scholarly journals Glucose Sensitivity and Metabolism-Secretion Coupling Studied during Two-Year Continuous Culture in INS-1E Insulinoma Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 667-678 ◽  
Author(s):  
Arnaud Merglen ◽  
Sten Theander ◽  
Blanca Rubi ◽  
Gaelle Chaffard ◽  
Claes B. Wollheim ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Cell Model ◽  
Cell Voltage ◽  
Resting Potential ◽  
Cell Phenotype ◽  
Similar Degree ◽  
Β Cell ◽  
Patch Clamp Technique ◽  
Insulinoma Cells

Abstract Rat insulinoma-derived INS-1 cells constitute a widely used β-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E β-cell phenotype over 116 passages (no. 27–142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40–100 with average insulin contents of 2.30 ± 0.11 μg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca2+, and mitochondrial Ca2+ levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A KATP current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native β-cells, tolbutamide induced electrical activity, indicating that the KATPconductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable β-cell model.

Electrophysiological differentiation of alpha-receptors on arteriolar smooth muscle

1983 ◽  
Vol 244 (4) ◽  
pp. H540-H545 ◽  
Author(s):  
K. G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Activity ◽  
Resting Potential ◽  
Vessel Size ◽  
Contraction Coupling ◽  
Alpha Receptors ◽  
Excitatory Junction Potentials ◽  
Coupling Mechanisms ◽  
Electrophysiological Differentiation

The effects of clonidine (a prototype of an alpha 2-agonist) and phenylephrine (a prototype of an alpha 1-agonist) on intracellularly recorded electrical activity and vessel size of feline submucosal arterioles were compared. Phenylephrine constricts the vessels and causes a depolarization and the initiation of oscillations of the membrane potential. These oscillations occasionally give rise to spike potentials. In contrast, clonidine produces no significant depolarization of the resting potential in spite of the simultaneous initiation of contraction. Neurally induced depolarizations (excitatory junction potentials) are not blocked and are sometimes augmented by the nonselective alpha-blocker phentolamine even though the depolarization induced by norepinephrine is blocked by phentolamine. Excitatory junction potentials are antagonized by the alpha 1-blocker prazosin. The contraction caused by clonidine is blocked to a greater degree by yohimbine (a relatively selective alpha 2-blocker) than by prazosin. The contraction caused by phenylephrine is blocked to a greater degree by prazosin than by yohimbine. These data indicate that phenylephrine and clonidine act by different mechanisms and, taken together with previous studies, suggest that alpha 1- and alpha 2-stimulation utilize different excitation-contraction coupling mechanisms.

Control of intracellular calcium by membrane potential in human melanoma cells

1993 ◽  
Vol 265 (6) ◽  
pp. C1501-C1510 ◽  
Author(s):  
B. Nilius ◽  
G. Schwarz ◽  
G. Droogmans
Keyword(s):  
Cell Proliferation ◽  
Membrane Potential ◽  
Intracellular Calcium ◽  
Reversal Potential ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Human Melanoma Cells

The modulation of intracellular calcium ([Ca2+]i) by the membrane potential was investigated in human melanoma cells by combining the nystatin-perforated patch-clamp technique with Ca2+ measurements. Voltage steps to -100 mV induced a rise in [Ca2+]i and a creeping inward current. These effects were absent in Ca(2+)-free solution and could be blocked by Ni2+ or La3+. Voltage ramps revealed a close correlation between [Ca2+]i and voltage, with the strongest voltage dependence around the resting potential. Long-lasting tail currents, closely correlated with the rise in [Ca2+]i and a reversal potential close to the K+ equilibrium potential, occurred if the membrane potential was clamped back to 0 mV. They were absent if intracellular K+ was replaced by Cs+ and blocked by extracellular tetraethylammonium (5 mM), Ba2+ (1 mM), or a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue. These observations are discussed in relation to cell proliferation. The enhanced expression of K+ channels during cell proliferation provides a positive-feedback mechanism resulting in long-term changes in [Ca2+]i required for the G1-S transition in the cell cycle.

scholarly journals Effect on Membrane Potential and Electrical Activity of Adding Sodium to Sodium-Depleted Cardiac Purkinje Fibers

1974 ◽  
Vol 64 (4) ◽  
pp. 473-493 ◽  
Author(s):  
Jay R. Wiggins ◽  
Paul F. Cranefield
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Resting Potential ◽  
Average Increase ◽  
Purkinje Fibers ◽  
Cardiac Purkinje Fibers ◽  
Sodium Extrusion

Canine cardiac Purkinje fibers exposed to Na-free solutions containing 128 mM TEA and 16 mM Ca show resting potentials in the range -50 to -90 mV; if the concentration of Na in the perfusate is raised from 0 to 4 to 24 mM, hyperpolarization follows. If the initial resting potential is low, the hyperpolarization tends to be greater; the average increase in the presence of 8 mM Na is 14 mV. Such hyperpolarization is not induced by adding Na to K-free solutions, is not seen in cooled fibers, or in fibers exposed to 10-3 M ouabain, nor is it induced by adding Li and thus may result from electrogenic sodium extrusion. Fibers exposed to Na-free solutions are often spontaneously active; if they are quiescent they often show repetitive activity during depolarizing pulses. Such spontaneous or repetitive activity is suppressed by the addition of Na. This suppression may or may not be related to the hyperpolarization.

scholarly journals A Minimum of Fuel Is Necessary for Tolbutamide to Mimic the Effects of Glucose on Electrical Activity in Pancreatic β-Cells*

Endocrinology ◽  
1998 ◽  
Vol 139 (3) ◽  
pp. 993-998 ◽  
Author(s):  
Jean-Claude Henquin
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Slow Waves ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
K Channels ◽  
Low Concentrations ◽  
Low Glucose ◽  
Intracellular Microelectrodes

Glucose stimulation of pancreatic β-cells triggers electrical activity (slow waves of membrane potential with superimposed spikes) that is best monitored with intracellular microelectrodes. Closure of ATP-sensitive K+ channels underlies the depolarization to the threshold potential and participates in the increase in electrical activity produced by suprathreshold (>7 mm) concentrations of glucose, but it is still unclear whether this is the sole mechanism of control. This was investigated by testing whether blockade of ATP-sensitive K+ channels by low concentrations of tolbutamide is able to mimic the effects of glucose on mouse β-cell electrical activity even in the absence of the sugar. The response to tolbutamide was influenced by the duration of the perifusion with the low glucose medium. Tolbutamide (25 μm) caused a rapid and sustained depolarization with continuous activity after 6 min of perifusion of the islet with 3 mm glucose, and a progressive depolarization with slow waves of the membrane potential after 20 min. In the absence of glucose, the β-cell response to tolbutamide was a transient phase of depolarization with rare slow waves (6 min) or a silent, small, but sustained, depolarization (20 min). Readministration of 3 mm glucose was sufficient to restore slow waves, whereas an increase in the glucose concentration to 5 and 7 mm was followed by a lengthening of the slow waves and a shortening of the intervals. In contrast, induction of slow waves by tolbutamide proved very difficult in the absence of glucose, because the β-cell membrane tended to depolarize from a silent level to the plateau level, at which electrical activity is continuous. Azide, a mitochondrial poison, abrogated the electrical activity induced by tolbutamide in the absence of glucose, which demonstrates the influence of the metabolism of endogenous fuels on the response to the sulfonylurea. The partial repolarization that azide also produced was reversed by increasing the concentration of tolbutamide, but reappearance of the spikes required the addition of glucose. It is concluded that inhibition of ATP-sensitive K+ channels is not the only mechanism by which glucose controls electrical activity inβ -cells.

Model of β-cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables

1998 ◽  
Vol 274 (4) ◽  
pp. C1174-C1184 ◽  
Author(s):  
Gerhard Magnus ◽  
Joel Keizer
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Adenine Nucleotide ◽  
Tricarboxylic Acid Cycle ◽  
Calcium Handling ◽  
Β Cell ◽  
Futile Cycle ◽  
Cyclic Changes

In the preceding article [ Am. J. Physiol. 274 ( Cell Physiol. 43): C1158–C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca2+ handling and electrical activity in the pancreatic β-cell. Here we describe further results of those simulations, focusing on mitochondrial variables, the rate of respiration, and fluxes of metabolic intermediates as a function of d-glucose concentration. Our simulations predict relatively smooth increases of O2consumption, adenine nucleotide transport, oxidative phosphorylation, and ATP production by the tricarboxylic acid cycle asd-glucose concentrations are increased from basal to 20 mM. On the other hand, we find that the active fraction of pyruvate dehydrogenase saturates, due to increases in matrix Ca2+, near the onset of bursting electrical activity and that the NADH/NAD+ ratio in the mitochondria increases by roughly an order of magnitude as glucose concentrations are increased. The mitochondrial ATP/ADP ratio increases by factor of <2 between thed-glucose threshold for bursting and continuous spiking. According to our simulations, relatively small changes in mitochondrial membrane potential (∼1 mV) caused by uptake of Ca2+ are sufficient to alter the cytoplasmic ATP/ADP ratio and influence ATP-sensitive K+ channels in the plasma membrane. In the simulations, these cyclic changes in the mitochondrial membrane potential are due to synchronization of futile cycle of Ca2+ from the cytoplasm through mitochondria via Ca2+ uniporters and Na+/Ca2+exchange. Our simulations predict steady mitochondrial Ca2+concentrations on the order of 0.1 μM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 μM during bursting. Abrupt increases in mitochondrial Ca2+concentration >5 μM may occur during continuous electrical activity.

scholarly journals Oscillatory Membrane Potential Response to Glucose in Islet β-Cells: A Comparison of Islet-Cell Electrical Activity in Mouse and Rat

Endocrinology ◽  
2006 ◽  
Vol 147 (10) ◽  
pp. 4655-4663 ◽  
Author(s):  
Jocelyn E. Manning Fox ◽  
Armen V. Gyulkhandanyan ◽  
Leslie S. Satin ◽  
Michael B. Wheeler
Keyword(s):  
Insulin Secretion ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Electrical Activity ◽  
Cell Membrane Potential ◽  
Dependent Manner ◽  
Β Cells ◽  
Action Potential Firing ◽  
Patch Clamp Technique ◽  
Elevated Glucose

In contrast to mouse, rat islet β-cell membrane potential is reported not to oscillate in response to elevated glucose despite demonstrated oscillations in calcium and insulin secretion. We aim to clarify the electrical activity of rat islet β-cells and characterize and compare the electrical activity of both α- and β-cells in rat and mouse islets. We recorded electrical activity from α- and β-cells within intact islets from both mouse and rat using the perforated whole-cell patch clamp technique. Fifty-six percent of both mouse and rat β-cells exhibited an oscillatory response to 11.1 mm glucose. Responses to both 11.1 mm and 2.8 mm glucose were identical in the two species. Rat β-cells exhibited incremental depolarization in a glucose concentration-dependent manner. We also demonstrated electrical activity in human islets recorded under the same conditions. In both mouse and rat α-cells 11 mm glucose caused hyperpolarization of the membrane potential, whereas 2.8 mm glucose produced action potential firing. No species differences were observed in the response of α-cells to glucose. This paper is the first to demonstrate and characterize oscillatory membrane potential fluctuations in the presence of elevated glucose in rat islet β-cells in comparison with mouse. The findings promote the use of rat islets in future electrophysiological studies, enabling consistency between electrophysiological and insulin secretion studies. An inverse response of α-cell membrane potential to glucose furthers our understanding of the mechanisms underlying glucose sensitive glucagon secretion.

scholarly journals Acetylcholine-Activated Ion Channels in Embryonic Cockroach Neurones Growing in Culture

1989 ◽  
Vol 142 (1) ◽  
pp. 337-355
Author(s):  
DAVID J. BEADLE ◽  
G. HORSEMAN ◽  
Y. PICHON ◽  
M. AMAR ◽  
T. SHIMAHARA
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Cell Voltage ◽  
Resting Potential ◽  
Corner Frequency ◽  
Fluctuation Analysis ◽  
Patch Clamp Technique ◽  
Single Channel Analysis ◽  
Channel Analysis

Application of acetylcholine and carbamylcholine to cultured cockroach neurones held under whole-cell voltage-clamp conditions evoked an inward current that was accompanied by an increase in current noise. Fluctuation analysis of the noise revealed the existence of two Lorentzian components in acetylcholine, of corner frequencies 10 ± 0.6 Hz and 116 ± 9 Hz, and one Lorentzian component in carbamylcholine, of corner frequency 35 ± 13 Hz. Single-channel analysis of the unitary currents evoked by acetylcholine or carbamylcholine in neurones held in the cell-attached mode of the patch-clamp technique revealed the presence of two categories of channel events. The large events had mean currents of 4.77 pA with acetylcholine and 5.09 pA with carbamylcholine, and the small events 1.92 pA (acetylcholine) and 1.72pA (carbamylcholine) for a hyperpolarization of 60 mV. The reversal potentials for these currents relative to the resting potential were estimated to be - 70 mV for acetylcholine and - 68 mV for carbamylcholine, and the conductance values calculated from the I/V curves were 37 pS (large) and 19 pS (small) for acetylcholine and 52 pS (large) and 15 pS (small) for carbamylcholine. It is concluded that embryonic cockroach neurones growing in vitro possess two populations of acetylcholine-activated ion channels, and the possibility that one of these represents an embryonic receptor and the other an adult receptor is discussed.

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic β-cell: a computational approach

2007 ◽  
Vol 293 (6) ◽  
pp. C1924-C1933 ◽  
Author(s):  
Leonid E. Fridlyand ◽  
Mark C. Harbeck ◽  
Michael W. Roe ◽  
Louis H. Philipson
Keyword(s):  
Membrane Potential ◽  
G Protein ◽  
Low Frequency ◽  
Receptor Activation ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Calcium Indicator ◽  
Insulinoma Cells ◽  
G Protein Coupled

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic β-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic β-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca2+) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca2+ oscillations in the β-cell. Slow Ca2+ oscillations (<1 min−1) produce low-frequency cAMP oscillations, and faster Ca2+ oscillations (>3–4 min−1) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca2+ oscillations. In contrast, observed antiphasic Ca2+ and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca2+-dependent AC and PDE activation in coupling of Ca2+ and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 β-cells.

scholarly journals A Ba2+-Sensitive K+ Current Contributes to the Resting Membrane Potential of Neurons in Rat Suprachiasmatic Nucleus

2002 ◽  
Vol 88 (2) ◽  
pp. 869-878 ◽  
Author(s):  
Marcel de Jeu ◽  
Alwin Geurtsen ◽  
Cyriel Pennartz
Keyword(s):  
Membrane Potential ◽  
Suprachiasmatic Nucleus ◽  
Voltage Dependence ◽  
Brain Slices ◽  
Resting Potential ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
M Current ◽  
Whole Cell Patch Clamp

A Ba2+-sensitive K+ current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba2+-sensitive K+ current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba2+ (500 μM) reversibly depolarized the membrane potential by 6.7 ± 1.3 mV ( n = 22) and concomitantly Ba2+ induced an increase in the spontaneous firing rate of 0.8 ± 0.2 Hz ( n = 12). The Ba2+-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba2+-sensitive current was observed. Voltage-clamp experiments showed that Ba2+ (500 μM) reduced a fast-activating, voltage-dependent K+ current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba2+-sensitive K+ current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 μM). A component of Ba2+-sensitive K+ current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba2+- and TEA-sensitive K+ current make it unlikely that this current is a delayed rectifier, Ca2+-activated K+ current, ATP-sensitive K+ current, M-current or K+ inward rectifier. Our data are consistent with the Ba2+- and TEA-sensitive K+ current in SCN neurons being an outward rectifying K+ current of a novel identity or belonging to a known family of K+ channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.

Electrophysiological response of rat ventricular myocytes to acidosis

2002 ◽  
Vol 283 (1) ◽  
pp. H412-H422 ◽  
Author(s):  
Kimiaki Komukai ◽  
Fabien Brette ◽  
Caroline Pascarel ◽  
Clive H. Orchard
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Ventricular Myocytes ◽  
Resting Potential ◽  
Disulfonic Acid ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Electrophysiological Response ◽  
Inwardly Rectifying

The effects of acidosis on the action potential, resting potential, L-type Ca2+( I Ca), inward rectifier potassium ( I K1), delayed rectifier potassium ( I K), steady-state ( I SS), and inwardly rectifying chloride ( I Cl,ir) currents of rat subepicardial (Epi) and subendocardial (Endo) ventricular myocytes were investigated using the patch-clamp technique. Action potential duration was shorter in Epi than in Endo cells. Acidosis (extracellular pH decreased from 7.4 to 6.5) depolarized the resting membrane potential and prolonged the time for 50% repolarization of the action potential in Epi and Endo cells, although the prolongation was larger in Endo cells. At control pH, I Ca, I K1, and I SS were not significantly different in Epi and Endo cells, but I K was larger in Epi cells. Acidosis did not alter I Ca, I K1, or I K but decreased I SS; this decrease was larger in Endo cells. It is suggested that the acidosis-induced decrease in I SS underlies the prolongation of the action potential. I Cl,ir at control pH was Cd2+ sensitive but 4,4′-disothiocyanato-stilbene-2,2′-disulfonic acid resistant. Acidosis increased I Cl,ir; it is suggested that the acidosis-induced increase in I Cl,ir underlies the depolarization of the resting membrane potential.

Sign in / Sign up

Export Citation Format

Share Document