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At least 667 records · Page 37Linked to original sources
PubMed1997

Assessment of insulin action on glucose uptake and production during a euglycemic-hyperinsulinemic clamp in dog: a new kinetic analysis.

We evaluated the validity of the traditional method of assessment of the speed of insulin action during a euglycemic-hyperinsulinemic clamp. We first estimated the error of Steele's model on glucose uptake in these experimental conditions. We tested the appropriateness of estimating the half-time of insulin action by expressing the glucose flux changes as a percent of the maximal change (normalization on a 0% to 100% scale). For this purpose, we performed a 390-minute euglycemic-hyperinsulinemic (2 mU.min-1.kg-1) clamp in five chronically catheterized conscious dogs. We used [3-3H]glucose to assess glucose kinetics. We used a novel analysis based on a circulatory model, which allowed us to overcome the limitations of compartmental analysis. We found that the primary effect of insulin (increased from 12.3 +/- 1.6 to 104 +/- 15 microU/mL) was to increase the whole-body fractional extraction of glucose (3.0% +/- 0.3% to 18% +/- 2%). Insulin did not alter the mean whole-body artery-vein transit time (3.1 +/- 0.2 v 2.9 +/- 0.4 minutes). In contrast to the assumptions of the Steele model, which assumes that glucose uptake and rate of appearance (Ra) are equal during the clamp, during the initial 30 minutes of the clamp the increase in glucose uptake preceded (by approximately 4 minutes) the increase in Ra. Thus, during this period uptake exceeded Ra by about 15%. The maximal difference between Ra and uptake (1 to 1.5 mg.min-1.kg-1) occurred approximately 15 minutes after insulin infusion. Finally, to estimate the half-time of the insulin signal that controls glucose uptake and production, we accounted for the nonlinear relationship between insulin concentration and glucose uptake and production. We found that the traditional normalization of the glucose flux changes on a 0% to 100% scale underestimated the half-time of onset of the insulin signal that controls glucose uptake (half-time, 20 v 54 minutes) and glucose production (half-time, 25 v 39 minutes). Accounting for the nonlinearity of the dose-response curves may thus be of crucial importance in the evaluation of the onset and offset of insulin action.

Animals↗
PubMed2001

The use of control theory for the design of voltage clamp systems: a simple and standardized procedure for evaluating system parameters.

Voltage clamp (VC) instruments are closed-loop control systems based on electronic feedback. Such feedback systems can be described in the framework of control theory. We used a mathematical approach based on control theory to improve the performance of VC systems. This approach considerably simplifies the design and optimal tuning of these systems, as is demonstrated for a standard two electrode and a time-sharing single electrode clamp system. The major advantage of this approach and the consequent optimization procedure is that only proportional-integral controllers for VC systems must be used. As a consequence, the design of such VC systems is solely based on the time constants of the clamp circuit. In our approach, the 'symmetrical optimum' rule was applied for the first time to VC systems. This yields optimized systems with respect to speed of response and clamp accuracy. An empirical procedure has been derived from this theoretical approach which allows the optimal tuning of VC instruments based on PI controllers while running an experiment.

Algorithms↗
PubMed2002

Patch clamp electrophysiology in nodose ganglia of adult rat.

The patch clamp technique is widely utilized for studying the electrophysiological properties of enzymatically isolated sensory neurons. Unfortunately, dissociation of the ganglion severs the afferent fibers. As a result, isolated neurons can only be broadly categorized according to somatic action potential waveforms, ion channel subtypes, chemical sensitivities and cell diameter. Such restricted classifications contrast with the continuum of conduction velocities (CVs), discharge patterns, sensory modalities and functional properties of visceral and spinal afferents. Previous reports of patch clamp recordings using intact ganglion have been limited to young animal preparations. This raises concerns regarding postnatal development and impedes the use of chronic models of disease or injury, which often necessitate the use of a more mature animal preparation. Here, we present a methodology for preparing nodose ganglion from adult rat (250-400 g) for study using the patch clamp technique. Successful whole cell recordings were obtained from approximately 50% of the cells selected for study, the majority of which had intact afferent fibers. Measures of somatic discharge and afferent fiber CV at both room and physiological temperatures were consistent with investigations using sharp microelectrodes. Voltage clamp recordings of whole cell Na(+), Ca(2+) and K(+) ion channel currents were comparable to those obtained using isolated neuron preparations. The ability to classify voltage- and ligand-gated ion channel type with afferent fiber CV in an adult preparation adds a valuable new dimension to cellular investigations of the diverse functional and chemical properties of the peripheral afferent nervous system.

Animals↗
PubMed1997

Effects of clamp rise-time on rat brain IIA sodium channels in Xenopus oocytes.

The kinetic properties of wild-type rat brain IIa sodium channels in excised macropatches were studied using step depolarizations and ramp depolarizations to imitate the slow settling-time of voltage in two-electrode voltage clamp. Ramp depolarizations longer than 1 ms produce an increasing suppression of peak sodium current (I[Na]). Two rates of inactivation can be seen in macroscopic sodium current records from excised patches following both step and ramp depolarizations. During slow ramp depolarizations, reduction in peak I[Na] is associated with selective loss of the fastest rate of test-pulse inactivation. This change can be interpreted as resulting from inactivation of a separate sub-population of 'fast mode' channels. The slow rate of test-pulse inactivation is relatively unaffected by changing ramp durations. These results are sufficient to explain the typically slow inactivation kinetics seen in two-electrode voltage clamp recordings of sodium channels in Xenopus oocytes. Thus, the kinetics of sodium channels expressed in Xenopus oocytes are not readily characterizable by two-electrode clamp because of the large membrane capacitance and resulting slow clamp settling time which artifactually selects for slow mode channels.

Animals↗
PubMed1998

Patch clamp recording from the intact dorsal root ganglion.

A method for patch-clamp recording from intact dorsal root ganglion (DRG) cells in rat is described. The L4 and L5 DRGs with sciatic nerve attached were excised from rats (10-15 days old) and placed in a recording chamber after removing the ganglion sheath and dissolving the connective tissue with dilute collagenase. The somata of individual cells were exposed by gentle surface cleaning through a perfusion micropipette. Somata were classified as Abeta, Adelta or C based on the cell size and the shape of the action potential (AP). Under current clamp, axonal conduction velocity (CV) was calculated from the distance between a stimulating electrode and the center of the ganglion divided by the latency of the AP elicited by stimulation of the sciatic nerve. CVs ranged from 0.2-0.8 m/s for C cells, 0.8-2.4 for Adelta and 3.2-5.0 for A/beta cells. AP threshold occurred at a significantly more positive potential in C cells than in Adelta and Abeta cells. Under voltage clamp, sodium currents were recorded from C cells. Both TTX-resistant (TTX-R) and TTX-sensitive (TTX-S currents) were demonstrated in the present study. The results demonstrate the feasibility of patch-clamp recording from intact, identified DRG cells in vitro.

Animals↗
PubMed2004

Insulin sensitivity assessment in uncomplicated obese women: comparison of indices from fasting and oral glucose load with euglycemic hyperinsulinemic clamp.

BACKGROUND AND AIM: Obesity is associated with a great variability to insulin sensitivity degree. Several formulae developed from measurements in the fasting state and during the oral glucose tolerance test (OGTT) have been proposed to assess insulin sensitivity. AIM: In this work we sought to compare the published insulin sensitivity indices with the metabolized glucose index obtained by hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. Uncomplicated obesity provides a good model in order to study insulin sensitivity per se. METHODS AND RESULTS: In this protocol, 65 obese women affected by uncomplicated obesity (without impaired glucose tolerance, diabetes, hypertension and dyslipidemia) underwent 2 h OGTT and euglycemic hyperinsulinemic clamp. Common formulae obtained in the fasting state and from a 2h OGTT were calculated. Simple linear regression analysis showed that ISI (r=0.592, p=0.01), 2 h OGIS (r=0.576, p=0.02), MCRest (r=0.507, p=0.02), 120 insulin (r=-0.494, p=0.03) and fasting insulin (r=-0.382, p =0.02) are significantly correlated to the M index obtained from the hyperinsulinemic euglycemic clamp. The Bland-Altman plot confirmed the good agreement between indices from OGTT and the clamp. CONCLUSION: OGTT-derived indices provide a good assessment of insulin sensitivity in obesity. OGTT could easily be applied in a large number of obese patients in order to obtain information on both glucose tolerance and insulin sensitivity.

Adult↗
PubMed2006

A high-performance elastomeric patch clamp chip.

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (>1 Gohms) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

Animals↗
PubMed1994

Metabolic effects of acute hyperketonaemia in man before and during an hyperinsulinaemic euglycaemic clamp.

1. The effects of acutely raising blood ketone body levels to those seen after 72 h of starvation were examined in 10 subjects after an overnight fast. Metabolic rate and respiratory exchange ratio were measured with indirect calorimetry before and during an insulin-glucose clamp. Arteriovenous differences were measured across forearm and subcutaneous abdominal adipose tissue. 2. In response to the clamp the respiratory exchange ratio rose from 0.82 to 0.83 during 3-hydroxybutyrate infusion and from 0.83 to 0.94 during control (saline) infusion (P < 0.001). 3. Forearm glucose uptake at the end of the clamp was 4.02 +/- 0.95 (3-hydroxybutyrate infusion) and 7.09 +/- 1.24 mmol min-1 100 ml-1 forearm (saline infusion). Whole body glucose uptake at the end of the clamp was 72.8 +/- 7.9 (3-hydroxybutyrate infusion) and 51.0 +/- 3.0 (saline infusion) mmol min-1 kg-1 body weight-1. 4. 3-Hydroxybutyrate infusion reduced the baseline abdominal venous-arterialized venous glycerol difference from 84 +/- 28 to 25 +/- 12 mmol/l and the non-esterified fatty acid difference from 0.60 +/- 0.17 to 0.02 +/- 0.09 mmol/l (P < 0.05 versus saline infusion). 5. Hyperketonaemia reduces adipose tissue lipolysis and decreases insulin-mediated forearm glucose uptake. Hyperketonaemia appears to prevent insulin-stimulated glucose oxidation, but does not reduce insulin-mediated glucose storage.

3-Hydroxybutyric Acid↗
PubMed2001

Calcium-activated potassium current clamps the dark potential of vertebrate rods.

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of approximately -35 mV. The present work investigates the physiological role of the Ca2+-activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light- and dark-adapted rods under voltage- or current-clamp conditions, respectively. In light-adapted rods of the salamander, selective blockade of Ca2+-activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current-clamped rods by approximately 30 mV, from a resting potential of approximately -35 mV. A similar depolarization was observed if external Ca2+ (1 mM) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to -100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of approximately -20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark-adapted current-clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady-state depolarizations of approximately 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+-activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.

Ambystoma mexicanum↗
PubMed1999

Glucose and fatty acid metabolism in type 2 diabetes mellitus: an assessment using low-dose insulin infusion and the hyperinsulinaemic euglycaemic clamp.

AIM: To assess whether resistance to the effects of insulin on lipid metabolism in type 2 diabetes mellitus parallels that demonstrated for glucose metabolism. METHODS: Thirteen patients with type 2 diabetes were studied (mean age 56.2 years, mean body mass index (b.m.i.) 28.7 kg m(-2)). The hyperinsulinaemic euglycaemic clamp (2 mU insulin kg(-1) min(-1)) and a low-dose insulin infusion technique were used in order to assess insulin effects on both glucose and lipid metabolism. RESULTS: Using the low-dose insulin infusion there were significant correlations between measures of insulin sensitivity for glucose metabolism and those for NEFA (r = 0.82, p < 0.01) and glycerol (r = 0.73, p < 0.01). Mean clamp glucose disposal was 33.4 micromol kgb.w.(-1) min(-1). There was no correlation between clamp glucose disposal and insulin sensitivity derived from the low-dose insulin infusion. CONCLUSIONS: At low insulin concentrations, insulin's effects on glucose metabolism (mainly suppression of hepatic glucose output) mirror those on inhibition of lipolysis. At the high insulin concentrations seen during the clamp, stimulation of glucose uptake provides a measure of maximal insulin action which does not parallel its physiological effects shown at lower concentrations.

Blood Glucose↗
PubMed2001

Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells.

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.

Adrenal Glands↗
PubMed1999

Increased forearm blood flow during glucose clamp is related neither to insulin sensitivity nor to hyperinsulinemia in borderline hypertensive young men.

It is controversial whether raised insulin within the physiological concentration range increases forearm blood flow (FBF). The aim of the present study was therefore to examine the effect of the isoglycemic hyperinsulinemic glucose clamp procedure on FBF and to relate the increase to the glucose disposal rate (GDR), i.e. insulin sensitivity. Borderline hypertensive young men were examined with the clamp technique or received saline infusion, and FBF was measured using plethysmography. It is of particular interest to study this group of subjects because their GDR correlates to a number of metabolic and hemodynamic variables, and these subjects hyperreact to stressful stimuli. There was no correlation between deltaFBF during clamp and GDR (r = -0.002, p = 0.99, n = 28). While serum insulin increased from 107 +/- 5 to 628 +/- 31 pmol/l in the hyperinsulinemic group and remained unchanged (135 +/- 11 vs 116 +/- 11 pmol/l) in the saline group, FBF increased from 3.5 +/- 0.3 to a maximum of 5.1 +/- 0.4 ml/min/100 ml (p < 0.001, n = 28) and from 2.8 +/- 0.5 to a maximum of 4.5 +/- 0.5 ml/min/100 ml (p = 0.01, n = 8), respectively. The increase in FBF (delta%) was similar in the two groups (p = 0.9). Thus, we could not demonstrate any relationship between insulin sensitivity and increments in FBF during hyperinsulinemic glucose clamp in borderline hypertensive young men. The moderate increases in FBF during insulin infusion with serum concentrations within the physiological range seem to be time-dependent and not caused by hyperinsulinemia.

Adult↗
PubMed2004

Dynamic ion channel activation scheduling in patch clamp on a chip.

In 2002, Fertig et al. made a remarkable invention: the first successful demonstration of a patch clamp on a chip--a planar quartz-based biological chip that contains up to several hundred ion channels. This patch-clamp chip can be used in massively parallel screens for ion channel activity, thereby providing a high-throughput screening tool for drug discovery efforts. In this paper, we propose computationally efficient dynamic stochastic scheduling algorithms for activating individual ion channels in the patch-clamp chip. By formulating the ion channel activation scheduling problem as a partially observed Markov decision process with a multiarmed bandit structure, near-optimal dynamic scheduling for activation of the individual channels is achieved to optimize the information gained from the patch-clamp chip. Numerical examples using state-of-the-art algorithms developed recently in artificial intelligence and operations research are presented to illustrate these dynamic ion channel (macromolecule) activation scheduling algorithms.

Algorithms↗
PubMed2001

Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings.

1. Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective I(h) blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. 2. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (C(m)), specific membrane resistance (R(m)) and intracellular resistivity (R(i)) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: C(m), 0.77 +/- 0.17 microF cm(-2) (mean +/- S.D.; range, 0.64-1.00 microF cm(-2)), R(m), 122 +/- 18 kOmega cm(2) (98-141 kOmega cm(2)) and R(i), 115 +/- 20 Omega cm (93-142 Omega cm). 3. The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. 4. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. 5. A model of I(h) was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. 6. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients such as synaptic potentials and action potentials are heavily filtered.

4-Aminopyridine↗
PubMed1994

Local, stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy.

1. Confocal microscopy and the fluorescent Ca2+ indicator fluo-3 (K+ salt) were used to measure cytosolic free calcium ion concentration ([Ca2+]) during excitation-contraction (E-C) coupling in single, voltage-clamped, rat cardiac ventricular cells. 2. Local [Ca2+]i transients were measured nearly simultaneously in different, separate, subcellular volumes of approximately 2.0 microns 3. During depolarization, local [Ca2+]i transients were distinctly different from each other and from whole-cell [Ca2+]i transients. These differences were particularly apparent during small depolarizations, and were substantially reduced by ryanodine. 3. Components of the local [Ca2+]i transients, particularly those evoked by small depolarizations, were closely similar, in time course and amplitude, to spontaneous local [Ca2+]i transients, or 'sparks' (which have been shown previously to be Ca2+ released from sarcoplasmic reticulum). 4. Analysis of local [Ca2+]i transients in the spatial frequency domain (power spectrum) revealed that high power at spatial frequencies of 0.05-0.2 microns-1 was always associated with spontaneous calcium 'sparks' and with local [Ca2+]i transients evoked by small depolarizing pulses (e.g. to -31 mV). Evoked local [Ca2+]o transients in the presence of ryanodine, and those evoked by depolarization to very positive clamp-pulse potentials (+45 mV), were associated with considerably lower power at this frequency. 5. The results suggest that whole-cell [Ca2+]i transients evoked by voltage-clamp depolarization, and thus by L-type Ca2+ current, are comprised of local [Ca2+]i transients that are similar to the spontaneous calcium 'sparks'. At very positive clamp-pulse potentials, however, the electrically evoked local [Ca2+]i transients may be smaller, perhaps as a result of smaller unitary L-type Ca2+ current.

Aniline Compounds↗
PubMed1995

Voltage-dependent clamp of intracellular pH of identified leech glial cells.

1. The intracellular pH (pHi) was measured in voltage-clamped, giant neuropile glial cells in isolated segmental ganglia of the leech Hirudo medicinalis, using double-barrelled, pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. The potential sensitivity of the pHi regulation in these glial cells was found to be due to an electrogenic Na(+)-HCO3- cotransporter (Deitmer & Szatkowski, 1990). 2. In the presence of 5% CO2 and 24 mM HCO3- (pH 7.4), pHi shifted by 1 pH unit per 110 mV, corresponding to a stoichiometry of 2HCO3-: 1 Na+ of the cotransporter, while in Hepes-buffered CO2-HCO3(-)-free saline (pH 7.4), pHi changed by 1 pH unit per 274 mV. The potential sensitivity of pHi decreased at lower pHo, being 1 pH unit per 216 mV at external pH (pHo) 7.0. 3. Changing pHo between 7.8 and 6.6 induced pHi shifts with a slope of 0.72 pHi units per pHo unit in non-clamped, and of 0.80 pHi units per pHo unit in voltage-clamped cells, indicating that pHi largely followed pHo. The electrochemical gradient of H(+)-HCO3- across the glial membrane was around 56 mV, and remained almost constant over this pHo range. 4. The membrane potential-dependent and pHo-sensitive shifts of pHi were unaffected by amiloride, an inhibitor of Na(+)-H+ exchange. 5. The intracellular acidification upon lowering pHo could be reversed by depolarizing the membrane as predicted from a cotransporter, whose equilibrium follows the membrane potential by resetting pHi. 6. The results indicate that the pHi of leech glial cells is dominated by the electrogenic Na(+)-HCO3- cotransporter, and is hence a function of the membrane potential, and the Na+ and H(+)-HCO3- gradients, across the cell membrane.

Amiloride↗
PubMed2005

Role of the transient outward current (Ito) in shaping canine ventricular action potential--a dynamic clamp study.

Transmural electrical dispersion determines the repolarization sequence across the ventricular wall, and plays an important role in the development of arrhythmias under pathological conditions. While it is clear that the transmural gradient of the transient outward current (I(to)) underlies the dramatic difference in phase 1 repolarization across the ventricle, its contribution to the transmural action potential duration (APD) dispersion is not clear. We investigated this problem using the dynamic clamp technique in canine ventricular myocytes. The dynamic clamp allows quantitative 'insertion' of simulated conductances in real, biological cells, bridging pure computer modelling and experimental electrophysiology. 'Insertion' of an epicardial level of I(to) in endocardial cells produced a prominent phase 1 repolarization and a 'spike-and-dome' action potential morphology, but did not significantly affect the APD. Increasingly larger I(to) densities prolonged, and then dramatically shortened the endocardial APD. We also used the dynamic clamp to subtract, or 'block' the native I(to) in epicardial cells. Such 'blockade' eliminated the epicardial action potential notch, but had no significant effect on the APD. We conclude that I(to), while being a key regulator of phase 1 repolarization, does not significantly affect the APD of canine ventricular myocytes, and that the I(to) gradient is not a significant contributor to the transmural APD dispersion in the canine ventricle. By allowing computer simulation on a biological background, the dynamic clamp is a new and effective tool to study the ionic basis of the electrical properties of cardiac cells.

Action Potentials↗
PubMed1998

Lactate clamp: a method to measure lactate utilization in vivo.

A lactate clamp method has been developed to quantify the whole body lactate utilization in conscious, unstressed rats. Dichloroacetate (DCA), a known lactate utilization enhancer, was used to validate the method. Fasting blood lactate concentrations before the clamps were identical for DCA-treated (1 mmol/kg) and control groups (1.65 +/- 0.37 vs. 1.65 +/- 0.19 mM). The animals received a primed continuous lactate infusion for 90 min at variable rates to clamp the blood lactate concentration at 2 mM. The steady-state (60-90 min) lactate infusion rate, which represents the whole body lactate utilization in DCA-treated animals, was 144% higher than that in the control animals (13.2 +/- 1.0 vs. 5.4 +/- 1.1 mg . kg-1 . min-1; P < 0.001). The markedly increased lactate infusion rate indicates an enhanced lactate flux by DCA. To determine whether the increased lactate infusion by DCA reflected reduced endogenous lactate production, lactate production was measured. The results indicate that endogenous lactate production was not affected by DCA. In conclusion, the lactate clamp provides a sensitive and reliable method to assess lactate utilization in vivo, a dynamic measurement that may not be clearly demonstrated by blood lactate concentrations per se.

Animals↗