Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

1991 ◽  
Vol 11 (3) ◽  
pp. 147-157 ◽  
Author(s):  
Krister Bokvist ◽  
Carina Ämmälä ◽  
Per-Olof Berggren ◽  
Patrik Rorsman ◽  
Karin Wåhlander
Keyword(s):  
Adrenergic Stimulation ◽  
Β Cells ◽  
Adrenergic Agonist ◽  
Cell Interior ◽  
Pancreatic Β Cells ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Maximal Activation ◽  
Cell Recording

The effects of α2-adrenergic stimulation on the Ca2+-current in mouse pancreatic β-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 μM) or the α2-adrenergic agonist clonidine (5 μM) failed to reduce the Ca2+-current, irrespective of whether intracellular GTP (or GTPγ S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca2+-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended to increase the Ca2+-current amplitude in the presence of the higher Ca2+-concentration. Ca2+-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at ≈−20 mV. In addition, when Ca2+-currents were recorded from intact β-cells, using the perforated patch whole-cell configuration, clonidine (1 μM) also failed to detectably affect the Ca2+-current. It is therefore suggested that the inhibition of β-cell electrical activity and insulin-secretion produced by α2-adrenoreceptor stimulation does not result from suppression of the L-type Ca2+-current.

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

scholarly journals Metabolic inhibition impairs ATP-sensitive K+ channel block by sulfonylurea in pancreatic β-cells

1998 ◽  
Vol 274 (1) ◽  
pp. E38-E44 ◽  
Author(s):  
Eri Mukai ◽  
Hitoshi Ishida ◽  
Seika Kato ◽  
Yoshiyuki Tsuura ◽  
Shimpei Fujimoto ◽  
...  
Keyword(s):  
Metabolic Inhibition ◽  
K Channel ◽  
High Dose ◽  
Dependent Manner ◽  
Channel Activity ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Dose Dependent

The effect of metabolic inhibition on the blocking of β-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 μM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 μM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATPchannel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic β-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

Cell coupling in mouse pancreatic β-cells measured in intact islets of Langerhans

2008 ◽  
Vol 366 (1880) ◽  
pp. 3503-3523 ◽  
Author(s):  
Quan Zhang ◽  
Juris Galvanovskis ◽  
Fernando Abdulkader ◽  
Christopher J Partridge ◽  
Sven O Göpel ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Total Charge ◽  
Charge Movement ◽  
Cell Coupling ◽  
Β Cells ◽  
Patch Clamp Technique ◽  
Intact Mouse ◽  
Whole Cell ◽  
Junctional Conductance ◽  
Gap Junctional

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified β-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent β-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between β-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell (due to the firing of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of β-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to −55 mV) gave rise to a slow capacitive current indicative of coupling between β-cells, but not in non-β-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial β-cell in an islet is in electrical contact with six to seven other β-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K ATP channel conductance ( G K,ATP ) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC 50 of approximately 4 mM. Theoretical considerations indicate that the coupling between β-cells within the islet is sufficient to allow propagation of [Ca 2+ ] i waves to spread with a speed of approximately 80 μm s −1 , similar to that observed experimentally in confocal [Ca 2+ ] i imaging.

Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons

1995 ◽  
Vol 73 (4) ◽  
pp. 1374-1382 ◽  
Author(s):  
L. A. Merriam ◽  
R. L. Parsons
Keyword(s):  
Calcium Channels ◽  
Patch Clamp Technique ◽  
Maximal Inhibition ◽  
Whole Cell ◽  
Recording Technique ◽  
Perforated Patch ◽  
Cell Recording ◽  
Parasympathetic Neurons ◽  
Current Activation ◽  
Patch Technique

1. We determined the effect of the neuropeptide galanin on barium currents (IBa) flowing through voltage-gated calcium channels. We voltage clamped parasympathetic neurons dissociated from mudpuppy cardiac ganglia using both the standard whole cell and the perforated-patch variations of the patch-clamp technique. 2. Galanin produced a concentration-dependent inhibition of IBa. The maximal inhibition was 50-60% and the concentration that produced half-maximal inhibition (IC50) was 0.42 nM. In mud-puppy parasympathetic neurons, omega-conotoxin-GVIA (CTX)-sensitive channels are the predominant type of calcium channels, and only a small portion of IBa is contributed by dihydropyridine-sensitive channels. Galanin preferentially inhibited a portion of the CTX-sensitive current. 3. In currents recorded with the standard whole cell technique, activation of IBa was slowed in the presence of galanin. In contrast, in the majority of neurons studied with the perforated-patch technique, galanin decreased IBa without altering the kinetics of current activation. With both recording methods, the decrease in IBa was greatest with voltage steps to 0 mV and persisted with steps to +50 mV. For control currents, large depolarizing voltage steps (+70 to +120 mV) did not markedly facilitate IBa when either recording technique was used. However, the degree of facilitation in galanin was significantly greater with the standard whole cell recording technique. 4. IBa exhibited inactivation under the conditions of these experiments. Inactivation of IBa recorded during a 900-ms depolarizing voltage step was fitted to a double exponential. Galanin decreased the amplitude of IBa but did not alter the time constants of inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of whole cell currents by cytosolic cAMP, Ca2+, and Cl- in rat fetal distal lung epithelium

1995 ◽  
Vol 269 (1) ◽  
pp. C156-C162 ◽  
Author(s):  
T. Nakahari ◽  
Y. Marunaka
Keyword(s):  
Lung Epithelium ◽  
Cellular Mechanisms ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Adrenergic Receptor Agonist ◽  
K Currents

The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.

scholarly journals Evidence That the Product of the Human X-Linked Cgd Gene, Gp91-phox, Is a Voltage-Gated H+ Pathway

1999 ◽  
Vol 114 (6) ◽  
pp. 771-786 ◽  
Author(s):  
Lydia M. Henderson ◽  
Robert W. Meech
Keyword(s):  
Cell Membrane ◽  
Time Course ◽  
Lucifer Yellow ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Histidine Residues ◽  
Voltage Gated

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H+ conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O2·2). Suspensions of CHO91 cells exhibit arachidonate-activatable H+ fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909–5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H+. As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199–216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590–1598), a lowered external pH (pHo) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 μM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10–20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2′7′ bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pHi) was ∼6.9, as though pHi was largely determined by endogenous cellular regulation. Arachidonate (20 μM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl− concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH2-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H+ permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H+ selectivity. Mechanisms of H+ permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319–1327) and the transfer of protons across an “H-X-X-X-H-X-X-X-H” motif lining a conducting pore.

Sodium nitroprusside alters dark voltage and light responses in isolated retinal rods during whole-cell recording

1992 ◽  
Vol 9 (2) ◽  
pp. 205-209 ◽  
Author(s):  
Karl-Friedrich Schmidt ◽  
Gottfried N. Nöll ◽  
Yoshihiko Yamamoto
Keyword(s):  
Sodium Nitroprusside ◽  
Rana Esculenta ◽  
Recovery Phase ◽  
Patch Clamp Technique ◽  
Light Responses ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Retinal Rods ◽  
Cell Recording ◽  
Nitric Oxide Releasing

AbstractDark voltage and light responses of isolated retinal rods of Rana esculenta were investigated by employing the whole-cell patch-clamp technique. When the recording pipette was filled with a medium devoid of nucleotides, a spontaneous hyperpolarization of the dark voltage partly due to a diffusional loss of cGMP and its precursor GTP and a retardation in the recovery of the light responses was observed. The larger part of the retardation of the light responses was prevented by 1 mM ATP. Addition of GTP attenuated the hyperpolarization, but did not abolish it completely. When the nitric-oxide-releasing substance sodium nitroprusside plus GTP was applied, the tendency of hyperpolarization disappeared and a stable dark voltage or even a slight depolarization was measured during the whole-cell recording period. Similar results were also obtained when GTP was given in combination with either EGTA or IBMX which are both known to interfere with the cGMP regulating enzymes in retinal rods. In addition to its effects on the dark voltage, an acceleration of the recovery phase of the light responses by sodium nitroprusside was also observed. Our observations strongly suggest that sodium nitroprusside activates guanylate cyclase in photoreceptors, as it does in other tissues, but we cannot exclude with certainty an effect on the phosphodiesterase.

scholarly journals Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

1998 ◽  
Vol 45 (2) ◽  
pp. 311-326 ◽  
Author(s):  
A Ayar ◽  
N M Thatcher ◽  
U Zehavi ◽  
D R Trentham ◽  
R H Scott
Keyword(s):  
Flash Photolysis ◽  
Reversal Potential ◽  
Neonatal Rat ◽  
Xenon Lamp ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Sensory Neurones ◽  
Whole Cell ◽  
Inward Currents ◽  
Current Activation

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

scholarly journals ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. C1372-C1383 ◽  
Author(s):  
Bret A. Hughes ◽  
Masayuki Takahira
Keyword(s):  
Retinal Pigment Epithelial ◽  
Atp Hydrolysis ◽  
Retinal Pigment ◽  
Underlying Mechanism ◽  
Retinal Pigment Epithelial Cells ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Pigment Epithelial ◽  
Cell Recording ◽  
Inwardly Rectifying

Inwardly rectifying K+ current ( I Kir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, I Kir ran down completely in <10 min [half time ( t 1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained I Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, I Kir ran down by ∼71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+( t 1/2 = 1.8 min). I Kir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5′-adenylylimidodiphosphate ( t 1/2 = 2.7 min) or 4 mM adenosine 5′- O-(3-thiotriphosphate) ( t 1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of I Kirin bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.

Whole cell membrane currents in cultured pig endocardial endothelial cells

1995 ◽  
Vol 268 (5) ◽  
pp. H2036-H2047 ◽  
Author(s):  
P. F. Fransen ◽  
M. J. Demolder ◽  
D. L. Brutsaert
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelium ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
K Current ◽  
Endocardial Endothelial Cells ◽  
Inwardly Rectifying

The whole cell mode of the patch-clamp technique was applied to cultured endocardial endothelial cells from the porcine right ventricle to study their electrophysiological properties. With isotonic pipette and bathing solutions (300-310 mosmol/kgH2O), single endocardial endothelial cells had resting membrane potentials ranging from -20 to -90 mV (mean = -55 +/- 20 mV, n = 48). In voltage-clamp experiments, the main membrane current was an inwardly rectifying K+ current with all characteristics described for the inwardly rectifying K+ current in vascular endothelium. Outward currents at positive clamp potentials were small, but when cell swelling was induced by means of a hypertonic pipette or hypotonic bathing solution and ATP (5 mM) was present in the pipette solution, a large outwardly rectifying current developed. This volume-activated current was insensitive to extracellular K+ or Na+ concentration variations but sensitive to changes in extracellular Cl- concentrations. It was inhibited in the presence of 4,4'-diisothiocyanostilbene-2,2 disulfonic acid (100-300 microM) and flufenamic acid (50-100 microM). Volume-activated Cl- channels are different from the stretch-activated cationic channels described in vascular endothelium and might be involved in the regulation of cell volume or the response to mechanical stretch.

Sign in / Sign up

Export Citation Format

Share Document