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At least 1,441 records · Page 80Linked to original sources
PubMed2000

Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity: studies in subjects with various degrees of glucose tolerance and insulin sensitivity.

OBJECTIVE: To evaluate whether the homeostasis model assessment (HOMA) is a reliable surrogate measure of in vivo insulin sensitivity in humans. RESEARCH DESIGN AND METHODS: In the present study, we compared insulin sensitivity as assessed by a 4-h euglycemic (approximately 5 mmol/l) hyperinsulinemic (approximately 300 pmol/l) clamp with HOMA in 115 subjects with various degrees of glucose tolerance and insulin sensitivity. RESULTS: We found a strong correlation between clamp-measured total glucose disposal and HOMA-estimated insulin sensitivity (r = -0.820, P<0.0001), with no substantial differences between men (r = -0.800) and women (r = -0.796), younger (aged <50 years, r = -0.832) and older (r = -0.800) subjects, nonobese (BMI <27 kg/m2, r = -0.800) and obese (r = -0.765) subjects, nondiabetic (r = -0.754) and diabetic (r = -0.695) subjects, and normotensive ( r = -0.786) and hypertensive (r = -0.762) subjects. Also, we found good agreement between the two methods in the categorization of subjects according to insulin sensitivity (weighted k = 0.63). CONCLUSIONS: We conclude that the HOMA can be reliably used in large-scale or epidemiological studies in which only a fasting blood sample is available to assess insulin sensitivity

Adult↗
PubMed1996

Cell-attached patch clamping of the intact rabbit ciliary epithelium.

Following thorough removal of adhering aqueous humor, we have succeeded in patch clamping the intact rabbit ciliary epithelium in the cell-attached and inside-out excised-patch modes. Rapidly fluctuating currents ("chatter activity') were observed during recordings conducted for as long as 1 h. Chatter activity did not reflect seal instability since interconversion was noted between chatter activity and transitions between stable open and closed states, excision of patches into the bath was associated with substantial shifts in the reversal potential, and chatter activity could be triggered by sustained hyperpolarization, but was insensitive to stretch. The chatter channel was identified as cation-nonselective from the reversal potentials both in the cell-attached and excised-patch modes. The channel's kinetics were similar to those of the cGMP-activated phototransduction channel. The results of PCR amplifications of fragments in cDNA libraries from both human ciliary body and human nonpigmented ciliary epithelial (NPE) cells indicated that human ciliary epithelial cells transcribe message for the retinal phototransduction channel. The possible role of the phototransduction channel in expressing chatter activity was further explored by perfusing preparations with a known activator of that channel (cGMP) and with a known inhibitor (L-cis-diltiazem). Neither agent significantly affected chatter behavior. We conclude that: (1) this is the first demonstration of the feasibility of patch-clamping the intact ciliary epithelium; (2) the NPE cells display chatter activity arising from rapidly fluctuating transitions of a cation-nonselective channel; (3) NPE cells can transcribe message for the cation-nonselective phototransduction channel; and (4) if the observed chatter activity is from a homologue of the photo-transduction channel, the homologue is pharmacologically distinct.

Animals↗
PubMed2005

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: a natural protocol and its use for patch clamp experiments.

AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine cells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic beta-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels (K(ATP)), voltage-dependent potassium channels (K(V)), and voltage-dependent calcium channels (K(CA)) in beta-cells were identified by patch clamp technique. RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024+/-0.003 to 0.028+/-0.003, insulin secretion from 2.6+/-0.6 to 3.1+/-0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028+/-0.003 to 0.052+/-0.008, insulin secretion from 3.1+/-0.8 to 18.3+/-2.6 mIU/L), then reached high plateau (MTT value >0.052+/-0.008, insulin secretion >18.3+/-2.6 mIU/L). In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029+/-0.01 to 0.031+/-0.011, insulin secretion from 13.9+/-3.1 to 14.3+/-3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031+/-0.011, insulin secretion <8.2+/-1.5 mIU/L), and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic beta-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from K(ATP), K(V), and K(CA). CONCLUSION: Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

Animals↗
PubMed1999

Sites and mechanisms of antibiotic-induced neuromuscular block: a pharmacological analysis using quantal content, voltage clamped end-plate currents and single channel analysis.

Since the original observation of Vital Brazil and Corrado (1957) concerning the antibiotic induced neuromuscular block produced by streptomycin, there has been considerable interest in the mechanisms responsible for not only neuromuscular block but also the effects of antibiotics on different systems. We used the voltage clamped end-plate of transacted skeletal muscle to examine the concentration-dependent actions of several groups of antibiotics. The aminoglycoside antibiotics, neomycin and streptomycin, were both more effective at reducing quantal release of acetylcholine (ACh) than interacting with the postjunctional ACh receptor-channel complex. Neomycin was approximately 10 X more potent prejunctionally than streptomycin and the prejunctional effects of each antibiotic were reversed competitively by raising extracellular calcium. Both neomycin and streptomycin also had postjunctional actions at higher concentrations. Neomycin interacted with the open state of the ACh receptor ion channel complex while streptomycin blocks the ACh receptor. The lincosamide antibiotics, lincomycin and clindamycin produced their neuromuscular block postjunctionally by interacting with the open state of the ACh-receptor channel complex. Clindamycin is approximately 20 X more effective at blocking the open channel than was lincomycin. Using cell attached patch clamp recordings in cultured rat myotubes, we demonstrated a lincosamide-induced block of open ion channels with clindamycin having a much slower unblocking rate than lincomycin. Using epimers of the lincosamides, we demonstrated that lipophilicity of the molecule, rather than stereochemical considerations, is important for open channel blockade affecting primarily the "off" rate of channel blocking. This mechanism appears important for not only the lincosamide antibiotics but also for the postjunctional actions of the aminoglycoside antibiotics, particularly neomycin.

Animals↗
PubMed1999

A patch clamp study on reconstituted calcium permeable channels of human sperm plasma membranes.

Ionic flux is thought to be important in the initiating process of gamete interaction such as acrosome reaction. However, modern electrophysiological methods, intracellular recording and patch-clamping, are difficult to approach the ion channels in mammal sperm membrane of an intact sperm due to its small size. In this work, by reconstituting the channel protein into lipid bilayer, Ca2+ channels in human spermatozoa were investigated with voltage clamp technique. Membrane proteins isolated from human sperm of 12 healthy donors were incorporated into lipid bilayer via fusion. In a cis 50//trans 10 mmol/L CaCl2 solution system, two types of channel events with similar reversal potential near the value of a perfect Ca2+ electrode, and sensitive to nifedipine and verapamil, were observed. Their unit conductance was 40 and 25 pS respectively. Percentage of channel open time was not dependent to holding potential for the former. However, for the channels of 25 pS, the percentage increased when the holding potential was changed from -20 to 100 mV. Ca(2+)-permeable channels were also detected from the spermatozoon samples of two infertile donors. Abnormal open time of these channels indicates that there are some defects in the conformation of the channel protein of infertile sperm membrane.

Adult↗
PubMed2001

A patch-clamp study on human sperm Cl- channel reassembled into giant liposome.

AIM: To record the single-channel currents and characterize the electrophysiological properties of the Cl- channels in human sperm membrane. METHODS: The membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10 microm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl- channel activities by patch-clamp technique. RESULTS: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100// pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 +/- 8.3) pS, (117.0 +/- 5.7) pS and (144.7 +/- 4.5) pS, respectively, were detected. Their activities were voltage-dependent and all were blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed. CONCLUSION: There exist three kinds of Cl- channels with different conductance in human sperm membrane at least.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2002

[Patch-clamp technique in investigations of potassium channel activation in T lymphocytes].

This review focuses on results of patch-clamp studies on modulation of T lymphocyte potassium channel activity by physiologically relevant factors. In the preface the patch-clamp technique is briefly presented and basic properties of potassium channels in T lymphocytes are characterised. The paper contains an overview of the data on modulatory effects of extracellular and intracellular pH, temperature, extracellular potassium, extracellular divalent and trivalent metal cations, channel phosphorylation processes and membrane lipid metabolities on potassium channel activity. Some still unresolved problems in that area are indicated.

Animals↗
PubMed1999

[The advances of the research work on lens cells using patch clamp technique].

The study of lens cells by patch clamp technique began in the early 90's. It shows that the membrane of lens cells possess different types of ionic channels. These channels are responsible for the modulation of the hydration state and transparency of lens. Several patch clamp configurations have been used to study the activity of ionic channels. The resting potential and capacitance as well as resistance of different lens cells were also measured.

Aerospace Medicine↗
PubMed2000

[Single channel and whole cell recordings using patch clamp technique].

Action of ion channel on membrane is the key events of messenger transduction for excitable cells, which can be detected by the patch clamp technique. The developments of patch clamp technique have brought a revolution of life science research. The theory, work modes, single channel and whole cell recording techniques in single cells are described here in detail.

Cells, Cultured↗
PubMed2002

A method of patch clamp recording in hyperbaric oxygen.

A method of patch clamp recording in hyperbaric oxygen is described which is derived from a method for patch clamping at high hydrostatic pressure. Excised patches equilibrate with hyperbaric oxygen within 10 minutes. Experiments with BK channels expressed in HEK 293 cells showed that 1 MPa O2 caused an increase in the ionic permeability of the patch, an effect accelerated in the presence of ferrous ions. Seal resistance was unaffected. Small negative currents, (VH - 40 to - 65 mV, symmetrical KCl solutions), apparent in skewed all-points amplitude histograms, reduced the signal to noise ratio. In some patches the BK channel activity was inhibited and in others it persisted with the currents becoming irresolvable in the increased noise.

Cell Line↗
PubMed2003

Flip the tip: an automated, high quality, cost-effective patch clamp screen.

The race for creating an automated patch clamp has begun. Here, we present a novel technology to produce true gigaseals and whole cell preparations at a high rate. Suspended cells are flushed toward the tip of glass micropipettes. Seal, whole-cell break-in, and pipette/liquid handling are fully automated. Extremely stable seals and access resistance guarantee high recording quality. Data obtained from different cell types sealed inside pipettes show long-term stability, voltage clamp and seal quality, as well as block by compounds in the pM range. A flexible array of independent electrode positions minimizes consumables consumption at maximal throughput. Pulled micropipettes guarantee a proven gigaseal substrate with ultra clean and smooth surface at low cost.

Animals↗
PubMed2003

CYTOCENTERING: a novel technique enabling automated cell-by-cell patch clamping with the CYTOPATCH chip.

Automats for patch clamping suspended cells in whole-cell configuration must (1) bring isolated cells in contact with patch contacts, (2) form gigaseals, and (3) establish stable intracellular access that allows for high quality recording of ionic currents. Single openings in planar substrates seem to be intriguing simple solutions for these problems, but due to the low rate of formation of whole-cell configurations we discarded this approach. Single openings are not suited for both attracting cells to the opening by suction and forming gigaseals with subsequent membrane rupture. To settle the three tasks with a mechanical microstructure we developed the socalled CYTOCENTERING technique to apply to suspended cells the same operation sequence as in conventional patch clamping. With this method we immobilized selected cells from a flowing suspension on the tip of a patch pipette by suction with a success rate of 97% and formed gigaseals with a success rate of 68%. Subsequent whole-cell recordings and intracellular staining with Lucifer yellow proved the stable access to the cytoplasm. Currently, a chip with an embedded suction opening in glass surrounding the microstructured contact pipette is under development. The processing of this CYTOPATCH chip is compatible to large-volume production. The CYTOPATCH automat will allow for fully automated, parallel, and asynchronous whole-cell recordings.

Animals↗
PubMed2004

Inward rectifying currents stabilize the membrane potential in dendrites of mouse amacrine cells: patch-clamp recordings and single-cell RT-PCR.

PURPOSE: To explore the possible existence of inward rectifying currents in the distal dendrites of amacrine cells. METHODS: Patch-clamp recordings were made from amacrine cells in a new horizontal slice preparation of mouse retina. Single-cell RT-PCR studies were performed after the patch-clamp recordings. RESULTS: In contrast to results from vertical slices or dissociated cells, all amacrine cells tested demonstrated inward rectifying currents, IIR. Within the limits of our sample, this current did not depend on the morphological and physiological type of the amacrine cell. Amacrine cells from which the dendrites had been removed did not possess detectable amounts of IIR. Pharmacological experiments with ZD7288 (100 microM) and single-cell RT-PCR from recorded cells revealed that IIR includes an h-current (I(H)) carried by hyperpolarization-activated cyclic nucleotide gated channels (HCN), HCN1 and/or HCN2 subtypes. In the presence of extracellular Cs+ (5 mM), which greatly suppressed IIR, the resting membrane conductance was reduced. IIR suppressed the generation of oscillatory potentials. Intracellular cAMP (8-cpt-cAMP, 1 mM) activated IIR. CONCLUSIONS: IIR appears to occur within dendrites of many amacrine cells, where it tends to stabilize the resting membrane potential. HCN1 and/or HCN2 channels contribute to IIR in amacrine cells. Dendritic IIR would be expected to contribute to functional independence of the distal dendrites of amacrine cells that express it.

Amacrine Cells↗
PubMedDate not supplied

[Inhibition of pancreatic beta-cell secretion during "glucose clamp" in patients with polycystic ovary syndrome].

Insulin-resistance is a well-known feature of polycystic ovary syndrome (PCOS) and the present paper investigates the comparative roles of the peripheral tissues and the pancreatic beta-cells in its pathogenesis. The study was conducted on 5 young women with PCOS, all of normal weight and glucose tolerance in whom the glucose-insulin clamp test had revealed reduced peripheral glucose uptake that was not influenced by other conditions typically associated with insulin-resistance. Beta-cell function was investigated in these patients and 5 healthy controls via the assessment of the efficiency of the insulin/insulin feedback which involved studying the suppression of plasma C peptide during the glucose clamp. The results suggested that the insulin/insulin beta-cell feedback mechanism had retained its efficiency despite proven peripheral insulin-resistance. These data do not therefore support the hypothesis advanced by others that there is some sort of beta-cell insulin resistance parallel to the insulin resistance of the peripheral tissues. On the basis of those results it is rather believed that in PCOS the insulin-resistance is generated peripherally while the insulin/insulin feedback in the beta-cell is unimpaired. This obliges us to rethink the role of the hyperinsulinism encountered in PCOS and suggests that the changed sensitivity of the peripheral tissues to insulin activity may constitute a primary event in the genesis of insulin-resistance. This type of behavior has been demonstrated in the obese and in people with acanthosis nigricans, all with normal glucose tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Effects of single and combined infusions of human biosynthetic proinsulin and insulin on glucose metabolism and on plasma hormone concentrations in euglycaemic clamp experiments.

Euglycaemic clamp experiments with single or combined infusions of human biosynthetic proinsulin and insulin were performed in 6 healthy, normal weight subjects in order to assess the possibility of antagonistic, additive, or synergistic effects on whole body glucose metabolism. Insulin (i: 2.02 pmol x kg-1 x min-1) or proinsulin (p: 9.26 pmol x kg-1 x min-1) were infused under euglycaemic clamp conditions over 240 min. After 120 min, the infusion rates were doubled (protocols ii and pp), or proinsulin (protocol ip) or insulin (protocol pi) infusions were added. After 240 min of infusing insulin or proinsulin alone, euglycaemia was maintained for an additional 60 min period without hormone infusions to measure the decay of hormone concentrations and of effects on glucose metabolism. Effects on glucose uptake were measured as the glucose infusion rate necessary to maintain euglycaemia. IR-insulin, IR-proinsulin, IR-C-peptide and IR-glucagon were determined by specific radioimmunoassays. After 240 min, similar steady state glucose infusion rates were reached for all protocols (mean +/- SEM, mg x kg-1 x min-1: ii: 10.6 +/- 1.0; ip: 9.1 +/- 0.4; pi: 10.0 +/- 0.9; pp: 8.4 +/- 0.7). The infusion rate with proinsulin alone (pp), however, was significantly smaller than with insulin alone (ii), indicating a somewhat lower effectiveness of the proinsulin dose employed. With all protocols, nonesterified fatty acid and IR-glucagon concentrations were decreased to a similar extent. Steady state hormone concentrations were reached within 30 min of each infusion period.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[Patch-clamp methods in research on the kidney tubules].

Patch-clamp studies in renal tubule cell have been very helpful in increasing our understanding of the tubule transport function. However, it has been a disappointing experience that the properties of cultured cells deviate grossly from the physiology of the intact tubule segment. Also, several important conductive pathways have not been characterized thus far on single channel level. Several ion channels, which have been found in the patch-clamp studies, such as the non-selective cation channel, do not find their equivalent in the intact tubule preparation. Therefore, much more efforts are required not only to characterize individual ion channels, but rather to integrate this knowledge for the understanding of tubule function.

Animals↗
PubMedDate not supplied

[Methods for the measurement of insulin resistance. Hyperinsulinemic euglycemic clamp].

Insulin resistance is defined as the decrease of the biological action of insulin, and it mainly presents as an hyperinsulinaemia. The clinical investigation of the states of insulin resistance requires expertise in sophisticated in vivo and in vitro methods. The hyperinsulinaemic euglycemic clamp is the reference method for quantifying insulin resistance. The clamp technique consists in infusing insulin at constant rate and, to prevent any decrease in the plasma glucose level, by infusing dextrose. The rate of dextrose infused to maintain euglycaemia is an estimate of the amount of glucose which is taken up by the tissues under the effect of a defined plasma insulin concentration. Using several rates of insulin infusion allows to establish the relationship between the whole body glucose disposal and plasma insulin levels, and to discriminate between the states of decreased insulin sensitivity and/or altered maximal capacity to dispose of glucose. The measurement of glucose fluxes, using the constant infusion of stable isotope labelled glucose, allows to differentiate the hepatic and the peripheral components of insulin resistance. Insulin sensitivity may be estimated using other methods, such as the minimal model which consists in a mathematical analysis of the intravenous glucose tolerance test. In vitro studies include the search for anti-insulin antibodies and anti-insulin-receptor antibodies, the study of insulin receptors (insulin binding, tyrosine-kinase activity and gene structure) and of the post-receptor glucose metabolism. Genetic syndromes of extreme insulin resistance, the pathophysiology of which is poorly known, are the main applications of these investigations. However, insulin resistance plays an important role in non-insulin-dependent diabetes mellitus, and also in insulin-dependent diabetes and various pathological or physiological endocrine disorders.

Glucose↗
PubMed1994

Single-channel inositol 1,4,5-trisphosphate receptor currents revealed by patch clamp of isolated Xenopus oocyte nuclei.

Patch clamp of the outer nuclear membrane of isolated Xenopus oocyte nucleus was used to measure the single-channel properties of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observed channel was activated by IP3, inhibited by heparin, and Ca(2+)-selective, with ion permeabilities PCa:PK:PCl = 8:1:0.05. In symmetric KCl buffer, the channel was ohmic (113 picosiemens in 140 mM KCl) at low channel currents but rectified at higher positive currents. The nuclear IP3R exhibited three conductance substates: a main substate occurring approximately 90% of channel open time, a double substate with twice the main substate conductance and a third substate with half the main substate conductance, which was observed rarely. Channel open probability fluctuated over time and among nuclei. Mean open channel durations of the main and double substates were approximately 5 and 1 ms, respectively. Many channels exhibited periods of closure lasting seconds, and most inactivated permanently within 5 min of IP3 stimulation. These results provide the first characterization of the single-channel properties of the IP3R in its native membrane environment and demonstrate that patch clamp electrophysiology of intact nuclei can be used to directly record currents through the IP3R.

Animals↗