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At least 937 records · Page 52Linked to original sources
PubMed1994

Lack of acute effect of amylin (islet associated polypeptide) on insulin sensitivity during hyperinsulinaemic euglycaemic clamp in humans.

It is suggested that amylin (islet associated polypeptide), co-secreted with insulin from the pancreatic beta cells acts as a circulating hormone which opposes the action of insulin on muscle and increases hepatic glucose production. We have tested the effect of amylin in human subjects on postabsorptive glucose homeostasis and on insulin sensitivity using the euglycaemic hyperinsulinaemic clamp. The amylin used opposed insulin-mediated glucose disposal in rat soleus muscle at concentrations of 10 nmol/l. Seven subjects were studied on two occasions and infused with either amylin or placebo for 6 h, initially when postabsorptive and then during a euglycaemic hyperinsulinaemic clamp. Mean plasma amylin concentrations during the first 3 h were 2006 +/- 327 pmol/l during amylin infusion and 20 +/- 9 pmol/l during the control infusion. Amylin infusion had no effect on postabsorptive plasma concentrations of insulin (control: 32 +/- 16 vs amylin: 25 +/- 8 pmol/l) or glucose (5.1 +/- 0.1 vs 5.3 +/- 0.1 mmol/l). During the clamp, amylin concentrations were 1636 +/- 422 pmol/l when it was infused and 24 +/- 6 during control infusions. Plasma glucose and insulin concentrations were well matched during the control and amylin infusions (glucose: 4.7 +/- 0.1 vs 4.8 +/- 0.1 mmol/l; insulin: 198 +/- 37 vs 195 +/- 22 pmol/l). Exogenous glucose infusion rates were a mean of 13% lower than control values during the amylin infusion but were not statistically different (p = 0.17).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

What can we learn about cell signalling by combining optical imaging and patch clamp techniques?

Optical imaging is a powerful technique with which to investigate the activity, distribution and movement of biomolecules. The increased resolution of images obtained with confocal microscopy now allows us to visualize the signalling events in individual intracellular organelles. Local photobleaching and uncaging of caged compounds enable investigators to control the activity of many biologically important molecules in small localized regions of both cytosol and internal spaces of cellular organelles. Uncaging and photobleaching conveniently complement laser scanning confocal microscopy. The whole-cell recording configuration of the patch-clamp technique has been widely used not only to measure ionic currents, but also to control the concentration of important molecules in the cytosol. The cell-attached configuration of patch clamp was utilized for local stimulation of the cell and local delivery of the second messengers. This paper describes the advantages of combining patch-clamp and optical imaging methods as well as some of the recent achievements using this approach.

Calcium Signaling↗
PubMed2004

The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes.

We developed a non-invasive technique for electrophysiological investigations of ion transport proteins endogenously or heterologously expressed in Xenopus laevis oocytes. We named this technique the transoocyte voltage clamp (TOVC). Whereas in the classical two-microelectrode voltage-clamp (TEVC) technique, the oocyte is impaled with two glass microelectrodes, we mount the egg in a modified Ussing chamber as used for transepithelial electrophysiological studies. The oocyte is introduced in a container that is positioned between the two chamber halves. Proper fixation of the oocyte in the aperture of the container is accomplished under a stereo binocular microscope and the electrical seal between the oocyte and the container is achieved with silicon grease. The new method allows measurement of transoocyte currents and conductances as well as the recording of membrane impedance and the fluctuation analysis of ion currents. We studied a K+ channel that resembles the inward rectifier K+ channel endogenously expressed in Xenopus laevis oocytes. K+ currents were obtained by exposing one side of the oocyte to K(+)-containing solutions and by the application of different voltages. Adding Cs+ and Ba2+ inhibited these currents. The analysis of the fluctuation in current demonstrated a Lorentzian component in the power density spectrum. With the transoocyte voltage clamped to zero, the corner frequency (fc) was 61+/-1.7 Hz. Imposed positive transoocyte potentials caused a downward shift of fc. These findings are consistent with previous data obtained using the TEVC technique, and extend the characterization of the channel with kinetic data obtained from noise analysis.

Animals↗
PubMed1998

A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels.

The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 microm and variable lengths of 100 microm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall.

Animals↗
PubMed2026

Sutureless versus renorrhaphy in robot-assisted off-clamp partial nephrectomy: a systematic review and meta-analysis.

BACKGROUND: The necessity of routine parenchymal renorrhaphy during off-clamp robot-assisted partial nephrectomy (RAPN) remains uncertain. This study aimed to compare perioperative, functional, safety, and oncological outcomes between sutureless and conventional renorrhaphy. METHODS: We conducted a systematic review and meta-analysis following PRISMA 2020 guidelines. Comparative studies evaluating sutureless versus conventional renorrhaphy during purely off-clamp RAPN were included. Trifecta achievement was the primary outcome. Random-effects models were used for pooled analyses, with subgroup analysis according to study design. RESULTS: Four studies involving 787 patients, including one randomized controlled trial (RCT) and three propensity score-matched (PSM) studies, were included. The overall pooled estimate showed no statistically significant difference in Trifecta achievement (RR 1.17, 95% CI 0.97-1.41), with substantial heterogeneity (I² = 86.5%). The PSM studies favored the sutureless approach (RR 1.26, 95% CI 1.05-1.52), whereas the RCT yielded an RR of 0.97 (95% CI 0.92-1.04) and met the prespecified noninferiority criterion without demonstrating superiority. The sutureless approach was associated with a smaller perioperative eGFR decline (MD - 3.89, 95% CI - 6.16 to - 1.62), while no significant difference was observed in eGFR at 3 months. No statistically significant differences were identified in major complications, blood transfusion, or positive surgical margins; urinary and vascular complications were sparsely reported. CONCLUSIONS: In selected patients undergoing purely off-clamp RAPN, randomized evidence supports the noninferiority of a strategy that omits routine parenchymal renorrhaphy while permitting clinically necessary selective repair, but does not demonstrate superiority. Favorable estimates from PSM studies remain vulnerable to intraoperative treatment-selection bias. Current evidence is insufficient to determine whether omission of renorrhaphy affects urinary complications, long-term renal function, or oncological outcomes. REGISTRATION: This systematic review was registered prospectively in PROSPERO (CRD420261435995).

Humans↗
PubMed1988

Androgen levels during sequential insulin euglycemic clamp studies in patients with polycystic ovary disease.

It is postulated that insulin may play a role in the regulation of ovarian androgen production. In order to test the possible interrelation between serum insulin levels and androgen production, sequential euglycemic insulin clamp (Mode 9:1 on Biostator, insulin infusion rate: 0.1; 0.2 and 0.4 U/kg b. wt/h, each rate for 90 min, BC = 80 mg/dl) was done in 6 patients with polycystic ovary disease and normal glucose tolerance. Insulin, C-Peptide, testosterone and dehydroepiandrosterone-sulphate were measured in 0, 70, 80, 90, 160, 170, 180, 250, 260 and 270 min. Significant suppression of C-Peptide levels were achieved (0 min vs 270 min = 0.81 + 0.25 vs 0.15 + 0.20 nmol/l; P less than 0.05). Basal insulin as well as the mean plateau for each insulin infusion rate were as follows: 28 + 9; 248 + 119; 427 + 69 and 524 + 77 microU/l. There was significant testosterone increase at the end of insulin infusion (0 vs 270 min = 4.8 + 1.2 vs 8.1 + 1.7 nmol/l; P less than 0.05). There were no significant changes in dehydroepiandrosterone-sulphate levels during clamp studies (0 vs 270 min = 1055 + 133 vs 913 + 114 ng/ml; P greater than 0.05). It is concluded that acute insulin infusion under the condition of sequential euglycemic clamp could increase androgen production in the ovaries of patients with PCO.

Adult↗
PubMed1991

Effect of insulin on the properties of liver carnitine palmitoyltransferase in the starved rat: assessment by the euglycemic hyperinsulinemic clamp.

The effect of insulin on the properties of liver carnitine palmitoyltransferase I (CPT I) was assessed in conscious starved rats with the euglycemic hyperinsulinemic clamp. A 24-hour clamp was necessary to fully reverse the effect of starvation on liver malonyl-CoA concentration, CPT I maximal activity, and apparent km and Ki for malonyl-CoA. Since glucagon was not decreased during the clamp, insulin is the major factor involved in the regulation of CPT I.

Animals↗
PubMed1993

Insulin resistance in the conscious spontaneously hypertensive rat: euglycemic hyperinsulinemic clamp study.

To determine whether spontaneously hypertensive rats (SHR) are insulin resistant when compared with their genetic control, Wistar-Kyoto rats (WKY), insulin-stimulated glucose utilization was studied in both strains with the euglycemic hyperinsulinemic clamp technique. This methodology can determine if insulin resistance is present and whether it is due to ineffective stimulation of peripheral glucose utilization, or to incomplete suppression of (hepatic) endogenous glucose production (EGP) by insulin, or both. Twelve WKY and 15 SHR (all male) had long-term catheters surgically placed. After surgical recovery, fasting metabolic parameters were measured in the conscious, unstressed state. Clamp studies were then performed on nine WKY and eight SHR. EGP was measured before and during euglycemic hyperinsulinemia with the tracer-dilution technique (6-3H-glucose). Indices of fasting metabolism (plasma glucose, insulin, and hepatic EGP) were not different between WKY and SHR. During the clamp studies, the glucose infusion rate (GIR) required to maintain euglycemia was significantly lower in SHR (SHR, 0.055 +/- 0.003 v WKY, 0.106 +/- 0.001 mmol/kg.min-1; P < .001). EGP was completely suppressed during euglycemic hyperinsulinemia in all WKY and in six of eight SHR. We conclude that conscious, nonstressed SHR are insulin resistant when compared with WKY. Attenuated insulin-stimulated peripheral glucose utilization implicates skeletal muscle, and not liver, as the primary site of insulin resistance in SHR.

Animals↗
PubMed1995

In situ regulation of lipolysis by insulin and norepinephrine: a microdialysis study during euglycemic-hyperinsulinemic clamp.

Lipolytic responsiveness of subcutaneous and epididymal adipose tissue to norepinephrine (NE) was measured by microdialysis before and during a euglycemic-hyperinsulinemic clamp in male Sprague-Dawley rats (280 +/- 7g, n = 8). Microdialysis probes were perfused with standard Krebs-Ringer buffer without (basal condition [BC]) or with NE 10(-6) mol/L to determine basal and stimulated rates of lipolysis. The dialysate concentration of glycerol was measured (lipolytic index). NE infusion resulted in 3.0- and 4.2-fold increases in glycerol release in abdominal subcutaneous and epididymal adipose tissues, respectively. A euglycemic-hyperinsulinemic clamp at 6 mU/kg.min increased by ninefold the insulinemia (120 +/- 9 U/L). Hyperinsulinemia suppressed basal glycerol release by 57% and 42% in subcutaneous and epididymal adipose depots, respectively (BC + I). Lipolytic responses to NE infusion during a euglycemic-hyperinsulinemic clamp (NE + I) were reduced by 45% and 33% in subcutaneous and epididymal adipose tissues, respectively, as compared with BC. Under BC, the lipolytic response to NE was greater in epididymal than in subcutaneous adipose tissue. Physiological levels of insulin regulated basal lipolysis and counteracted adrenergic stimulation of lipolysis to a similar extent in both superficial (subcutaneous) and intraabdominal (epididymal) adipose tissue. Our findings show that lipolysis is more responsive to NE in epididymal than in subcutaneous adipose tissue. The antilipolytic effects of insulin are similar in both superficial and deep intraabdominal adipose tissues. Furthermore, physiological plasma insulin levels cannot fully antagonize the lipolytic effects of NE.

Adipose Tissue↗
PubMed1994

Measuring complex admittance and receptor current by single electrode voltage-clamp.

Studying the membrane properties of small excitable cells like sensory receptors in situ is often difficult. Two new techniques are described here which utilize white noise during single-electrode voltage-clamp. Cells are impaled with a single microelectrode and voltage-clamped to a given holding potential, using a time-sharing technique. The first method, based on modulating the voltage command with repeated sequences of a pseudorandom stimuli, allows measurements of cell conductance (complex admittance) in the frequency domain. The second method is designed to characterize the dynamics of the receptor current in the frequency domain. In both cases, R1-6 type blowfly photoreceptors were used as experimental models. The photoreceptor was first light-adapted to a steady light background and then clamped to the resulting potential. A pseudorandomly modulated light stimulus was then superimposed on the steady light background and the resulting receptor current was recorded. The frequency response was then calculated from the light modulation and the receptor current via fast Fourier transform (FFT). By using intracellularly applied ion channel blockers, the effects of active and passive membrane properties in modulating the transmitted signals could also be studied.

Animals↗
PubMedDate not supplied

A Windows software package to record from voltage-clamped Xenopus oocytes.

We have written a software package to record, display and analyze membrane currents elicited by neurotransmitter receptors or voltage-activated channels in voltage-clamped Xenopus oocytes. This suite, which consists of 4 applications, runs on IBM-PC compatible microcomputers under Windows 3.1. The recording programs use Direct Memory Access (DMA) to access the analog-digital board. The first program, NicPulse, is aimed at studying voltage-activated channels. It delivers voltage steps to the voltage-clamp and records the resulting membrane current. The second program, NicScope, emulates a dual-trace digital oscilloscope. It operates either in continuous or triggered mode, and is used chiefly to display neurotransmitter-induced responses in oocytes. The third recording program, VRamp, automatically determines the voltage-current relationship of drug-activated responses (I/V curve), by applying a voltage ramp and recording the subsequent clamping current. The last program, NicView, is designed to analyze records taken with NicScope and NicPulse. The present paper will discuss several issues regarding the design of these programs, and will give a brief description of each application.

Animals↗
PubMed1994

Patch-clamp recordings from subpopulations of autonomic and afferent neurons identified by axonal tracing techniques.

This study determined whether axonal tracing methods can be used in combination with patch-clamp techniques to examine the electrical properties of identified populations of autonomic and afferent neurons in the adult rat. Fluorescent dyes (Fast Blue, FB and Fluoro-Gold, FG) were injected into the wall of the urinary bladder or colon and into various somatic structures to label postganglionic neurons in the major pelvic ganglia (MPG) as well as visceral and somatic afferent neurons in the lumbosacral dorsal root ganglia (DRG) and trigeminal ganglia (TG). One to 3 weeks after dye injection, neurons were isolated from ganglia by enzymatic dissociation. Following dissociation, single neurons labelled with FB were identified in the three types of ganglion preparations; however FG was only identified consistently in TG neurons. FB was retained in neurons during short-term culture (1-5 days). Following 10 to 20 s exposure to UV light which was required for identification of the cells, whole-cell patch-clamp recordings revealed that the electrophysiological properties of FB-labelled cells did not differ from those of unlabelled cells. However, a more prolonged exposure (1-5 min) of the neurons to UV light produced irreversible damage to the cells which was evident as changes in the action potential, sodium current and resting membrane potential. These results indicate that patch-clamp recording in combination with axonal tracing is a useful approach for studying the electrical properties of identified populations of autonomic and afferent neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

Insulin antibody does not cause insulin resistance during glucose clamping in rats.

Although it has often been stated that insulin antibodies cause insulin resistance, this concept is still controversial. The effect of insulin antibody GP30, commonly used in insulin radioimmunoassay, on insulin action was investigated in Wistar rats in vivo by the euglycemic glucose clamp technique. As a preliminary experiment, the equilibrium time required for insulin antibody to bind with endogenous insulin was examined. One hundred microliters/kg insulin antibody took 60 min or more to attain equilibrium, but 10 microliters/kg insulin antibody almost immediately equilibrated with endogenous insulin. During a 60-min glucose clamp study, 2 mU/kg/min porcine insulin was infused with 100 microliters/kg insulin antibody. At steady state, during the last 20-min period, the mean glucose infusion rate was 2.10 +/- 0.85 mg/kg/min (n = 5, mean +/- SD), significantly lower than the 5.77 +/- 1.61 mg/kg/min of the control, indicating insulin resistance before equilibrium was reached. However, the glucose infusion rates during the clamp with 10 microliters/kg insulin antibody and 100 microliters/kg insulin antibody infused 75 min before the insulin were 6.10 +/- 1.44 and 7.12 +/- 1.19 mg/kg/min, respectively, no different from the control. In these instances, free insulin levels measured by radioimmunoassay using the polyethyleneglycol method were 43.8 +/- 20.4 and 15.4 +/- 6.1 microU/ml, respectively, lower than the control (77.0 +/- 16.1 microM/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effect of porcine somatotropin on the response of growing pigs to acute challenges of glucose, insulin and epinephrine and during a hyperinsulinemic-euglycemic clamp.

Response of tissues to homeostatic signals may play a role in the mediation of nutrient partitioning effects of somatotropin. To investigate this, the effects of exogenous porcine somatotropin (pST) on the metabolic responses to a series of intravenous challenges with dextrose, insulin and epinephrine were examined in twelve crossbred barrows (65 kg). In addition, the hyperinsulinemic-euglycemic clamp technique was used to further explore effects of pST on insulin resistance in eight of these animals. Pigs received daily sc injections of either pituitary-derived pST (120 micrograms/kg bw) or an equivalent volume of excipient for 28 d. Treatment with pST resulted in a chronic elevation of plasma glucose, insulin and non-esterified fatty acid concentrations and lowered glucagon concentrations. Acute iv challenges of dextrose (100 mg/kg bw), insulin (1.0 micrograms/kg bw), and epinephrine (2.2 micrograms/kg bw) were administered on days 21, 22, and 23 of the treatment period, respectively. Hyperinsulinemic-euglycemic clamps were carried out on day 28. Effects of pST were most dramatic for responses associated with insulin. In pST-treated pigs, insulin response to dextrose infusion was enhanced, while glucose response to insulin was attenuated and glucose clearance rate was reduced. During the hyperinsulinemic-euglycemic clamp, dextrose infusion rate required to maintain euglycemia during physiologic elevations of insulin was reduced in pST-treated pigs to 28% of control. In pST-treated pigs, glucose response to epinephrine challenge was halved, while insulin response was increased three-fold. Therefore, one mechanism by which pST shifts the nutrient partition is by altering metabolic responses to homeostatic signals. In growing pigs, this is especially evident for glucose response to insulin.

Animals↗
PubMed2006

Hourglass SiO2 coating increases the performance of planar patch-clamp.

Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.

Animals↗
PubMed2004

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons.

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.

Animals↗
PubMed2004

The dynamic clamp comes of age.

The dynamic clamp uses computer simulation to introduce artificial membrane or synaptic conductances into biological neurons and to create hybrid circuits of real and model neurons. In the ten years since it was first developed, the dynamic clamp has become a widely used tool for the study of neural systems at the cellular and circuit levels. This review describes recent state-of-the-art implementations of the dynamic clamp and summarizes insights gained through its use, ranging from the role of voltage-dependent conductances in shaping neuronal activity to the effects of synaptic dynamics on network behavior and the impact of in vivo-like input on neuronal information processing.

Animals↗
PubMed2000

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.

Animals↗