Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I(K,Ca)) were investigated in ramified murine brain macrophages. In order to induce I(K,Ca) the intracellular concentration of nominal free Ca2+ was adjusted to 1 microM. The Ca2+-activated K+ current of brain macrophages did not show any voltage dependence at test potentials between -120 and +30 mV. A tenfold change in extracellular K+ concentration shifted the reversal potential of I(K,Ca) by 51 mV. The bee venom toxin apamin applied at concentrations of up to 1 microM did not affect I(K,Ca). Ca2+-activated K+ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3 nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I(K,Ca) at concentrations between 1 and 50 nM. Tetraethylammonium (TEA) blocked 8.0% of I(K,Ca) at a concentration of 1 mM, whereas 31.4% of current was blocked by 10 mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2 mM for their ability to block I(K,Ca). La3+ reduced I(K,Ca) by 72.8%, whereas Cd2+ decreased I(K,Ca) by 17.4%; in contrast, Ni2+ did not have any effect on I(K,Ca). Ba2+ applied at a concentration of 1 mM reduced I(K,Ca) voltage-dependently at hyperpolarizing potentials.     

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Delayed rectifier K+ channel overexpression in transgenic islets and beta-cells associated with impaired glucose responsiveness

Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.     

JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.     

Role of potassium channels in amyloid-induced cell death

Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid beta-peptide (A beta) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to A beta. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (+/-19; p < 0.02) and 44% (+/-12; p < 0.02) in K+ current density were noted 6-12 and 12-18 h following the addition of A beta to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 +/- 4 and 39 +/- 4% of SN56 cells were dead after 48- and 96-h exposures to A beta, respectively. Perfusion of SN56 cells with 10-20 mM TEA blocked 71 +/- 6 to 92 +/- 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 +/- 14 and 68 +/- 14% of the A beta toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from A beta toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, A beta was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by A beta and MES23.5 cells were not protected by TEA from A beta toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of A beta. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in A beta-mediated toxicity for septal cholinergic cells.     

Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at -80, -40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately -61 and -22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30-35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute approximately 20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.     

Electrophysiological effects of ketamine on Ca(2+)-activated K+ channel in single rabbit portal vein cell

The effects of ketamine on Ca(2+)-activated K+ channel currents were studied in dispersed single smooth muscle cells from rabbit portal vein using inside-out patch clamp technique. In a near physiological K+ and Ca2+ gradient, three populations of outward rectangular single currents were recorded in isolated cell membrane of rabbit portal vein at +60 mV membrane potential. These currents were judged as Ca(2+)-activated K+ channel currents since application of EGTA or Apamin in the internal solution inhibited these currents. Application of 10(-5)M or 10(-4)M ketamine inhibited the number of occurrences of channel opening and decreased open times, but did not reduce the amplitudes. When the 10(-3)M ketamine was applied, the Ca(2+)-activated K+ channel currents were abolished. We suggest that the depression of Ca(2+)-activated K+ channel currents may explain the continuous contraction observed in rabbit portal vein at a clinical concentration of ketamine from a point of electrophysiological K+ current study.     

Design and synthesis of small molecule interleukin-1 receptor antagonists based on a benzene template

Effects of the antiepileptic drug carbamazepine on nerve action potential and transmitter release in mouse neuroblastoma-glioma hybrid cells (NG108-15) and the frog neuromuscular junction were studied. Carbamazepine within a concentration range of 0.1-0.5 mmol/L reduced the peak height of the action potential of the NG108-15 cells, whereas the membrane potential and membrane resistance were unaffected. Voltage clamp revealed that the decrease in the action was due to the blockage of the Na+, delayed K+ and transient Ca2+ currents. Carbamazepine did not affect Ca(2+)-activated and A type K+ currents and long-lasting Ca2+ current. In the frog neuromuscular junction, carbamazepine decreased the mean quantal content by a parallel shift in the frequency augmentation-potentiation (FAP) relation. It is concluded that carbamazepine blocks the voltage-dependent Na+, delayed K+, and transient Ca2+ currents and quantal transmitter release through a decrease of nerve excitation.     

Hypotonically induced whole-cell currents in A6 cells: relationship with cell volume and cytoplasmic Ca2+

We investigated changes in whole-cell currents, cell volume, and intracellular calcium concentration ([Ca2+]i) during hypotonic stimulation in whole-cell clamped cultured amphibian renal cells (A6 cells). Upon being exposed to hypotonic solution (80% osmolality), the A6 cells swelled and peaked in the first 5 min, which was followed by a progressive decrease in cell volume termed regulatory volume decrease (RVD). Following the cell swelling, there were large increases in both outward- and inward-currents, which seemed to be carried by K+ efflux and Cl- efflux, respectively. A K+ channel blocker (TEA or quinine) or a Cl- channel blocker (NPPB or SITS) significantly inhibited both currents and RVD, suggesting that the inward- and outward-currents are highly correlated with each other and essential to RVD. Hypotonic stimulation also induced a transient [Ca2+]i increase, of which the time course was essentially similar to that of the currents. When internal and external Ca2+ were deprived to eliminate the Ca2+ transient increase, whole-cell currents and RVD were strongly inhibited. On the other hand, channel blockers TEA and NPPB, which inhibited whole-cell currents and RVD, did not inhibit the [Ca2+]i increase. It is concluded that hypotonic stimulation to A6 cells first induces cell swelling, which is followed by [Ca2+]i increase that leads to the coactivation of K+ and Cl- channels. This coactivation may accelerate K+ and Cl- effluxes, resulting in RVD.     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.     

The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.     

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Two different types of action potentials were observed among the pyramidal cells and interneurons in cat motor cortex: the narrow action potentials and the wide action potentials. These two types of action potentials had similar rising phases (528.8 +/- 77.0 vs 553.1 +/- 71.8 mV/ms for the maximal rising rate), but differed in spike duration (0.44 +/- 0.09 vs 1.40 +/- 0.39 ms) and amplitude (57.31 +/- 8.22 vs 72.52 +/- 8.31 mV), implying that the ionic currents contributing to repolarization of these action potentials are different. Here we address this issue by pharmacological manipulation and using voltage-clamp technique in slices of cat motor cortex. Raising extracellular K+ concentration (from 3 mM to 10 mM), applying a low dose of 4-aminopyridine (2-200 microM) or administering a low concentration of tetraethylammonium (0.2-1.0 mM) each not only broadened the narrow action potentials, but also increased their amplitudes. In contrast, high K+ medium or low dose of tetraethylammonium only broadened the wide action potentials, leaving their amplitudes unaffected, and 4-aminopyridine had only a slight broadening effect on the wide spikes. These results implied that K+ currents were involved in the repolarization of both types of action potentials, and that the K+ currents in the narrow action potentials seemed to activate much earlier than those in the wide spikes. This early activated K+ current may counteract the rapid sodium current, yielding the extremely brief duration and small amplitude of the narrow spikes. The sensitivity of the narrow spikes to 4-aminopyridine may not be mainly attributed to blockade of the classical A current (IA), because depolarizing the membrane potential to inactivate IA did not reproduce the effects of 4-aminopyridine. Blockade of Ca2+ influx slowed the last two-thirds repolarization of the wide action potentials. On the contrary, the narrow action potentials were not affected by Ca(2+)-current blockers, but if they were first broadened by 4-aminopyridine or tetraethylammonium, subsequent application of Ca(2+)-free medium caused further broadening, suggesting that the narrow action potentials were too brief to activate the Ca(2+)-activated potassium currents for their repolarization. Therefore, the effects of low concentrations of tetraethylammonium on the narrow spikes appeared to be mainly due to blockade of an outward current that was different from the tetraethylammonium-sensitive Ca(2+)-activated potassium current (IC). In the neurons with the narrow spikes, voltage-clamp experiments revealed two voltage-gated outward currents that were sensitive to tetraethylammonium and 4-aminopyridine, respectively. Both currents were activated rapidly following the onset of depolarizing steps. Interestingly, the tetraethylammonium-sensitive current was a transient outward current that inactivated rapidly (tau < or = 5 ms), while the 4-aminopyridine-sensitive current was relatively persistent during maintained depolarization. The 4-aminopyridine-sensitive current did not show obvious inactivation even at membrane potential of -40 mV, which completely inactivated the transient tetraethylammonium-sensitive, current. The results indicate that different potassium currents are involved in the repolarization of the narrow and wide action potentials in cat motor cortex. A novel tetraethylammonium-sensitive transient outward current and a 4-aminopyridine-sensitive outward current are responsible for the short duration and small amplitude of the narrow action potentials in the interneurons and some of the layer V pyramidal cells. These two currents are voltage-gated and Ca(2+)-independent. For the wide action potentials that characterize most pyramidal neurons, a Ca(2+)-independent tetraethylammonium-sensitive outward current and a Ca(2+)-activated potassium current are the main contributors to their repolarization.     

GABAB receptor activation causes a depression of low- and high-voltage-activated Ca2+ currents, postinhibitory rebound, and postspike afterhyperpolarization in lamprey neurons

1. Activation of gamma-aminobutyric acid-B (GABAB) receptors during N-methyl-D-aspartate (NMDA)-induced fictive locomotor activity in the lamprey spinal cord reduces the burst frequency and changes the intersegmental coordination. Presynaptic inhibition of both the excitatory and inhibitory synaptic transmission from spinal premotor interneurons occurs through GABAB receptor activation. To further analyze the cellular mechanisms underlying the GABABergic modulation of the locomotor network, the present study investigates somatodendritic effects of GABAB receptor activation on interneurons and motoneurons in the lamprey spinal cord in vitro using single-electrode current- and voltage-clamp techniques. 2. High- (HVA) and low- (LVA) voltage-activated calcium currents were studied with single-electrode voltage clamp when Na+ and K+ currents were blocked--using tetrodotoxin, tetraethylammonium (TEA), and CsCl electrodes--after substituting Ca2+ with Ba2+. Cobalt-sensitive inward barium currents, activated at -50 mV, became larger when the holding potential was set to a more hyperpolarized level, thus suggesting the existence of an LVA calcium current. The presence of cobalt-sensitive inward barium currents activated at -30 and -10 mV suggests the existence of an HVA calcium current. GABAB receptor activation (baclofen) reduced the peak amplitude of both the LVA and HVA Ca2+ component. 3. The late phase of the afterhyperpolarization (AHP), which follows the action potential, was reduced in amplitude by cobalt, thus lending further support to the notion that the Ca2+ influx, and the subsequent activation of Ca(2+)-dependent K+ channels (KCa2+), constitutes the major part of the AHP generation. Application of the GABAB agonist baclofen also reduced the peak amplitude of the AHP in interneurons and motoneurons, and this reduction was counteracted by the GABAB antagonist 2(OH)saclofen. Baclofen reduced the duration of action potentials broadened by TEA, thus suggesting that the Ca2+ inflow was reduced. Intracellular injection of the GTP analogue GTP gamma S also reduced the duration of the action potential and the peak amplitude of the AHP in TEA, thus supporting the notion that a GTP-binding protein (G-protein)-mediated GABAB receptor activation reduced the calcium inflow, leading to less activation of KCa channels and, consequently, to a smaller peak amplitude of the AHP. 4. Baclofen suppressed the subthreshold depolarization induced by a depolarizing current pulse injection without affecting either the spike threshold or the resting membrane conductance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Physiological and theoretical analysis of K+ currents controlling discharge in neonatal rat mesencephalic trigeminal neurons

Whole cell voltage- and current-clamp recordings were obtained from mesencephalic trigeminal sensory (Mes 5) neurons identified visually in thin brain stem slices of neonatal rats with the use of infrared video microscopy. These cells exhibited accommodation in spike discharge responses to depolarizing current injection protocols whose duration differed as a function of holding potential (-50 vs. -65 mV). Several spikes were elicited before the membrane response accommodated from -50 mV, whereas from -65 mV only single action potentials were evoked. In response to similar protocols, application of the K+ channel blocker 4-aminopyridine (4-AP) (50 microM to 2 mM) caused sustained repetitive spiking whereas tetraethylammonium (TEA) (10-30 mM) did not cause repetitive spiking. In voltage clamp, 4-AP application (100 microM) revealed a sustained outward current (I4-AP) that was active between -60 and -30 mV. I4-AP was responsible for suppressing sustained repetitive spiking behavior, producing accommodation under normal circumstances. TEA application in voltage clamp revealed a sustained outward current evoked positive to -40 mV. Two transient outward currents (TOCs) were identified by prepulse protocols typically used to characterize A-type currents: a 4-AP-insensitive fast TOC, and a slow TOC (ITOC-S) sensitive to 4-AP (> 500 microM). A Ca(2+)-dependent outward current that activated positive to -30 mV was also characterized. A mathematical model of a Mes 5 neuron was assembled from our voltage-clamp records to simulate the dynamic interaction of outward currents during membrane excitation. We conclude that in Mes 5 neurons, the 4-AP-sensitive currents ITOC-S and I4-AP determine the duration of spike trains. In particular, the noninactivating I4-AP determines whether cells exhibit sustained repetitive discharge or accommodate in response to depolarizing current. Neurotransmitter modulation of this current or modulation of the resting membrane potential could modify the output properties of Mes 5 neurons, and therefore the properties of these currents must be incorporated into our current understanding of how these cells contribute to shaping oral-motor pattern generation.     

Functional and ionic properties of a slow afterhyperpolarization in ferret perigeniculate neurons in vitro

Intracellular recordings from spontaneously spindling GABAergic neurons of the ferret perigeniculate nucleus in vitro revealed a fast afterhyperpolarization after each action potential, a medium-duration afterhyperpolarization after each low-threshold Ca2+ spike, and a slow afterhyperpolarization after the cessation of spindle waves. The slow afterhyperpolarization was associated with an increase in membrane conductance, and the reversal potential was sensitive to extracellular [K+]o, indicating that it is mediated at least in part by the activation of a K+ conductance. However, the block of Ca2+ channels did not block the slow afterhyperpolarization, whereas the block of Na+ channels did block this event, even after the generation of repetitive Ca2+ spikes, indicating that it is mediated by a Na+-activated K+ current. Application of apamin reduced the afterhyperpolarization and enhanced a plateau potential after each low-threshold Ca2+ spike. This plateau potential could result in a prolonged depolarization of perigeniculate neurons, even before the application of apamin, resulting in the generation of tonic discharge. The plateau potential was blocked by the local application of tetrodotoxin, indicating that it is mediated by a persistent Na+ current. The activation and interaction of these slowly developing and persistent currents contributes significantly to low-frequency components of spindle wave generation. In particular, we suggest that the activation of the slow afterhyperpolarization may contribute to the generation of the spindle wave refractory period in vitro.     

Deinstitutionalization in psychiatry: revising our principles of community psychiatry or failure of deinstitutionalization!

The involvement of large conductance Ca(2+)-activated K+ channels (BK) and ATP-sensitive K+ (KATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca2+ concentration ([Ca2+]i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca2+]i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca2+]i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca2+]i with force in the presence of serotonin at different extracellular Ca2+ concentration ([Ca2+]o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca(2+)-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.