Decrease In Membrane Conductance Research Articles

ChemInform Abstract: EPC Syntheses and Structure-Activity Relationships of Hypoglycaemic Semicyclic Amidines.

Abstract A series of homochiral sterically hindered mono- and bicyclic amidines was prepared as hypoglycaemic agents by lethargic reaction of O-methylcaprolactim and 3-ethoxy-2-azabicyclo[2.2.2]oct-2-ene, respectively, with homochiral cis -2-substituted cyclopentane amines provided by asymmetrical reductive amination of racemic 2-substituted cyclopentanones. All compounds, except the cyclohexylmethyl-isoquinuclidone derivative which inhibited secretion at 100 μM, significantly stimulated insulin secretion 2–8-fold at 10 μM and 100 μM in INS-1 cells. The most potent activator was the 2-cyclopentyl-substituted caprolactam derivative 5e . The stimulatory effects on secretion increased with rising steric hindrance of both the amidine α-carbon and the bicyclic amidine moiety itself. Enantiomeric discrimination was observed for the 2-[( cis -2-bulkysubstituted cyclopentyl)imino]hexahydroazepine halides 5e and 5f and for the 3-[( cis -2-substituted cyclopentyl)imino]-2-azabicyclo[2.2.2]octane halides 6a and 6c . The amidines depolarized INS-1 cells and generated action potentials, accompanied by a decrease of membrane conductance. Simultaneously [Ca 2+ ] i increased, probably due to Ca 2+ -entry through voltage-dependent Ca 2+ -channels. At high concentrations, where inhibition of secretion was observed, [Ca 2+ ] i still rose upon application of the amidines, indicating an additional inhibitory pathway downstream to the elevation of [Ca 2+ ] i . Even at high concentrations (100 μM), the amidines had no toxic effects on insulin secreting INS-1 cells.

EPC syntheses and structure–activity relationships of hypoglycaemic semicyclic amidines

A series of homochiral sterically hindered mono- and bicyclic amidines was prepared as hypoglycaemic agents by lethargic reaction of O-methylcaprolactim and 3-ethoxy-2-azabicyclo[2.2.2]oct-2-ene, respectively, with homochiral cis-2-substituted cyclopentane amines provided by asymmetrical reductive amination of racemic 2-substituted cyclopentanones. All compounds, except the cyclohexylmethyl-isoquinuclidone derivative which inhibited secretion at 100 μM, significantly stimulated insulin secretion 2–8-fold at 10 μM and 100 μM in INS-1 cells. The most potent activator was the 2-cyclopentyl-substituted caprolactam derivative 5e. The stimulatory effects on secretion increased with rising steric hindrance of both the amidine α-carbon and the bicyclic amidine moiety itself. Enantiomeric discrimination was observed for the 2-[( cis-2-bulkysubstituted cyclopentyl)imino]hexahydroazepine halides 5e and 5f and for the 3-[( cis-2-substituted cyclopentyl)imino]-2-azabicyclo[2.2.2]octane halides 6a and 6c. The amidines depolarized INS-1 cells and generated action potentials, accompanied by a decrease of membrane conductance. Simultaneously [Ca 2+] i increased, probably due to Ca 2+-entry through voltage-dependent Ca 2+-channels. At high concentrations, where inhibition of secretion was observed, [Ca 2+] i still rose upon application of the amidines, indicating an additional inhibitory pathway downstream to the elevation of [Ca 2+] i. Even at high concentrations (100 μM), the amidines had no toxic effects on insulin secreting INS-1 cells.

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

Asbestos induces cytogenetic and genotoxic effects in cultured cell lines in vitro. For further investigations of the fiber-induced cellular changes, electrorotation (ROT) measurements can be used to determine early changes of surface properties and dielectric cellular changes. In the present study, human mesothelial cells (HMC) were exposed to nontoxic concentrations of crocidolite asbestos (1 microg/cm(2)) for 12, 24, 30, 50, and 72 hr, and were investigated for changes in dielectric properties, morphologic and biochemical changes using ROT measurements, electron microscopy, and flow cytometry, respectively. The results of ROT measurements revealed slightly increased internal conductivity and decreased membrane conductance of HMC during the first 12 hr of exposure to crocidolite. This may be due to functional changes of ion channels of the cellular membrane. However, after exposures of >= 30 hr, reduced internal conductivity and increased membrane conductance of HMC occurred. These effects may be caused by permeabilization of the cell membrane and the leakage of ions into the surrounding medium. The membrane capacitance of HMC is always decreased during exposure of cells to crocidolite fibers. This decreased membrane capacitance may result from the observed reduction in the number of microvilli and from the shrinkage of cells as observed by electron microscopy and flow cytometry. Changes in composition of the plasma membrane were also observed after the labeling of phosphatidylserines (PS) on the cell surface. These observed changes can be related to apoptotic events. Whereas during the first 50 hr of exposure only a small number of HMC with increased exposure of PS on the cell surface was detected by flow cytometry, the dielectric properties of HMC showed marked changes during this time. Our results show that surface property changes of the cellular membrane of HMC as well as interior dielectric changes occur after the exposure of cells to crocidolite fibers. The observed changes are discussed in terms of complex combined cellular effects after amphibole asbestos exposure.

Distinct Subtypes of Metabotropic Glutamate Receptors Mediate Differential Actions on Excitability of Spinal Respiratory Motoneurons

Metabotropic glutamate receptors (mGluRs) modulate neuronal function by affecting excitability and altering synaptic transmission. We have shown that the mGluR agonist (1S,3R)-1-amino-1, 3-cyclopentanedicarboxylic acid (1S,3R-ACPD) has multiple actions on phrenic motoneurons (PMNs), including reduction of inspiratory-modulated synaptic currents and an increase of neuronal excitability. We hypothesized that these actions were mediated by different mGluR subtypes. We have now identified the involvement of mGluR subtypes and their roles in modulating the excitability of PMNs and the consequent inspiratory motor output in an in vitro neonatal rat brainstem-spinal cord preparation. Activation of postsynaptic group-I mGluRs increases PMN excitability, associated with the production of an inward current and a decrease in membrane conductance, whereas activation of group-II or group-III mGluRs decreases PMN inspiratory-modulated synaptic current, probably via a presynaptic mechanism. To confirm further the distinction and the involvement of group-I and group-II/-III receptor subtypes affecting PMN excitability, we used the membrane permeable cAMP analog 8-bromo-cAMP (8-Br-cAMP) to elevate intracellular cAMP concentration to mask or occlude any effects mediated via the cAMP cascade. 8-Br-cAMP attenuated the reduction of the inspiratory-modulated activity of PMNs by both (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), agonists for group-II and group-III mGluRs, respectively, but did not affect the actions of 3,5-dihydroxyphenylglycine (DHPG), an agonist for group-I mGluRs. These three groups of mGluRs are all endogenously activated during the inspiratory phase. We conclude that three groups of mGluRs are functionally expressed in the phrenic nucleus and that their activation modulates PMN excitability via distinct mechanisms, with group-I acting at postsynaptic sites and group-II and group-III acting at presynaptic sites.

5-HT modulates multiple conductances in immature rat rostral ventrolateral medulla neurones in vitro.

1. Whole-cell patch-clamp recordings were made from rostral ventrolateral medulla (RVLM) neurones of brainstem slices from 8- to 12-day-old rats. In the presence of tetrodotoxin (0.5 microM), 5-HT (50 microM) elicited an outward current (I5-HT,outward) (10/44 neurones) associated with an increase in membrane conductance, and an inward current (I5-HT,inward) (29/44 neurones) accompanied by a decrease or no significant change in membrane conductance. 2. The steady-state I-V relationship of I5-HT,outward showed an inward rectification; the 5-HT-induced current, which reversed at -87.9 +/- 3.0 mV, was suppressed by 0.1 mM Ba2+. 3. Two types of steady-state I-V relationship for I5-HT,inward were noted: type I I5-HT,inward was characterized by a significant decrease in membrane conductance and reversed at a potential close to or negative to the theoretical K+ equilibrium potential (EK), -94 mV, in 8/17 neurones; type II I5-HT,inward was not associated with a significant change in membrane conductance and was relatively independent of membrane potential. 4. Both type I and type II I5-HT,inward were significantly reduced in a low [Na+]o solution. In this solution, I5-HT,inward decreased with hyperpolarization and had a linear steady-state I-V relationship with a reversal potential of approximately -110 mV. The reversal potential of type I I5-HT,inward shifted to about -80 mV as the [K+]o was increased from 3.1 to 7.0 mM in low [Na+]o solution. The type II I5-HT,inward did not reverse at the estimated EK in the same solution. 5. While not affected by externally applied Cs+ (1 mM), I5-HT,inward was significantly smaller in RVLM neurones patched with Cs+-containing electrodes; the current reversed at -11.9 +/- 6.4 mV in 8/15 responsive neurones. 6. It may be concluded that in rat RVLM neurones 5-HT increases an inwardly rectifying K+ conductance which may underlie the I5-HT, outward and that a combination of varying degrees of K+ conductance decrease and a Cs+-insensitive, non-selective cation conductance increase may account for the two types of conductance change associated with I5-HT,inward.

IL-1beta and IL-6 excite neurons and suppress nicotinic and noradrenergic neurotransmission in guinea pig enteric nervous system.

Conventional intracellular microelectrodes and injection of biocytin were used to study the actions of IL-1beta and IL-6 on electrical and synaptic behavior in morphologically identified guinea pig small intestinal submucous neurons. Exposure to nanomolar concentrations of either IL-1beta or IL-6 stimulated neuronal excitability. The excitatory action consisted of depolarization of the membrane potential, decreased membrane conductance, and increased discharge of action potentials. Excitatory action of IL-1beta was suppressed by the natural IL-1beta human receptor antagonist. Electrical stimulation of sympathetic postganglionic axons evoked inhibitory postsynaptic potentials (IPSPs), and stimulation of cholinergic axons evoked nicotinic fast excitatory postsynaptic potentials (EPSPs). Both kinds of synaptic potentials occurred in neurons with uniaxonal morphology believed to be secretomotor neurons. Either IL-1beta or IL-6 suppressed the noradrenergic IPSPs and the fast EPSPs, and the two acted synergistically when applied in combination. Suppression of the IPSP resulted from presynaptic inhibition of the release of norepinephrine from sympathetic nerves. The results suggest that the presence of either or both inflammatory cytokines will release the sympathetic brake from secretomotor neurons to the intestinal crypts and from nicotinic synapses in the integrative microcircuits, where norepinephrine is known to have a presynaptic inhibitory action. This, in concert with excitation of secretomotor neurons, may lead to neurogenic secretory diarrhea.

Vasopressin acting at V1-type receptors produces membrane depolarization in neonatal rat spinal lateral column neurons

Vasopressin-immunoreactive fibers have been visualized in the area of spinal lateral horn cells, including spinal sympathetic preganglionic neurons. The presence and nature of vasopressin receptors on neurons in this area were addressed using whole-cell patch-clamp techniques in transverse spinal cord slice preparations from neonatal rat. Bath applications of Arg8-vasopressin (VP) induced a slow-onset membrane depolarization accompanied by spike discharges and membrane oscillations. In voltage-clamp, applications of VP induced a reversible, tetrodotoxin-resistant and dose-dependent inward current in 90% of tested cells. This effect was blocked by a V1 receptor antagonist [D-(CH2)5 Tyr (Me)-VP], whereas a V2 receptor agonist [desamino-(D-Arg8)-vasopressin] was ineffective. Furthermore the applications of oxytocin produced significantly smaller depolarizations when compared with VP suggesting that, at least in the neonatal lateral horn cells, vasopressin rather than oxytocin is more effective ligand. Both the amplitude and duration of the VP effect were enhanced after intracellular dialysis with GTP-gamma-S, a non-hydrolyzable GTP analogue, whereas the inward current was significantly reduced after intracellular dialysis with GDP-beta-S, a stable analogue of GDP that competitively inhibits G-proteins. The observation that the VP-induced net inward current reversed at a potential close to the equilibrium for potassium ions and was associated with a decrease in membrane conductance in a majority of tested cells suggest mediation through closure of a leak potassium conductance. These data indicate that SPNs and other lateral horn cells possess functional G-protein-coupled V1-type vasopressin receptors that, in adult spinal cord, may contribute to CNS regulation of autonomic nervous system function.

Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action.

Receptor cells of the vomeronasal organ (VNO) are thought to detect pheromone-like molecules important for reproductive physiology. Several compounds derived from male mouse urine have been demonstrated to affect endocrine events in female mice. In the present study, the ability of these compounds to affect VNO activity was tested. In dissociated VNO cells held under voltage clamp conditions, application of dehydro-exo-brevicomin (DHB) evoked an outward current at negative holding potentials and an inward current at positive holding potentials. Under current clamp, DHB reduced action potential firing. Since DHB application caused a decrease in membrane conductance, this compound appeared to act by reducing inward current through closing an ion channel. Biochemical experiments tested the effects of DHB and 2-(sec-butyl)-4,5-dihydrothiazole (SBT) on cAMP levels in the VNO. A mixture of DHB and SBT decreased cAMP levels in VNO sensory tissue and had no effect on VNO non-sensory tissue. The results suggest that pheromones have an inhibitory influence on action potential generation and on cAMP levels in receptor cells of the VNO.

Substance P-induced inward current in identified auditory efferent neurons in rat brain stem slices.

The effects of substance P (SP) on whole cell currents were studied in neurons of the medial olivocochlear efferent system (MOCS) in the ventral nucleus of the trapezoid body (VNTB) of brain stem slices from neonatal rats. Each neuron was identified by retrograde labeling with Fast Blue injected into the cochlea. Bath application of SP (0.1-10 microM) reversibly induced an apparent inward current in 49 of 63 labeled neurons when voltage clamped at near resting voltages. This apparent inward current was consistent with the SP-induced membrane depolarization observed in current-clamp mode. The SP-induced change in current was dose dependent with a half-maximal response dose of 200 nM. It was mimicked by [Cys3,6, Tyr8, Pro9]-SP, a neurokinin (NK1) receptor selective agonist, whereas [Succinyl-Asp6, MePhe8]-SP 6-11 (Senktide), a NK3 receptor agonist, had no detectable effect. The SP effect was not blocked by 10(-6) M tetrodotoxin (TTX) and persisted when the perfusate contained 30 mM tetraethylammonium (TEA) or 100 microM Cd2+ or was in a 0-Ca solution. In a TTX-containing solution, SP caused a voltage-dependent decrease of membrane conductance, and the SP-evoked current reversed at a potential at around -105 mV. The predicted K+ equilibrium potential was -93.8 mV under the experimental conditions. The SP-induced inward current was attenuated by 66% when the perfusate contained 3 mM Cs+. We conclude that the apparent inward current is partly caused by SP decreasing an outward current normally maintained by the inward rectifier K+ channels in these cells. In the presence of Cs solution in the recording pipette and with a perfusate containing 3 mM Cs+, 0.1 mM Cd2+ and 10(-6) M TTX, a residual SP-induced inward current was observed at test voltages ranging from -120 to 40 mV. This subcomponent reversed its polarity at approximately 20 mV. This inward current was reduced substantially (but not abolished) when all NaCl in the external solution was replaced by TEA-Cl. The results indicate that SP also opens an unknown cation channel, which the available data suggests may be relatively nonselective. The results suggest that MOCS neurons are subject to modulation by SP, which depolarizes the cell membrane by decreasing the activity of inward rectifier K+ channels as well as concurrently activating a separate cation conductance. It also was found that in MOCS neurons responsive to both SP and norepinephrine, the norepinephrine effect was abolished by TTX, suggesting that an interneuronal population excited by norepinephrine converges selectively onto SP-sensitive MOCS neurons in the VNTB.

Muscarinic receptor activation in guinea-pig chromaffin cells causes decreased membrane conductance and depolarization

Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. The local pressure ejection of muscarinic agonists, acetylcholine (in the presence of hexamethonium) or bethanecol, caused a transient depolarization that was relatively slow (1–2 s) in onset compared with the depolarization associated with the activation of nicotinic receptors. Muscarinic receptor-induced depolarization was associated with an increase in input resistance and the firing of action potentials. Repetitive stimulation of splanchnic nerve fibers within the gland, in the presence of hexamethonium, caused a maintained depolarization that was slow in both onset and decay and in many cells caused the repetitive firing of action potentials. It is suggested that, in this species, the exocytosis of catecholamines caused by the activation of muscarinic receptors, described by others, may be due to the initiation of tetrodotoxin-sensitive action potentials and consequent opening of voltage sensitive Ca 2+ channels.

Vasopressin's depolarizing action on neonatal rat spinal lateral horn neurons may involve multiple conductances.

Vasopressin-immunoreactive fibers have been visualized in the area of spinal lateral horn cells, including spinal sympathetic preganglionic neurons (SPNs). The presence and nature of vasopressin receptors on 125 neurons in this area were addressed using whole-cell patch-clamp techniques in transverse spinal cord slice preparations from neonatal rat (11-21 days). Local pressure applications of Arg-vasopressin (AVP, 1 microM) induced a slow-onset membrane depolarization accompanied by spike discharges and membrane oscillations. In voltage-clamp, applications of AVP (10 nM-1 microM) induced a reversible, tetrodotoxin-resistant and dose-dependent inward current in 90% of tested cells. This effect was blocked by a V1 receptor antagonist [D-(CH2)5 Tyr (Me)-AVP], whereas a V2 receptor agonist [desamino-(D-Arg8)-vasopressin] was ineffective. Both the amplitude and duration of the AVP effect were significantly modified after intracellular dialysis of non-hydrolysable G-protein modulators. I-V relationships, examined in 75 cells, suggested two conductances. In 36 cells the net AVP current reversed approximately -102 mV, was associated with a decrease in membrane conductance and yielded linear I-V plots, suggesting mediation through closure of a resting potassium conductance. In a further 26 cells the I-V lines remained almost parallel in the voltage range used in this study (-130 to -40 mV), while the membrane conductance was decreased in a majority of these cells. In the remaining 13 cells the net AVP current was estimated to reverse approximately -30 mV and was associated with a small increase in membrane conductance, suggesting mediation through opening of a nonselective cationic conductance. These data indicate that the majority of SPNs and other lateral horn cells possess functional G-protein-coupled V1-type vasopressin receptors in the neonatal spinal cord. In the adult spinal cord, some of these receptors are likely to participate in CNS regulation of autonomic nervous system function.

Apoptosis dependent decrease of the intramembrane ion traffic in cultured mouse fibroblasts shown by conductivity dispersion.

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.

Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors.

Olfactory bulb mitral cells express group I (mGluR1), group II (mGluR2), and group III (mGluR7 and mGluR8) metabotropic glutamate receptors. We examined the role of these mGluRs on excitatory synaptic transmission in cultured mitral cells with the use of whole cell patch-clamp recordings. The effects of group-selective mGluR agonists and antagonists were tested on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate (ACPD) or the group-I-selective agonist 3,5-dihydroxyphenylglycine evoked an inward current accompanied by a decrease in membrane conductance, consistent with the previously described closure of potassium channels by group I agonists. The increased cellular excitability was accompanied by an increase in mEPSC frequency in some cells. When calcium entry was blocked by cadmium, ACPD or the group-II-selective agonist 2-(2,3-dicarboxycyclopropyl)-glycine reduced the mEPSC frequency. L-2-amino-4-phosphonobutyric acid (L-AP4), a group-III-selective agonist, caused a similar decrease. The concentration-dependence of L-AP4-mediated inhibition was most consistent with activation of mGluR8. We investigated two possible effector mechanisms for the group III presynaptic receptor. Bath application of forskolin or 3-isobutyl-1-methylxantine had no effect on mEPSC frequency. Increasing calcium influx by raising extracellular K+ caused a large increase in the mEPSC frequency but did not enhance L-AP4-mediated inhibition. Thus inhibition of mEPSCs involves a mechanism downstream of calcium entry and appears to be independent of adenosine 3',5'-cyclic monophosphate. Our results indicate that both group II and III receptors can inhibit glutamate release at mitral cell terminals. Although group II/III receptors had a similar effect on mEPSCs, differences in location on nerve terminals and in glutamate sensitivity suggest that each mGluR may have discrete actions on mitral cell activity.

Voltage Oscillations in Xenopus Spinal Cord Neurons: Developmental Onset and Dependence on Co‐activation of NMDA and 5HT Receptors

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 microM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2-5 microM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg2+ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT1alpha receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT1, a selective 5HT1alpha receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg2+, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4-8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an 'apparent' decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg2+ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg2+ block. When the membrane potential was depolarized by approximately 20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg2+ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg2+ through activation of receptors with 5HT1alpha-like pharmacology.

Enhancement of rat trigeminal ganglion neuron responses to piperine in a low-pH environment and block by capsazepine

Both trigeminal and spinal ganglion neurons show a strong potentiation of responses to the irritant capsaicin in an acidic environment. The present study revealed that there is also a strong interaction between protons and piperine, another vanilloid irritant. We studied the mechanism of the interaction between protons and piperine. Whole-cell patch clamp recordings were performed on cultured adult rat trigeminal ganglion (TG) neurons voltage-clamped near their resting membrane potential (−60 mV). Piperine (10 μM) caused a sustained net inward current associated with either an increase or decrease in membrane conductance. When protons and piperine were co-applied, the membrane currents evoked in piperine-sensitive TG neurons far exceeded the algebraic sum of the responses to the two stimuli applied in isolation. Capsazepine blocked the response of TG neurons to piperine at both physiological and acidic pH. In the presence of capsazepine, responses to the mixture of piperine and protons resembled the response to the low pH stimulus applied alone. Capsazepine had no effect on the sustained proton-induced current. These findings suggest that protons enhance the piperine current by altering the vanilloid receptor/channel complex or increasing the length constant of the space clamp.

Reduction of K+Uptake in Glia Prevents Long-Term Depression Maintenance and Causes Epileptiform Activity

Extracellular cesium causes synchronous, interictal-like bursting and prevents maintenance of long-term depression (LTD) in the CA1 hippocampal region. We have investigated the cellular mechanisms underlying cesium actions. Whole-cell recordings showed that brief (2 min) bath exposures to cesium caused pyramidal cell hyperpolarization associated with decreased membrane conductance attributable to blockade of an inward h-type current. After prolonged (>2 min) exposures, a late depolarizing response was observed; this effect was not associated with changes in cell membrane conductance. Recordings from interneurons revealed that Ih is expressed in a subpopulation of cells and that cesium effects on interneurons expressing Ih are comparable to those observed in pyramidal cells. Consistent with this effect, cesium decreased the early component of the IPSP recorded in pyramidal cells. Interneurons lacking Ih were not affected by cesium but developed a depolarizing response when drug applications were paired to orthodromic stimulation. We concluded that cesium actions on LTD and cesium-induced epileptiform activity were not attributable exclusively to its direct effects on neurons. Recordings from hippocampal slice astrocytes revealed that cesium interfered with glial electrical responses during LTD induction. Cesium blocked glial inwardly rectifying potassium channels and increased the amplitude and duration of stimulation-evoked [K+]out increases. Thus, the effects of cesium on CA1 synchronization and synaptic plasticity appear to be mediated predominantly by blockade of glial voltage-dependent potassium uptake.

Neuromedin C decreases potassium conductance and increases a non-specific conductance in rat suprachiasmatic neurones in brain slices in vitro

Whole-cell recordings were made, in both current and voltage clamp, from suprachiasmatic neurones maintained in coronal rat brain slices. In current clamp doses of 10 and 100 nM neuromedin C (NMC) were shown to increase basal firing rate in 9 out of 32 neurones. The excitatory responses to 100 nM NMC were accompanied by small increases in neuronal input resistance (25.0±9.9% in 4 out of 7 neurones tested) and depolarisations of membrane potential (9.8±3.4 mV in 4 out of 7 neurones tested). However, 10 nM NMC caused no changes in either neuronal input resistance or membrane potential despite the clear increases in neuronal firing rate. When voltage-clamped at −60 mV, 100 nM NMC induced an inward current of 14.8±1.2 pA in 46 of 210 neurones. The NMC-induced inward current was shown to be unaffected by perfusion with 1 μM tetrodotoxin (TTX). The inward current recorded at −60 mV was typically associated with a decrease in membrane conductance. Construction of current–voltage relationships in the absence and presence of 100 nM NMC showed that with the majority of the NMC-sensitive neurones the inward current either reversed polarity close to the potassium reversal potential or decreased at hyperpolarised potentials. This reversal potential was shifted to more depolarised potentials when the extracellular concentration of potassium was increased. The NMC-induced inward current was unaffected by reduction of the extracellular concentration of sodium or by addition of 0.2 mM cadmium. In potassium-free conditions, in both the dialysing pipette solution and perfusing saline, NMC was still able to induce an inward current. The additional reduction of the extracellular concentration of sodium, whilst recording in potassium-free conditions, was also unable to abolish the inward current. Recordings made with an electrode containing the non-hydrolysable guanosine triphosphate analogue, guanosine 5′-thio-triphosphate, resulted in NMC-induced inward currents which failed to recover to baseline. It is concluded that NMC excites a subpopulation of suprachiasmatic neurones by decreasing a resting potassium conductance and increasing a non-specific conductance, via a G-protein link.

Inhibition of K ATP channel activity by troglitazone in CRI-G1 insulin-secreting cells

Patch-clamp recording techniques were used to examine the effect of troglitazone on K ATP channel activity in Cambridge rat insulinoma-G1 (CRI-G1) insulin-secreting cells. In both inside-out and outside-out patch recordings, bath application of troglitazone reduced K ATP channel activity. This inhibition was independent of the membrane voltage and was poorly reversible. In whole-cell studies, troglitazone inhibited K ATP channel currents with an IC 50 of 697 ± 92 nM and an associated Hill coefficient of 1.2 ± 0.2. In current clamp recordings 10 μM troglitazone depolarised the CRI-G1 cell membrane by 36.8 ± 3.9 mV with a concomitant decrease in membrane conductance. However, in contrast to the rapid depolarisation produced by tolbutamide, the effects of troglitazone developed more slowly, usually taking 15–20 min to develop.

Tetraethylammonium-sensitive calcium-sensitive potassium current in a subclass of the bullfrog dorsal root ganglion cells

Bullfrog dorsal root ganglion (DRG) cells were classified into three types, As, Ar and C, according to their electrophysiological properties. Actions of tetraethylammonium (TEA; 100 μM−100 mM) on As-type cells were examined using current- and voltage-clamp methods; TEA caused a membrane depolarization or an inward current, associated with a decrease in membrane conductance. These TEA-induced responses reversed in polarity at −85 to −90 mV, and the change in reversal potential followed the Nernst equation as extracellular K + concentration was changed. The TEA-induced responses were reversibly inhibited by Ca ++ -free/high-Mg ++ solutions and inorganic Ca blockers. It is concluded that bullfrog DRG As-type cells might be also endowed with Ca-sensitive K channels which may be open at rest and blocked by TEA.

Suppression of K+ conductance by metabotropic glutamate receptor in acutely dissociated large cholinergic neurons of rat caudate putamen.

1. Responses to metabotropic glutamate receptor (mGluR) activation were investigated in acutely dissociated rat neostriatal (caudate putamen, CP) large cholinergic neurons, with the use of a nystatin-perforated patch-clamp technique. 2. Application of L-glutamate (Glu) in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) induced a slow inward current accompanied by a decrease in membrane conductance at a holding potential (VH) of -44 mV. 3. The ratio of the maximal amplitude of the slow inward metabotropic glutamate (mGlu) response to that of the ionotropic glutamate response for large cholinergic CP neurons was larger than that for the hoppocampal CA1, CA3, and dentate gyrus neurons the nucleus tractus solitarii neuron, cerebellar Purkinje neuron, and granule cell of the main olfactory bulb. The threshold and half-maximal effective concentration values of these mGlu responses were 10- to 30-fold lower than those of the respective ionotropic responses. 4. Specific agonists of the mGluR, quisqualate (QA), (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD], and (2S,3S,4S)-alpha-carboxycyclopropyl-glycine [L-CCG-1), similarly induced slow inward currents at the same VH. The relative affinities of the mGlu agonists were QA > Glu > L-CCG-1 > (1S,3R)-ACPD. L-CCG-1 did not induce any current at concentrations < 10(-6) M. 5. DL-2-amino-3-phosphonopropionic acid or DL-2-amino-4-phosphonobutyric acid did not block the mGlu response, whereas (RS)-alpha-methyl-4-carboxyphenyl-glycine, a selective mGluR antagonist, partially reduced the mGlu response. 6. The reversal potential of the mGlu response was close to the K+ equilibrium potential, and it shifted by 56.4 mV for a 10-fold change in extracellular K+ concentration. In 90.6% of the neurons tested, the instantaneous current induced by hyperpolarizing voltage steps was markedly suppressed during the mGlu response. In 9.4% of the neurons, the currents elicited by step pulses showed a voltage-dependent slow relaxation that was not affected by mGluR activation. 7. Under the current-clamp mode, the slow afterhyperpolarization (AHP) following a spontaneous discharge was not affected by tACPD. The AHP current was not blocked under the voltage-clamp mode, either. 8. (1S,3R)-ACPD or Glu in the presence of CNQX elicited membrane depolarization accompanied by increased rate of action potentials under the current-clamp mode. Tetrodotoxin had no effect on the membrane depolarization. 9. These results indicate that the mGluR on large cholinergic CP neurons is mainly of the mGluR1 and/or mGluR5 type, and it plays a significant role in controlling the membrane potential by way of suppressing the leak K+ conductance.