SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,243 records · Page 69Linked to original sources

PubMed2001

Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure.

OBJECTIVE: Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS: Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS: A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS: Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Adrenergic beta-Agonists↗

PubMed2001

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance.

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.

8,11,14-Eicosatrienoic Acid↗

PubMed2000

Expression and function of endothelial Ca(2+)-activated K(+) channels in human mesenteric artery: A single-cell reverse transcriptase-polymerase chain reaction and electrophysiological study in situ.

Ca(2+)-activated K(+) (K(Ca)) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase-polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K(Ca) channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K(Ca) channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non-cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K(Ca) channel (hIK1) was detected in non-cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K(Ca) currents, whereas respective K(Ca) currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non-cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P:<0.05). However, K(Ca) current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K(Ca) channel (hSlo) was detected in single ECs from CA patients. The increased K(Ca) channel expression in CA patients resulted in a 2. 7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P:<0.05). This increased expression and function of K(Ca) channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Adenocarcinoma↗

PubMed2002

The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans.

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.

Action Potentials↗

PubMed2004

Early expression of sodium channel transcripts and sodium current by cajal-retzius cells in the preplate of the embryonic mouse neocortex.

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Na(v)1.3 (SCN3) Na channel isoform.

Animals↗

PubMedDate not supplied

Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.

INTRODUCTION: In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells. METHODS: A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration-response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern. RESULTS: Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC(50) values for hERG block differed by less than twofold for 15 drugs. DISCUSSION: Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.

Cells, Cultured↗

PubMed1996

Growth hormone-releasing hormone augments voltage-gated Na+ current in cultured rat pituitary cells.

The effect of human growth hormone-releasing hormone (hGHRH), a potent stimulator of adenylate cyclase in somatotrophs on the voltage-gated sodium current was determined by perforated patch clamp of cultured rat somatotrophs The amplitude of the voltage-gated sodium current was augmented by 65.3 +/- 20.6% (mean +/- SE, n = 7) by 10 nM hGHRH. This augmentation was reversibly blocked by 10 microM H-89 a specific inhibitor for adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The membrane-permeant analogue of cAMP, dibutyryl cAMP (5 mM), also augmented the voltage-gated sodium current by 39.6 +/- 7.4% (n = 10). There were no effects of hGHRH or dibutyryl cAMP on steady-state inactivation of the sodium current. In contrast, in the whole cell configuration of patch clamp, no augmentation of the sodium current was observed by hGHRH or by the membrane-permeant analogue of cAMP. These results suggest that hGHRH augments the peak amplitude of the voltage-gated sodium current in rat somatotrophs via phosphorylation by cAMP-dependent protein kinase. For this augmentation, the intracellular environment must be kept relatively intact.

Animals↗

PubMed2002

Involvement of postsynaptic EP4 and presynaptic EP3 receptors in actions of prostaglandin E2 in rat supraoptic neurones.

We have reported that supraoptic nucleus (SON) neurones are excited by prostaglandin E2 (PGE2) presumably via dual postsynaptic PG receptors, FP receptors and unidentified EP receptors, and that presynaptic EP receptors may also be involved in the excitation. In the present study, to clarify the receptor mechanism of the PGE2-mediated actions on SON neurones, we studied the pre- and postsynaptic effects of four newly developed EP agonists that are selective for each of the four EP receptors, EP1-4, on rat SON neurones using extracellular recording and whole-cell patch-clamp techniques. The EP4 agonist ONO-AE1-329 mimicked the excitatory effects of PGE2, whereas the EP1 agonist ONO-DI-004, the EP2 agonist ONO-AE1-257 and the EP3 agonist ONO-AE-248 had little or no effect. The effects of ONO-AE1-329 were unaffected by the EP1/FP/TP antagonist, ONO-NT-012, which potently suppressed the excitation caused by the FP agonist fluprostenol and PGE2. ONO-AE1-329 caused marked excitation when responses to fluprostenol were desensitized by repeated applications of fluprostenol. Patch-clamp analysis in SON neurones showed that ONO-AE1-329 induced inward currents at a holding potential of -70 mV and the reversal potential of the currents was -35.1 +/- 2.3 mV. On the other hand, the frequency of spontaneous inhibitory postsynaptic currents recorded from SON slice preparations was suppressed by ONO-AE-248, but unaffected by the other three EP agonists. These results suggest that SON neurones possess postsynaptic EP4 receptors and that gamma-aminobutyric acid neurones innervating SON neurones possess presynaptic EP3 receptors in their terminals. Activation of the two EP receptors may be involved in the excitatory regulation of SON neurones by PGE2.

Animals↗

PubMed2004

Hypoxia-induced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal KATP channels.

The opening of sarcolemmal and mitochondrial ATP-sensitive K(+) (KATP) channels in the heart is believed to mediate ischemic preconditioning, a phenomenon whereby brief periods of ischemia/reperfusion protect the heart against myocardial infarction. Here, we have applied digital epifluorescent microscopy, immunoprecipitation and Western blotting, perforated patch clamp electrophysiology, and immunofluorescence/laser confocal microscopy to examine the involvement of KATP channels in cardioprotection afforded by preconditioning. We have shown that adult, stimulated-to-beat, guinea-pig cardiomyocytes survived in sustained hypoxia for approximately 17 min. An episode of 5-min-long hypoxia/5-min-long reoxygenation before sustained hypoxia dramatically increased the duration of cellular survival. Experiments with different antagonists of KATP channels, applied at different times during the experimental protocol, suggested that the opening of sarcolemmal KATP channels at the beginning of sustained hypoxia mediate preconditioning. This conclusion was supported by perforated patch clamp experiments that revealed activation of sarcolemmal KATP channels by preconditioning. Immunoprecipitation and Western blotting as well as immunofluorescence and laser confocal microscopy showed that the preconditioning is associated with the increase in KATP channel proteins in sarcolemma. Inhibition of trafficking of KATP channel subunits prevented preconditioning without affecting sensitivity of cardiomyocytes to hypoxia in the absence of preconditioning. We conclude that the preconditioning is mediated by the activation and trafficking of sarcolemmal KATP channels.

Adenosine Triphosphate↗

PubMed1992

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Calcium↗

PubMed2003

Effects of tumor necrosis factor-alpha on calcium movement in rat ventricular myocytes.

AIM: To study the effects of tumor necrosis factor-alpha (TNF-alpha) on calcium movement in rat ventricular myocytes. METHODS: Intracellular free Ca2+ concentration was measured with calcium fluorescent probe Fluo-3/AM and laser confocal microscope. L-type calcium current (ICa,L) was recorded with the whole-cell configuration of the patch-clamp techniques. RESULTS: At 2, 20 and 200 microg/L, TNF-alpha was found to increase intracellular free Ca2+ concentration in a dose-dependent manner illustrated by the increment of calcium fluorescence density with laser confocal microscope. Nicardipine 0.5 micromol/L slightly attenuated TNF-alpha-induced response. When the cardiac myocytes were exposed to caffeine (100 mmol/L) for 30 min, TNF-alpha failed to induce any change of intracellular free calcium. However, it was found that TNF-alpha inhibited I(Ca,L) in whole-cell patch-clamp experiments. At 2, 20, and 200 microg/L, TNF-alpha decreased peak I(Ca,L) by 3.9 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.9 pA/pF+/-0.2 pA/pF, n=9, P>0.05), 15.7 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.3 pA/pF+/-0.3 pA/pF, n=9, P<0.05) and 19.6 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.1 pA/pF+/-0.4 pA/pF, n=9, P<0.01), respectively. It shifted the steady-state inactivation curve of I(Ca,L) to the left (V1/2 shifted from -28.7 mV+/-0.3 mV to -37.8 mV+/-1.4 mV, n=7, P<0.05), while it took no effects on steady-state activation and recovery from inactivation. CONCLUSION: TNF-alpha inhibited I(Ca,L) in rat ventricular myocytes, while increasing the intercellular free Ca2+ level due to the release of Ca2+ from intracellular stores.

Animals↗

PubMed2002

Trachynilysin, a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom, forms nonselective pores in the membrane of NG108-15 cells.

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about -3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.

Acetylcholine↗

PubMed1996

A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.

Caenorhabditis elegans spermatogenesis is especially suited for studies of nonrandom cytoplasmic segregation during cellular differentiation. Spermatocytes separate from an anuclear cytoplasmic core and undergo two sequential divisions. During the second division, intracellular organelles segregate specifically to spermatids as they bud from an anuclear residual body. We have applied patch-clamp techniques in order to investigate membrane protein distribution during these asymmetric divisions. We show that membrane components, as assayed by voltage-dependent ion channel activity, follow a specific distribution pattern during sperm development. Several voltage-sensitive ion channel activities are observed in spermatocytes and residual bodies, but only a single-channel type can be detected in spermatids, indicating that other channel activities are excluded from or inactivated within these cells as they form. The channel that is observed in spermatids is an inward-rectifying chloride channel (Clir), as indicated by its sensitivity to chloride channel inhibitors and Cl-dependent shifts in its conductance. Treatment of spermatids with Cl channel blockers induce their differentiation into spermatozoa, suggesting that Clir plays a role during this developmental step. These studies are the first application of patch-clamp electrophysiology to C. elegans development.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

D2 receptor activation in distinct striatal neurons in comparison with D3 receptors.

The role of D2/D3 receptors in striatum was electrophysiologically examined in vitro in chloralose-anesthetized rats. In addition, in vitro patch clamp method with rat brain slices was followed. Stimulations of the substantia nigra pars compacta (SN) in vivo elicited spike generation which was inhibited by microiontophoretically applied domperidone, a D2 antagonist. These domperidone-sensitive neurons were activated by microiontophoretic application of D2 agonists such as talipexole, quinpirole and bromocriptine as well as the D2 agonist, 7-OH-DPAT. They were also excited by either intravenous injection of bromocriptine or talipexole in a dose-dependent manner. Furthermore, the SN-induced increases in neuronal firing were blocked during microiontophoretic application of domperidone. In patch clamp whole-cell recording large-sized cells, identified visually under Ramanosky microscope, were depolarized with repetitive firing on bath application of talipexole and 7-OH-DPAT at a current clamp mode. Talipexole-induced depolarization in the large-sized cell was similarly observed in the presence of TTX and high Mg2+ in Ca(2+)-free physiological solution. In contrast, the medium-sized cells were hyperpolarized on bath application of talipexole without being affected by 7-OH-DPAT. These findings suggest that the large-sized cells, which were presumably cholinergic interneurons, are activated by dopamine derived from the SN via D2 and/or D3 receptors, while the medium-sized cells are inhibited by dopamine via D2 receptors.

Animals↗

PubMed2002

Angiotensin II evokes calcium-mediated signaling events in isolated dog pancreatic epithelial cells.

INTRODUCTION: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. AIMS: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. METHODS: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 microM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. RESULTS: Western blots demonstrated presence of both AT and AT AngII receptors in DPDE and CFPAC cells; the density of AT receptors appeared lower than that of AT receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 +/- 11 nM) and CFPAC (103 +/- 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 +/- 98 nM) and CFPAC (879 +/- 207 nM) cells, as did ATP (DPDE = 1722 +/- 228 nM; CFPAC = 1522 +/- 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels. CONCLUSION: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca ]. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.

Angiotensin II↗

PubMed2002

Chloride conductance determining membrane potential of rabbit articular chondrocytes.

Membrane conductance of cultured rabbit articular chondrocytes was characterized by means of the patch-clamp technique. The resting membrane potential of the articular chondrocytes was about -42 mV. The membrane potential shifted in accordance with the prediction by the Nernst equation for Cl- when intracellular and extracellular concentrations of Cl- were changed. On the other hand, change in extracellular concentration of K+ produced no shift in the membrane potential of chondrocytes. The Cl- channel blocker 4-acetamido-4'-isothiocyanatostilbene-2'2-disulfonic acid (SITS) depolarized the membrane potential. These findings suggest that the membrane potential of the chondrocytes is determined mainly by Cl- conductance. Using the cell-attached patch-clamp method, a large unitary conductance of 217 pS was observed in the articular chondrocytes. The unitary current was reversibly blocked by SITS. Therefore, the unitary current was carried by Cl-. The Cl- channel showed voltage-dependent activation and the channels exhibited long-lasting openings. Therefore, the membrane potential of rabbit cultured articular chondrocytes was mainly determined by the activities of the large-conductance and voltage-dependent Cl- channels.

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

PubMed1996

Electrophysiological characterization of a motilin agonist, GM611, on rabbit duodenal smooth muscle.

Effects of motilin and a newly synthesized erythromycin derivative, GM611, on membrane potential and currents of rabbit duodenal smooth muscle have been investigated by intracellular potential recording and whole cell patch-clamp technique and compared with results from contractile experiments. Motilin and GM611 (0.01-100 nM) dose dependently produced slowly sustained depolarizations (half-maximal effective dose = 0.15 and 3.9 nM for motilin and GM611, respectively) but exhibited biphasic effects on spike activities superimposed on slow waves. With small depolarizations, the number of spike discharges increased, whereas larger depolarizations markedly reduced spike amplitude. Motilin-induced (or GM611-induced) depolarization appeared to be associated with the activation of monovalent cation-selective channels, and the reduction in the spike amplitude appeared mainly to be associated with inhibition of voltage-dependent Ca2+ channels. Furthermore, data from patch-clamp experiments suggested that Ca2+ release occurred from heparin-sensitive internal stores upon stimulation of motilin receptors by these agonists. Possible implications of these electrophysiological effects in motilin- or GM611-induced tonic and phasic contractions have been discussed.

Animals↗

PubMed1998

Organotypic brain slice cultures for functional analysis of alcohol-related disorders: novel versus conventional preparations.

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.

Alcoholism↗