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At least 955 records · Page 53Linked to original sources
PubMed2003

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy.

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.

Anti-Bacterial Agents↗
PubMed1997

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices.

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (IH) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of IH if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of IH by Cs+ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that IH, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that IH does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of IH were modulated in a circadian manner. However, no substantial day-night differences in IH were found. We conclude that IH, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in 'rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.

Action Potentials↗
PubMed2001

Digitalis-like factor response to hyperinsulinemia accompanying a euglycemic hyperinsulinemic clamp or oral glucose tolerance test.

Many studies of essential hypertension find evidence of insulin resistance in the same individuals, leading some to postulate a hypertensive role for insulin. However, the mechanisms by which insulin might exert a hypertensive effect are not fully resolved. An endogenous sodium pump inhibitor or digitalis-like factor (DLF) has been proposed as a hypertensive agent and its plasma concentrations are elevated in hypertension and in Type II diabetes, where insulin levels are elevated. Hence, we studied the effect of insulin on DLF using two approaches to achieve hyperinsulinemia. Normotensive men and women underwent a hyperinsulinemic, euglycemic clamp (40 mU/m2/min insulin, 40 mU = 1.6 x 10(-6) g) in which plasma insulin concentration was kept at high, but physiologic levels. Serum DLF (measured as inhibition of [Na,K]ATPase activity) and insulin levels were measured at baseline and every 30 min throughout the 2 hr clamp. Additionally, other subjects underwent an oral glucose tolerance test (OGTT) as a second means of increasing insulin levels. Insulin and DLF levels were measured prior to and hourly for 3 hours after receiving 100 gm of oral glucose. Serum DLF increased significantly during the clamp from a baseline of 4.6 +/- 0.81 to a peak of 8.7 +/- 1.2% inhibition (p=0.001). Comparison of the baseline and peak DLF levels with concomitant plasma insulin levels revealed a significant correlation (R=0.60, p=0.003). During the OGTT, DLF levels rose from a baseline of 2.4 +/- 1.0 to a peak level of 5.0 +/- 0.4%, p = 0.04. These results suggest that DLF, a factor that can cause vascular smooth muscle contraction and potentially influence blood pressure, is increased by hyperinsulinemia and provides a mechanism by which insulin may increase blood pressure.

Adolescent↗
PubMed2003

Scientific and technological aspects of oxygen-sensitive electrodes for measurements of oxygen partial pressure in patch-clamp experiments.

Anoxia/hypoxia induces dramatic changes in brain activity leading to the damages in brain structure. Several minutes of decrease in environmental oxygen partial pressure (P(O2)) can irreversibly destroy nerve cells. Therefore, investigations of intracellular mechanisms responsible for hypoxia-induced changes of mammalian nerve system are very important. On-line adjustment and measurements of P(O2) in bath solution during patch-clamp experiments are especially topical. At the recent time, a special interest on the on-line measurements of oxygen contents in bath solution has appeared as a result of application of old approaches previously used for polarographic measurements of oxygen contents in the blood and tissues. Here we describe the simple method of manufacturing oxygen-sensitive microelectrodes, which can be used with standard patch-clamp amplifier. We also describe the main principles of polarographic method and properties of oxygen-sensitive electrodes used in patch-clamp experiments.

Biophysics↗
PubMed2001

Extended dynamic clamp: controlling up to four neurons using a single desktop computer and interface.

The dynamic clamp protocol allows an experimenter to simulate the presence of membrane conductances in, and synaptic connections between, biological neurons. Existing protocols and commercial ADC/DAC boards provide ready control in and between < or =2 neurons. Control at >2 sites is desirable when studying neural circuits with serial or ring connectivity. Here, we describe how to extend dynamic clamp control to four neurons and their associated synaptic interactions, using a single IBM-compatible PC, an ADC/DAC interface with two analog outputs, and an additional demultiplexing circuit. A specific C++ program, DYNCLAMP4, implements these procedures in a Windows environment, allowing one to change parameters while the dynamic clamp is running. Computational efficiency is increased by varying the duration of the input-output cycle. The program simulates < or =8 Hodgkin-Huxley-type conductances and < or =18 (chemical and/or electrical) synapses in < or =4 neurons and runs at a minimum update rate of 5 kHz on a 450 MHz CPU. (Increased speed is possible in a two-neuron version that does not need auxiliary circuitry). Using identified neurons of the crustacean stomatogastric ganglion, we illustrate on-line parameter modification and the construction of three-member synaptic rings.

Algorithms↗
PubMed1996

Action potentials recorded with patch-clamp amplifiers: are they genuine?

A growing number of experimental studies have used patch-clamp amplifiers (PCAs) in the current-clamp (CC) mode to investigate classical excitability. In this paper we show that the measurements obtained in this way are affected by errors due to the electronic design of the PCA input section. We present experimental evidence of such errors, and demonstrate that they derive from PCA current absorption. Moreover, we propose a new PCA input-circuit configuration for the CC mode, which is suitable for accurately recording physiological voltage signals and is perfectly compatible with the standard voltage-clamp mode.

Action Potentials↗
PubMed2001

Peripheral blood mitochondrial DNA content is inversely correlated with insulin secretion during hyperglycemic clamp studies in healthy young men.

Abnormalities in mitochondrial DNA(mtDNA) have been implicated in the pathogenesis of diabetes mellitus. We recently reported that decreased mtDNA content precedes the development of diabetes mellitus and is associated with parameters of insulin resistance. In this study, we examined whether there is any relation between mtDNA content and insulin secretion. We compared the mtDNA content of peripheral blood leukocytes with the parameters of insulin secretion measured by hyperglycemic clamp in a group of healthy young men. There were statistically significant correlations between mtDNA content in peripheral blood and fasting plasma insulin (r=-0.43, P<0.05) and C-peptide levels (r=-0.44, P<0.05). MtDNA content also correlated negatively with acute insulin response(r=-0.48, P<0.05), late insulin response (r=-0.50, P<0.05) during hyperglycemic clamp and insulin secretion after glucagon stimulation (r=-0.60, P<0.01). mtDNA content in peripheral blood correlated negatively with homeostasis model (HOMA) insulin resistance (r=-0.45, P<0.05) although it did not correlate with the insulin insensitivity index (M/I) during hyperglycemic clamp. In summary, the mtDNA content of peripheral blood correlated negatively with indices of insulin resistance and insulin secretion in healthy young men. The compensatory response of pancreas beta cells to insulin resistance might contribute in part to increased insulin secretion in these subjects.

Adult↗
PubMed1999

Peripheral blood mitochondrial DNA content correlates with lipid oxidation rate during euglycemic clamps in healthy young men.

Both qualitative and quantitative changes in mitochondrial DNA (mtDNA) have been implicated in the pathogenesis of diabetes mellitus. It was previously found that decreased mtDNA content preceded the development of diabetes and mtDNA content correlated with the clinical parameters of insulin resistance syndrome, including diastolic blood pressure and waist-hip ratio. These results prompted one to look whether there are correlations between mtDNA content and the biochemical parameters of insulin resistance in non-diabetic subjects. MtDNA content of peripheral blood leukocytes was measured in Korean healthy young men, and this was correlated with various parameters of fuel metabolism at baseline and during euglycemic hyperinsulinemic clamps with indirect calorimetry. MtDNA content in peripheral blood leukocytes did not correlate with insulin sensitivity index or other metabolic variables such as body mass index (BMI), waist-to-hip ratio (WHR) and blood pressure. However, mtDNA content showed a positive significant correlation with fat oxidation rate during euglycemic clamps (r = 0.61, P < 0.05). Changes in fat oxidation rate and carbohydrate oxidation rate during the clamps were significantly correlated with mtDNA content (r = 0.65, P < 0.05, r = -0.65, P < 0.05, respectively). These results suggest that mtDNA content in peripheral blood may not correlate with insulin resistance per se but with some aspect of insulin resistance in healthy young men.

Adult↗
PubMed2001

Correlation between the glucose clamp technique and the homeostasis model assessment in hypertension.

The homeostasis model assessment (HOMA) has been extensively used as a reliable surrogate marker for measuring insulin resistance in patients with diabetes mellitus and in normal subjects. Comparative data in another insulin resistance state, hypertension, is lacking. The goal of the present study was to obtain that information by testing the correlation of HOMA with the euglycemic hyperinsulinemic clamp in 36 hypertensive and 27 normotensive subjects. Clamp-derived insulin sensitivity was calculated as the glucose disposal rate over steady-state plasma insulin concentration. Homeostasis model assessment was computed using the formula (fasting glucose x fasting insulin)/22.5. There was significant correlation between the clamp and HOMA for both the hypertensive (r = -0.64, P < .0001) and normotensive subjects (r = -0.58, P = .002). The HOMA can be used reliably as a less expensive and less cumbersome alternative for measuring insulin resistance in hypertension.

Adult↗
PubMedDate not supplied

Additional efficient computation of branched nerve equations: adaptive time step and ideal voltage clamp.

Various improvements are described for the simulation of biophysically and anatomically detailed compartmental models of single neurons and networks of neurons. These include adaptive time-step integration and a reordering of the circuit matrix to allow ideal voltage clamp of arbitrary nodes. We demonstrate how the adaptive time-step method can give equivalent accuracy as a fixed time-step method for typical current clamp simulation protocols, with about a 2.5 reduction in runtime. The ideal voltage clamp method is shown to be more stable than the nonideal case, in particular when used with the adaptive time-step method. Simulation results are presented using the Surf-Hippo Neuron Simulation System, a public domain object-oriented simulator written in Lisp.

Cell Compartmentation↗
PubMed2000

Electrophysiological characterization of human keratinocytes using the patch-clamp technique.

The living, not-cornified part of the epidermis consists mainly of keratinocytes. The control mechanisms of proliferation and differentiation are only partly understood. Similarly, the influence of ionic channels of the cell membrane on the proliferation of keratinocytes remains unclear. Preliminary investigations point to a relation between the opening of ionic channels and keratinocyte proliferation. Therefore, voltage clamp experiments were performed to gain further knowledge of the electrophysiological characteristics of human keratinocyte cellular membranes. In-vitro cultured keratinocytes of the cell line HaCaT were characterized by means of the voltage-clamp technique. As measured in the whole-cell configuration, changing the extracellular K+ or Cl--concentration shifted the membrane potential of HaCaT cells. Application of the patch-clamp-technique in the cell-attached and inside-out configuration revealed an ionic channel with a conductance of multiples of 200 pS. The reversal potential of the single channel current was shifted by substituting of intracellular Cl- by aspartate-. Channel openings disappeared after addition of 0.1 mM of the anion channel blocker 4',4' diisothiocyanato-stilbene-2-2' disulfonic acid (DIDS). It is concluded that this channel contributes to the Cl--conductance of the cellular membrane and is a determinant of the membrane potential of human keratinocytes. This channel may represent a target for pharmacological manipulation of the membrane potential and possibly the growth of human keratinocytes in dermatological proliferation disorders.

Cell Division↗
PubMed1997

Seal-promoting solutions and pipette perfusion for patch clamping plant cells.

Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (G omega) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl(-)concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.

Arabidopsis↗
PubMed2001

Glucose disposal rates calculated from 60- to 90-minute isoglycemic hyperinsulinemic glucose clamp correlate with cardiovascular risk factors in borderline hypertensive young men.

The hyperinsulinemic glucose clamp is generally performed for at least 120 minutes, due to assumptions of steady-state. We were interested in relationships between glucose disposal rate (GDR) and cardiovascular risk factors, rather than a standard measure of insulin sensitivity per se. Therefore, we analyzed 120-minute clamps performed on borderline hypertensive, but otherwise healthy young men (n = 19). GDR was calculated at different time points and related to baseline cardiovascular risk factors and responses to a mental stress test (MST). The 60-, 90-, and 120-minute GDR correlated significantly with serum high-density lipoprotein (HDL) cholesterol (r=.59, r=.50, and r=.53, respectively), heart rate (HR) during MST (r = -.65, r = -.64, and r = -.58, respectively) and plasma epinephrine (Epi) (r = -.55, r= -.58, and r = -.56, respectively) and norepinephrine (NE) (r = -.52, r = -.49, and r = -.48, respectively) 1 minute after announcement of the MST (all P <.05). Although not statistically significant at all time points, similar relationships were observed between GDR and resting HR, systolic blood pressure (BP) at rest and during mental stress, body mass index (BMI), serum total cholesterol (Chol), serum triglycerides (TG), and blood hemoglobin (HgB), with remarkable consistency from about 40 to 50 minutes onwards. HDL cholesterol and Epi remained independent in stepwise multiple regression analyses with the 60-, 90-, and 120-minute GDR as dependent variables (all P <.05). We suggest that 60- to 90-minute glucose clamps may provide information about the relationship between insulin sensitivity and various cardiovascular risk factors in borderline hypertensive young caucasian men.

Adult↗
PubMed2001

Modified hyperinsulinaemic, eu- and hypoglycaemic clamp technique using lispro-insulin for insulinoma diagnostic.

SUMMARY: Characterization of metabolically inadequate insulin secretion is essential for insulinoma diagnostics. Hyperinsulinaemic, eu- and hypoglycaemic clamp procedures have been used to suppress endogenous insulin secretion in healthy subjects. The use of exogenous insulin precluded the use of insulin as a parameter to be measured. We now suggest to use exogenous insulin lispro and an insulin-specific ELISA not cross reacting with insulin lispro. Thus, determination of insulin by ELISA in this experimental setting reflects endogenous insulin. A 39-year-old man with a surgically confirmed pancreatic insulinoma was studied under hyperinsulinaemic [lispro insulin 40 mU x m(-2) body surface x min(-1)] clamp conditions. Euglycaemia was achieved (3.8 +/- 0.5 mmol/L) for 1 h and hypoglycaemia (2.36 +/- 0.49 mmol/L) was achieved for another 30 min. Insulin was evaluated by ELISA (cross-reaction with lispro insulin < 0.006%, C-peptide < 0.01%, proinsulin < 0.001%) and by a nonselective RIA (cross-reaction with proinsulin 40%). In control subjects the euglycaemic hyperinsulinaemia suppressed C-peptide to 0.36 +/- 0.03 ng/ml and hypoglycaemic hyperinsulinaemia to 0.29 +/- 0.03 ng/ml. Endogenous insulin was suppressed to 2.8 +/- 0.03 mU/L under euglycaemia and to 2.6 +/- 0.03 mU/L under hypoglycaemia in control subjects. In the insulinoma patient apparently irregular but small changes in both C-peptide (1.43 +/- 0.1 ng/ml) and more pronounced changes in endogenous insulin concentrations 4.41 +/- 0.1 mU/l under euglycaemia and 5.35 +/- 0.3 mU/l under hypoglycaemic conditions, were observed. The basal level of insulin (ELISA insulin 4.6 mU/L) and C-peptide (1.7 ng/ml) were not markedly elevated. Determination of insulin allowed better characterization of irregular pulses because of the shorter half-life of insulin relative to C-peptide. The new modification of sequential eu- and hypoglycaemic clamp procedures should also be useful in pharmacological studies of insulinotropic substances. Direct measurement of peripheral insulin may be more sensitive than C-peptide to detect low levels of autonomous insulin secretion in small insulinomas.

Adult↗
PubMed2005

A comparison of the steady-state pharmacokinetics and pharmacodynamics of a novel rapid-acting insulin analog, insulin glulisine, and regular human insulin in healthy volunteers using the euglycemic clamp technique.

Insulin glulisine is a new rapid-acting insulin analog. The aim of this study was to assess the glucodynamic efficacy of insulin glulisine compared with regular human insulin (RHI) using a manual euglycemic clamp technique. Steady-state pharmacokinetics of insulin glulisine, and its cardiac safety (ECG) and tolerability after intravenous administration, were also determined. This was a single center, randomized, open-label, two-way crossover study in healthy male subjects (n = 16). At the treatment visits subjects received an intravenous infusion of the study drug at a rate of 0.8 mU kg (-1) . min (-1) for 2 hours. Individual baseline glucose concentrations were targeted for euglycaemia and maintained with a manual adjusted 20 % glucose solution over the clamp period of a maximum 6 hours. A glulisine-specific antibody was used to quantify glulisine concentrations by radioimmunoassay, while a non-specific insulin antibody and C-peptide based correction for endogenous insulin was used to estimate exogenous human insulin (RHI). At steady state (90 - 120 min), insulin glulisine and RHI had equivalent glucose utilization (GIR-AUC (SS), 209 [corrected] mg . kg (-1) for glulisine, 214 [corrected] mg . kg (-1) for RHI) and infusion rates (GIR (SS), 7.0 and 7.2 [corrected] mg . kg (-1) . [corrected] min (-1) . kg (-1)). Both insulins also presented equal total glucose disposal (GIR-AUC (0 - clamp end), 995 and 1050 [corrected] mg . kg (-1)) and onset of activity within 20 min. Insulin glulisine and RHI showed parallel time concentration profiles with similar distribution and elimination, but the different antibodies employed for radioimmunoassay impeded a quantitative comparison. There were no noteworthy individual or within-group changes in cardiac repolarisation parameters measured by 12-lead ECG during insulin glulisine infusion. In conclusion, insulin glulisine and RHI show similar distribution and elimination profiles and equivalent glucodynamic efficacy on a molar, unit-per-unit basis.

Adult↗
PubMed1996

[Possible applications of the "patch-clamp" method in anesthesiologic research; comment].

The patch clamp technique has gained considerable significance during the past two decades since it was developed. Many mechanisms of cell function have been elucidated by the application of the patch clamp method because it was now possible to demonstrate and investigate directly single ion channels in excitable cell membranes. An important discovery, as far as medicine was concerned, was that various clinically used substances interact directly with ion channels. Local anaesthetics, antiarrhythmics, antidiabetics, muscle relaxants are a few examples. For these reasons, more and more clinical physicians use the patch clamp method for their specific investigations. Collaboration between a clinical institute and a basic research department can often be found, which gives clinical research more background and basic science more practical aspects.

Anesthetics↗
PubMedDate not supplied

Safety and feasibility of cardiac FDG SPECT following oral administration of Acipimox, a nicotinic acid derivative: Comparison of image quality with hyperinsulinemic euglycemic clamping in nondiabetic patients.

BACKGROUND: Image quality of cardiac fluorine-18-deoxyglucose (FDG) studies is highly dependent on the metabolic conditions during the study; hyperinsulinemic euglycemic clamping ensures adequate image quality. However, the approach is time-consuming. Data in a small number of patients suggest that oral administration of a nicotinic acid derivative (Acipimox, 250 mg; Byk, The Netherlands) results in good image quality. METHODS AND RESULTS: The safety and image quality of cardiac FDG single photon emission computed tomography studies after Acipimox administration were evaluated (21 patients, group 2); the results were compared with studies performed during hyperinsulinemic euglycemic clamping (69 patients, group 1). Image quality was assessed visually and quantitatively with use of heart-to-lung, heart-to-liver, and myocardium-to-background ratios. Blood samples were drawn at baseline and at the time of FDG injection to determine levels of glucose, free fatty acids, and insulin. Baseline characteristics of group 1 and 2 patients were comparable. No side effects occurred in group 1. Four patients in group 2 (19%) had paroxysmal flushing. Image quality, assessed visually, was good in 100% of group 1 patients and in 86% of group 2 patients. Images were uninterpretable in only 1 patient in group 2 (5%). All quantitative parameters of image quality (heart-to-lung, heart-to-liver, and myocardium-to-background ratios) were comparable between group 1 and 2 patients. Baseline plasma levels of all substrates were comparable between groups. At the time of FDG injection, plasma levels of glucose and free fatty acids were comparable between groups; insulin was higher in group 1 patients. CONCLUSIONS: Cardiac FDG single photon emission computed tomography after Acipimox is a simple and safe approach that renders comparable image quality to that obtained during hyperinsulinemic euglycemic clamping.

Administration, Oral↗
PubMed2000

The euglycemic hyperinsulinemic clamp examination: variability and reproducibility.

The aim was to examine the reproducibility of the euglycemic hyperinsulinemic clamp method. From a random population sample of 60-year-old clinically healthy men, 32 subjects with varying degrees of insulin sensitivity were recruited. Conventional 2-h clamp examinations were carried out at an interval of 2 weeks. Insulin was infused intravenously (priming for 10 min and thereafter 1.0 mU/kg body wt/min). Glucose was infused concomitantly aiming at a whole blood glucose of 5 mmol/L. The glucose infusion rate (GIR) was adjusted for body weight or fat free mass (FFM), the latter measured with dual-energy X-ray absorptiometry. During the final hour of each examination (60-120 min) the mean whole blood glucose concentrations were 5.06+/-0.15 and 5.09+/-0.17 mmol/L, respectively. Of the different time intervals studied, the glucose infusion rate during the final hour (GIR60-120) showed the highest correlation and lowest coefficient of variation (GIR60-120 adjusted by FFM: r=0.70, coefficient of variation=14.7%). Adjustment of GIR for weight instead of FFM underestimated insulin sensitivity in obese men. GIR60-120 adjusted for FFM tended to increase during the second examination. The measurement error was constant across all GIR. In summary, the euglycemic hyperinsulinemic clamp method has a coefficient of variation around 15%. The glucose infusion rate should be adjusted for fat free mass.

Body Mass Index↗