incipient molecular behavior of Au140 cores in the spacing of energies at which further electrons are added or removed.

Journal Article↗

Alteration of peripheral vasodilatory reserve capacity after cardioversion of atrial fibrillation.

In atrial fibrillation, exercise capacity is often reduced. This is usually ascribed to a decreased cardiac output as compared with sinus rhythm. Very few studies, however, have focused on changes in the peripheral blood flow during atrial fibrillation as a potential mechanism for exercise limitation. The aim of the present study was to determine the effect of conversion of atrial fibrillation to sinus rhythm on peripheral blood flow. Calf blood flow, using an electrocardiogram-triggered venous occlusion plethysmograph, and peak oxygen consumption (peak VO2), using treadmill exercise testing, were studied in 28 patients with chronic atrial fibrillation eligible for electrical cardioversion. Measurements were performed before cardioversion, and repeated 1 day and 1 month thereafter. Calf blood flow at rest, maximal calf blood flow, and minimal calf vascular resistance during the hyperaemic response immediately following 700 J of calf exercise were determined plethysmographically. One day and 1 month after cardioversion, 23 and 14 patients were still in sinus rhythm, respectively. In patients who still had sinus rhythm after 1 month, maximal calf blood flow increased from 33.7 +/- 12 to 40.0 +/- 13 ml. 100 ml-1.min-1 (P < 0.01) and minimal calf vascular resistance fell from 3.2 +/- 0.9 to 2.7 +/- 0.7 mmHg. ml-1. 100 ml-1. min-1 (P < 0.01); peak VO2 increased from 21.3 +/- 4 to 24.2 +/- 5 ml. min-1. kg-1 (P < 0.001). Calf blood flow at rest did not improve. In contrast, no significant changes in maximal calf blood flow, minimal calf vascular resistance and peak VO2 occurred in patients who had atrial fibrillation 1 month after cardioversion. A significant correlation was found between changes in maximal calf blood flow and peak VO2 1 month after cardioversion (r = 0.53, P < 0.01). One day after cardioversion, no changes in calf blood flow or peak VO2 were found, either in patients with sinus rhythm or atrial fibrillation. In conclusion, transition from chronic atrial fibrillation to sinus rhythm is associated with a (delayed) improvement in maximal calf blood flow, minimal calf vascular resistance, and peak VO2. Our findings suggest that increase in vasodilatory reserve capacity may contribute to the improvement of exercise capacity after cardioversion of atrial fibrillation.

Aged↗

Developmental expression of Na+ currents in mouse Purkinje neurons.

As Purkinje neurons mature during postnatal development, they change from electrically quiescent to active and exhibit high frequency spontaneous action potentials. This change in electrical activity is determined by both alteration in ion channel expression and the acquisition of synaptic input. To gain a better understanding of the development the intrinsic electrical properties of these neurons, acutely isolated Purkinje neurons from mice aged postnatal day 4 (P4) to P18 were examined. This included recording action potential frequency, threshold, height and slope, and input resistance and capacitance. Changes in a number of these properties were observed, suggesting significant changes in voltage-gated Na(+) currents. Because voltage-gated Na(+) currents, including the transient, resurgent and persistent currents, are known to play important roles in generating spontaneous action potentials, the developmental changes in these currents were examined. A large increase in the density of transient current, resurgent current and persistent current was observed at times corresponding with changes in action potential properties. Interestingly, the developmental up-regulation of the persistent current and resurgent current occurred at rate which was faster than the up-regulation of the transient current. Moreover, the relative amplitudes of the persistent and resurgent currents increased in parallel, suggesting that they share a common basis. The data indicate that developmental up-regulation of Na(+) currents plays a key role in the acquisition of Purkinje neuron excitability.

Action Potentials↗

Bipolar resistivity profiling of 3D tissue culture.

We describe a new low-cost technique for continuous monitoring of the thickness of biofilms and tissue cultures, and we demonstrate the advantage of using electrodes of different dimensions to probe different depths of a sample. We have used electric impedance spectroscopy to monitor keratinocyte stem cells (YF29) growing on an array of Ti/Pt coplanar microelectrodes. The thickness of the sample was reconstructed by fitting the measurements to theoretical curves. We have developed an algorithm for the rapid calculation of the resistance through a multilayered sample. This algorithm is based on conformal mapping and the serial partial capacitance technique. The validity of the technique was tested by measuring the sedimentation rate of an alumina powder. Sample thicknesses between 10 and 80 microm could be measured with a resolution of a few microns using the device.

Biological Assay↗