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Results for “Chloride Channel Agonists”

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PubMed2006

Functional characterization of sodium channel blockers by membrane potential measurements in cerebellar neurons: prediction of compound preference for the open/inactivated state.

Voltage-gated sodium channel (VGSC) blockers are widely used in the therapy, but most currently available blockers have suboptimal profile. However, discovery of new drug candidates has been hampered by the lack of appropriate in vitro assays. We established a fluorometric, plate reader-based membrane potential assay for testing the inhibitory potency of various VGSC blocking drugs, using primary cultures of cerebellar neurons, and veratridine, as activator of VGSCs. Since inhibition was strongly dependent on the depolarizing effect of veratridine, the EC(80) value of veratridine was determined on each experimental day, and this concentration was used for drug testing. This strict control on agonist effect seems to improve the reliability of the dose-inhibition measurements with antagonists. Veratridine responses could be completely inhibited by tetrodotoxin (TTX, IC(50)=17 nM), consistent with the exclusive expression of TTX-sensitive VGSCs. A variety of compounds known to block sodium channels inhibited veratridine-induced membrane depolarization concentration-dependently. Furthermore, inhibitory potencies of drugs strongly depended on whether their administration preceded or followed veratridine application. Potency of lamotrigine, carbamazepine, phenytoin and lidocaine was approximately 10-fold higher when applied after a steady-state depolarization had been achieved by a supramaximal veratridine dose, compared with those from a different protocol, where cells were preincubated with the antagonists prior to veratridine application. On the contrary, there was only relatively small difference between the IC(50) values of GBR 12909 obtained from the two different protocols (0.51 microM versus 1.23 microM). In contrast with most sodium channel blockers, this compound lacks binding preference to inactivated channels. We suggest that comparison of the results obtained with a particular blocker in the pretreatment and post-treatment schedules may be suitable for drawing conclusions regarding the state-dependency of its action. Thus, relevant information can be obtained about the potential therapeutic utility of different drugs by applying non-electrophysiological methods.

Animals↗

PubMed2004

Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells.

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Angiogenesis Inhibitors↗

PubMed2002

Basolateral adrenoceptor activation mediates noradrenaline-induced Cl- secretion in M-1 mouse cortical collecting duct cells.

Noradrenaline (NA) released from efferent renal sympathetic nerves may directly affect renal tubular transport. Here we examined the effect of NA on transepithelial ion transport of cultured M-1 mouse cortical collecting duct cells using equivalent short-circuit current ( I(SC)) measurements. Steady-state I(SC) averaged 87.5+/-2.9 microA cm(-2) (n=185) and was reduced by 97.1+/-0.1% (n=80) by apical amiloride (100 microM) confirming that the predominant electrogenic transport across M-1 monolayers is sodium absorption via the epithelial sodium channel (ENaC). Basolateral addition of NA (10 microM) induced a biphasic change in I(SC) characterized by an initial transient peak increase of 18.1+/-0.9 microA cm(-2) with a subsequent decline to a plateau 1.4+/-0.3 microA cm(-2) (n=20) above baseline. Apical application of NA had no effect. The response to basolateral NA was concentration dependent and was preserved in the presence of apical amiloride. In contrast, the response was largely reduced in the presence of apical diphenylamine-2-carboxylic acid (1 mM) and in the absence of extracellular Cl(-). The peak response to NA was reduced in the presence of the alpha-adrenoceptor antagonist phentolamine (100 microM), whereas the beta-antagonist propranolol (100 microM) reduced the secondary plateau phase while failing to influence the peak response. The alpha(1)-adrenoceptor-selective antagonists prazosin (10 nM) and corynanthine (1 microM) reduced the NA-induced peak response by about 75% and 70%, respectively, while the alpha(2)-adrenoceptor antagonist yohimbine (100 nM) was ineffective. We conclude that in M-1 cells NA stimulates Cl(-) secretion probably involving both alpha(1)- and beta-adrenoceptors located basolaterally.

Absorption↗

PubMed2000

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes.

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.

Anesthetics↗

PubMed1993

Spatial aspects of Ca2+ signalling in pancreatic acinar cells.

Secretory cells do not only respond to an agonist with a simple rise in [Ca2+]i. It is now clear that complex patterns of [Ca2+]i elevation in terms of space and time are observed in many cell types and that these patterns may be a cellular mechanism for the regulation of different responses. Ca2+ signalling in exocrine cells of the pancreas promotes the secretion of digestive enzymes and fluid. It has been shown that at high concentrations of agonist (acetylcholine or cholecystokinin) the [Ca2+]i response is initiated in the secretory pole of the cell before spreading across the whole cell. This site of initiation of the [Ca2+]i elevation is in the region where exocytotic release of enzymes occurs and is also the site of a Ca(2+)-dependent chloride channel thought to be crucially important for fluid secretion. Lower concentrations of agonist elicit [Ca2+]i oscillations with complex repetitive patterns characteristic of each agonist. At physiological agonist concentrations, we have recently described repetitive short-lasting Ca2+ spikes that are spatially restricted to the secretory pole of the cell. In addition to these spikes, cholecystokinin also promotes slow transient Ca2+ rises that result in a global rise in Ca2+. The inositol trisphosphate (InsP3) receptor plays a crucial role in all of these various agonist responses, most of which can be reproduced by the infusion of InsP3 into the cell. The high InsP3-sensitivity of the secretory pole is postulated to be due to a localization of high-affinity InsP3 receptors. We speculate that in response to cholecystokinin the short-lasting spikes elicit exocytosis from a small 'available pool' of vesicles and that the broader oscillations induce both exocytosis and cell changes that involve movement of vesicles into this 'available pool'.

Acetylcholine↗

PubMed1996

Early action potential shortening in hypoxic hearts: role of chloride current(s) mediated by catecholamine release.

We tested the hypothesis that the early action potential shortening induced by hypoxia in perfused hearts is attributable to chloride currents activated or modulated by endogenous catecholamine release. Rabbit hearts perfused at 33 degrees C and paced at 2.5-2.8 Hz were used for membrane potential recordings with microelectrodes. Catecholamine depletion was induced with reserpine treatment. The effects of nadolol (10 microM), the stilbenedisulfonic acid derivatives DIDS (10 microM) and SITS (1 mM), and diphenylamine-2 carboxylate (DPC, 100 microM) on action potential characteristics were determined at different times during hypoxia. The effect of chloride transport blockers on the outward currents induced by 200 nM carbonyl cyanide (CCCP) or by 1 microM isoproterenol in isolated cells was also tested. In control hearts, action potential duration (APD) at 25 and 95% repolarization decreased by 50 +/- 9% and 32 +/- 7% respectively after 5 min of hypoxia. This effect was fully antagonized by reserpine pretreatment, by respiratory acidosis, and by nadolol when present from the beginning of hypoxia. None of these agents affected action potential characteristics in normoxia and nadolol had no effect when added after 15 min of hypoxia. Lowering the chloride concentration to 17.5 mM reproduced the effects of nadolol and reserpine. DIDS and SITS lengthened APD in normoxia and attenuated the early APD shortening in hypoxia. DPC had no effect in normoxia but fully counteracted APD shortening produced by isoproterenol or early hypoxia. In isolated cells, DIDS did not affect the glibenclamide sensitive outward current induced by CCCP and DPC blocked the isoproterenol induced current. The data suggest that in whole hearts, chloride currents mediated by endogenous catecholamine release are involved in the early action potential shortening induced by hypoxia with preservation of glycolysis.

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

PubMed2006

Involvement of muscarinic acetylcholine receptors in chloride secretion by cultured rat epididymal epithelium.

The aim of our present study was to investigate the short-circuit current response to carbachol in cultured rat cauda epididymal epithelia and the signal transduction mechanisms involved. Carbachol added basolaterally induced a concentration-dependent increase in short-circuit current (Isc) across the epididymal epithelium consisting of a rapidly rising phase and a long term sustained response. The response was almost abolished by removing Cl(-) from the extracellular medium and blockable by pretreating the tissues with DPC, indicating a substantial contribution of Cl(-) secretion to the carbachol-induced response. The muscarinic acetylcholine receptor antagonist atropine inhibited the response, but the nicotinic acetylcholine receptors antagonist curarine had no effect, suggesting that only the muscarinic acetylcholine receptors mediated the secretory response of the basolateral side of rat cauda epididymal epithelium to carbachol. Addition of carbachol to the apical side of the tissue was found not to elicit an Isc response. These results suggested that muscarinic receptors are present in the basolateral side of rat cauda epididymal epithelium. Activation of these receptors by acetylcholine released from the nerve endings regulates epididymal transepithelial Cl(-) secretion. Cholinergic stimulation therefore contributes to the formation of luminal fluid microenvironment.

Animals↗

PubMed1998

Regulation of Ca2+-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca2+ current.

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH3) cells, using the whole cell configuration with Cs+ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of -50 mV activated the voltage-dependent L-type Ca2+ current (ICa,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba2+ replacement of external Ca2+ as the charge carrier through Ca2+ channels, removal of Ca2+ from the bath solution, or buffering intracellular Ca2+ with EGTA (10 mM). The reversal potential of inward tail current was approximately -25 mV. When intracellular Cl- was changed, the reversal potential of the Ca2+-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate ICa,L and allow Ca2+ influx, and is referred to as Ca2+-activated nonselective cationic current (ICAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca2+ spike was also observed with a hybrid current-voltage protocol. Thus, the present studies clearly indicate that Ca2+-activated nonselective cationic channels are expressed in GH3 cells, and can be elicited by the depolarizing stimuli that lead to the activation of ICa,L.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2001

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Adrenergic beta-Agonists↗

PubMed2001

The testosterone metabolite and neurosteroid 3alpha-androstanediol may mediate the effects of testosterone on conditioned place preference.

Testosterone (T) and pregnane neurosteroids can enhance conditioned place preference (CPP). The present experiment examined CPP produced by T and its androgenic metabolite dihydrotestosterone (DHT) and 3alpha-Androstanediol (3alpha-diol; an androstane neurosteroid). Administration of 3alpha-diol (>DHT>T) to intact male Long-Evans rats, 1.0 mg daily for six days, 30 min prior to exposure to the non-preferred side of the CPP chamber significantly increased preference for the non-preferred side of the chamber compared to that seen in home cage controls. Levels of circulating 3alpha-diol were increased significantly in 3alpha-diol>DHT>T-administered rats, compared to rats that had vehicle administered or androgen-administration discontinued. Androgen administration decreased seminal vesicle weight and intrahypothalamic androgen receptor (AR) binding compared to that seen in rats that had vehicle administered or androgen-administration discontinued. Testosterone, DHT, and 3alpha-diol decreased GABA-stimulated chloride influx in cortical synaptoneurosomes, and muscimol binding in the hippocampus compared to that seen in rats with vehicle administered or that had androgen-administration discontinued. These data indicate that administration of 3alpha-diol is more effective at enhancing CPP and increasing circulating 3alpha-diol levels than is DHT or T administration, and that all of the androgen regimens employed decreased peripheral and hypothalamic androgen receptor binding and cortical and hippocampal GABA(A) receptor function. Hence, whether the effects of 3 alpha-diol on CPP are mediated by differential actions at ARs or GABA(A) receptors in particular brain regions needs to be determined.

Androgens↗

PubMed2000

Differential distribution of ionic channels and muscarinic receptors at the cat tracheal neuromuscular junction.

The ionic and pharmacological properties of atropine-sensitive excitatory junction potentials (EJPs) were investigated by the double sucrose gap and microelectrode membrane potential recording methods, and compared with those of inward currents evoked by carbachol (I(CCh)), in cat tracheal smooth muscle. A single and repetitive field stimulation (10-30 V, 50 micros, 20 Hz) evoked atropine-inhibitable EJPs and associated twitch-like contractions. Reduction in external Na+ concentration strongly, but in the external Cl- concentration, decreased the EJP amplitude after 5 min superfusion, although prolonged exposure to low Cl- solutions attenuated the EJPs modestly. Chloride channel blockers such as 9-AC and niflumic acid (each 100 microM), at concentrations high enough to inhibit I(CCh) almost completely, failed to abolish the EJPs. Pirenzepine, AF-DX116 and 4-DAMP all effectively inhibited the EJPs at their concentrations to block respective muscarinic receptor subtypes relatively specifically, while depletion of internal stores by 10 mM caffeine and/or 3 microM ryanodine caused only a partial decrease in the EJP amplitude. These properties are considerably different from those of I(CCh) which is activated exclusively through activation of the M3 receptor/IP3-mediated Ca2+ release pathway and reflects mostly a Ca2+-dependent Cl- current, and suggests the differential distribution of muscarinic receptors and ionic channels inside and outside of the cholinergic neuromuscular junction of this muscle.

Acetylcholine↗

PubMed2003

Activation by N-acetyl-L-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors.

PURPOSE: We previously reported that an increase in the N-acetyl-L-aspartate (NAA) level due to the lack of aspartoacylase gene was found in the brain of the tremor rat (tm/tm), which is a mutant with a causative gene named tm that shows epileptic seizures. Therefore, NAA is suggested to be one of the factors involved in the induction of epileptic seizures. Patch-clamp studies were performed to determine whether NAA produces an excitatory effect on acutely dissociated rat hippocampal neurons. METHODS: Acutely dissociated hippocampal neurons were prepared from normal Wistar rats aged 3-4 weeks. NAA-induced currents were investigated by using the whole-cell voltage-clamp recording technique. RESULTS: Application of NAA at concentrations of 100 nM to 1 mM through a U-tube for 2 s produced an inward current in a concentration-dependent manner at a holding potential of -60 mV. When the current-voltage relation was examined, the reversal potential of the NAA-induced current was found to be approximately 0 mV. The NAA-induced current was inhibited by bath application of the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG) and by intracellular application of guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), a nonhydrolyzable GDP analogue. However, the NAA-induced current remained unaffected by glutamic acid diethyl ester, a non-N-methyl-D-aspartate (NMDA)-subtype ionotropic glutamate receptor antagonist, or the voltage-dependent ion channel blockers tetrodotoxin, CdCl2, and tetraethylammonium-chloride. Conversely, the mGluR agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) also induced an inward current, with a reversal potential of 0 mV. The ACPD-induced current also was inhibited by MCPG. CONCLUSIONS: These results suggest that NAA acts on the G protein-coupled mGluRs to induce an inward current that results in excitation of the neurons, thereby contributing to the occurrence of epileptic seizures.

Action Potentials↗

PubMed1992

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance.

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.

Acetylcholine↗

PubMed1998

Astrocytes regulate developmental changes in the chloride ion gradient of embryonic rat ventral spinal cord neurons in culture.

1. Embryonic rat ventral spinal cord neurons were dissociated at day 15 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of type I astrocytes; or (iii) PDL in astrocyte-conditioned medium (ACM) to examine the influence of astroglia on the regulation of GABAA receptor/Cl- channel properties. 2. Potentiometric oxonol dye recordings of intact cells indicated that embryonic neurons were uniformly depolarized by muscimol. The depolarizing effects disappeared in cells dissociated during the early postnatal period and recovered in culture for 24 h. Similar recordings using the calcium-imaging dye fura-2 AM revealed that GABA or muscimol triggered a sustained rise in cytosolic Ca2+ (Ca2+c ) in embryonic neurons that was dependent on extracellular Ca2+, blocked by bicuculline and nifedipine and sensitive to changes in extracellular chloride. The incidence and amplitude of the Ca2+ response decreased with time in vitro and was accelerated in neurons cultured on astrocytes compared with those on PDL. 3. Perforated patch-clamp recordings revealed that GABA depolarized neurons in a Cl--dependent and bicuculline-sensitive manner. Both the resting membrane potential and the GABA equilibrium potential became more hyperpolarized with time in vitro. 4. Astrocytes and ACM accelerated the transformation of GABAergic potential responses from depolarizing to hyperpolarizing. The change occurred over the first 4 days in co-culture or in ACM but took more than 2 weeks in neurons cultured on PDL alone. 5. The intrinsic, elementary properties of GABAA receptor/Cl- channels including open time and unitary conductance changed independently of the presence of astrocytes or ACM. Mean open time of the dominant kinetic component decreased and conductance increased with time in vitro. 6. In sum, astrocytes accelerate the developmental change in the Cl- ion gradient extrinsic to GABAA receptor/Cl- channels, which is critical for triggering Ca2+ entry, without influencing parallel changes in the intrinsic properties of the channels.

Animals↗

PubMed1993

Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells.

Smooth muscle cells in the intact guinea pig gallbladder had a resting membrane potential of about -45 mV and had spontaneous action potentials that consisted of a rapid depolarization, a transient repolarization, a plateau phase, and a complete repolarization. These action potentials lasted approximately 570 ms and occurred at a frequency of approximately 0.4 Hz. Action potentials were abolished by the dihydropyridine (DHP)-sensitive Ca2+ channel blocker nifedipine (1.0 microM) and were enhanced by the DHP-sensitive Ca2+ channel agonist BAY K 8644 (0.5 microM). The K+ channel blockers tetraethylammonium chloride (5.0 mM) and 4-aminopyridine (4-AP; 2.0 mM) prolonged the action potential, whereas charybdotoxin (100 nM), a blocker of calcium-activated potassium channels, had no effect. Whole cell currents were characterized in enzymatically isolated smooth muscle cells from the same preparation. 4-AP, a blocker of voltage-dependent K+ channels, suppressed 70% of the outward current at 0 mV. Charybdotoxin (100 nM) reduced an additional 15% of the current at 0 mV. Single calcium-activated potassium channels were identified. The potential for half-activation of these channels, at a cytosolic Ca2+ concentration of 100 nM, was 66.8 mV. A fivefold increase in cytosolic Ca2+ resulted in a shift of the activation curve by -53 mV. External tetraethylammonium chloride (200 microM) reduced the mean single channel current by 48% at 0 mV. The whole cell outward current was abolished by replacement of intracellular K+ for Cs+. Ca2+ currents were inhibited by nifedipine and were increased by BAY K 8644. We conclude that DHP-sensitive voltage-dependent Ca2+ channels are responsible for the depolarization of the action potentials and that the repolarization is due to primarily 4-AP-sensitive K+ current.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Effects of magnesium on human umbilical arteries: role of intracellular calcium stores.

The purpose of this investigation on human umbilical arteries was: (1) to analyze the effects of magnesium (Mg2+) in the prevention of contractions induced by 5-hydroxytryptamine (5-HT), KCl, Bay K 8644 and BaCl2; (2) to determine whether the presence of Mg2+ could modulate the response to caffeine in Ca2+-free medium at 25 and 37 degreesC, and (3) to examine the influence of Mg2+ and temperature in the release of intracellular calcium induced by 5-HT in Ca2+-free medium. Human umbilical arteries were dissected and mounted in organ baths for isometric measurement of force. Our results showed: (1) Mg2+ is a potent inhibitor of smooth muscle contraction of human umbilical arteries caused by 5-HT, KCl, Bay K 8644 and BaCl2, and that this effect might be due to the inhibition of the transmembrane calcium influx. (2) The relaxant effect of Mg2+ did not seem to be mediated by a blockade of the release of calcium from an intracellular store. (3) Moreover, our results suggest an effect of temperature independent from the action of Mg2+ on 5-HT-induced contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2005

Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity.

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.

Animals↗

PubMed2001

Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium.

PURPOSE: To identify the apical and basolateral membrane mechanisms and intracellular signaling pathways in human fetal retinal pigment epithelium (HRPE) that mediate membrane voltage and resistance changes caused by apical membrane adrenergic receptor activation. METHODS: Intact sheets of RPE-choroid from human fetal eyes were mounted in a modified Ussing chamber. Ringer's solution composition changes on the retina-facing and choroid-facing sides of the tissue were separately controlled. Intracellular microelectrodes recorded the membrane voltage and resistance changes after the addition of pharmacologic agents to the apical or basal baths. RESULTS: Apical adrenergic agonists, isoproterenol and epinephrine (10(-8) M), depolarized the basolateral membrane, decreased total tissue resistance (R:(t)) and increased the ratio of apical-to-basolateral membrane resistance (R:(A)/R:(B)). Experiments using antagonists for alpha(1) and ss adrenergic receptors, prazosin and propranolol, respectively, indicated that both receptor types were present. The epinephrine responses were inhibited by apical bumetanide and basal 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS). A cocktail of cyclic adenosine monophosphate (cAMP)-elevating agents produced basolateral membrane voltage and resistance changes very similar to the isoproterenol responses. The cAMP-induced electrical responses were strongly inhibited by basal 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). Ionomycin (to elevate intercellular Ca(2+), [Ca(2+)](i)) produced electrical responses similar to those caused by epinephrine. The Ca(2+) responses were unaffected by NPPB but were inhibited by 3 mM DIDS in the basal bath. CONCLUSIONS: The results provide evidence for two apical membrane adrenergic receptors, alpha(1) and ss, activated by epinephrine and isoproterenol, respectively. The membrane voltage and resistance changes produced by these two agonists mimic those produced by elevating [Ca(2+)](i) and [cAMP](i), suggesting that these ubiquitous signaling molecules activate separate basolateral membrane Cl channels inhibited by DIDS and NPPB, respectively. These two receptors, the apical membrane NaK2Cl cotransporters and the basolateral membrane Cl channels form a complex of proteins that help mediate fluid absorption across human RPE.

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