Sigma-binding site ligands inhibit K+ currents in rat locus coeruleus neurons in vitro

The effect of triphenyltin on the activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated. Triphenyltin inhibits the hydrolysis of chromatophore membrane-bound pyrophosphatase in a pH-dependent pattern, being maximal at pH 9-10. At basic pH values, the inhibition produced by this organotin on membrane-bound pyrophosphatase is very similar to that produced on the chromatophore H+ATPase (I50 = 14.4 and 10 microM, respectively). Detergent-solubilized membrane-bound pyrophosphatase is also inhibited by triphenyltin, but the cytoplasmic enzyme of R. rubrum is inhibited only slightly. The inhibitory effect of triphenyltin on membrane-bound pyrophosphatase is the same with Mg-PPi or Zn-PPi, and is dependent on the chromatophore membrane concentration. Triphenyltin modified mainly the Vmax of the enzyme, and only slightly its Km. Free Mg2+ does not reverse the inhibition. Reducing agents prevent triphenyltin inhibition of the membrane-bound pyrophosphatase, but their effect is due to an alteration of the inhibitor, and not to a modification of thiol groups of the enzyme. The most likely site for triphenyltin inhibition in chromatophore membrane-bound pyrophosphatase is a component either within or closely associated with the membrane.     

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons. J. Neurophysiol. 78: 2309-2320, 1997. Whole cell, Ca2+-independent, depolarization-activated K+ currents were characterized in identified callosal-projecting (CP) neurons isolated from postnatal day 7-16 rat primary visual cortex. CP neurons were identified in vitro after in vivo retrograde labeling with fluorescently tagged latex microbeads. During brief (160-ms) depolarizing voltage steps to potentials between -50 and +60 mV, outward K+ currents in these cells activate rapidly and inactivate to varying degrees. Three distinct K+ currents were separated based on differential sensitivity to 4-aminopyridine (4-AP); these are referred to here as IA, ID, and IK, because their properties are similar (but not identical) K+ currents termed IA, ID, and IK in other cells. The current sensitive to high (>/=100 mu M) concentrations of 4-AP (IA) activates and inactivates rapidly; the current blocked completely by low (相似文献   

Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.     

Intracellular calcium stores modulate miniature GABA-mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.     

Properties and the cytoskeletal control of Ca(++)-independent large conductance K+ channels in neonatal rat hippocampal neurons

ABC transporters are a large superfamily of integral membrane proteins involved inATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful. To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models.     

Survival and neurite formation of mesencephalic trigeminal neurones of the rat in vitro

In order to study the development and functional properties of single, isolated, rat mesencephalic trigeminal neurones, a cell-culture procedure was developed for these specific primary sensory neurones. Mesencephalic trigeminal neurones were isolated from the brainstem of 16-day-old rat embryos. Various factors thought to promote the survival and growth of these neurones in vitro were examined. Outgrowth and maintenance of mesencephalic trigeminal neurones in vitro appeared to be stimulated by a muscle-derived factor, present in muscle-conditioned medium or in muscle extract. Of the neurotrophic factors examined, brain-derived neurotrophic factor and neurotrophin-3, but not nerve-growth factor, promoted the survival of rat mesencephalic trigeminal neurones. Optimal survival of these neurones was found to occur on a monolayer of astrocytes, an effect mediated through direct cell-to-cell interactions.     

Whole cell recording of sodium, potassium and calcium currents of cultured neonatal rat sympathetic neurons

Na, K and Ca currents and other electrophysiological characteristics of cultured neonatal rat superior cervical sympathetic neurons were studied using whole cell clamp technique. The mean passive and active membrane properties measured are as follows: resting membrane potential, -51 +/- 6 mV; input resistance, 1432 +/- 389 M omega; time constant, 130 +/- 32 ms; amplitude of action potential, 96 +/- 10 mV; overshoot, 42 +/- 6 mV. Na, K and Ca currents were isolated upon pharmacological manipulations. The predominant type of K current was a noninactivating delayed rectifier. Voltage-clamp studies also showed the presence of a high voltage-activated sustained inward Ca current, while low voltage could not elicit any transient Ca current.     

ATP-regulated K+ channel and cytosolic Ca2+ mobilization in cultured rat spinal neurons

ATP activated the K+ channel responsible for outwardly rectifying currents via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein in cultured rat spinal neurons. The evoked currents were inhibited by a selective protein kinase C inhibitor, GF109203X, whereas a phospholipase C inhibitor, neomycin had no effect. These indicate that the currents are regulated by phospholipase C-independent protein kinase C activation. In addition, ATP enhanced intracellular free Ca2+ concentration. The increase in intracellular free Ca2+ concentration was inhibited by a broad G-protein inhibitor, GDP beta S, but not affected by neomycin or an inositol 1,4,5-triphosphate receptor antagonist, heparin, suggesting that the cytosolic Ca2+ mobilization is regulated by a mechanism independent of a phospholipase C-mediated phosphatidylinositol signaling. These results, thus, demonstrate that ATP has dual actions on the coupled K+ channel and cytosolic Ca2+ release.     

The distribution of afferent neurons in the trigeminal mesencephalic nucleus and the central projection of afferent fibers of the mylohyoid nerve in the rat

Horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) was injected into the proximal cut ends of three branches of the mylohyoid nerve in rats: the branch to the mylohyoid muscle (BrMh), the branch to the anterior belly of the digastricus muscle (BrDg), and the cutaneous branch (BrCu). HRP-labeled cells were detected in the ipsilateral caudal portion of the trigeminal mesencephalic nucleus (Vmes) and the ipsilateral ventromedial division of the trigeminal motor nucleus, except when HRP:WGA was applied to the BrCu. Morphologically, all labeled Vmes cells were of the pseudounipolar type. Projections of the primary afferents of the BrMh were observed in the ipsilateral trigeminal nucleus caudalis, the upper cervical dorsal horns of laminae I-III, and the dorsolateral recticular formation (Rf), whereas the primary afferents of the BrDg terminated in the ipsilateral trigeminal nucleus principalis and Rf. These observations suggest that the role of the afferent inputs of the mylohyoid muscle differs from that of those of the anterior belly of the digastricus muscle in terms of several functions associated with jaw-closing and infrahyoid muscles.     

Tetrahydroberberine blocks membrane K+ channels underlying its inhibition of intracellular message-mediated outward currents in acutely dissociated CA1 neurons from rat hippocampus

In the patch-clamp perforated whole-cell recording mode, tetrahydroberberine (THB), a novel dopamine (DA) receptor antagonist, inhibits not only DA-induced outward K+ currents, but also acetylcholine-, caffeine- or strychnine-induced outward current. However, THB does not affect either GABA- or glycine-induced Cl- currents, or non-NMDA receptor agonist-induced cation currents. As expected for a K+ channel blocker, THB evokes a downward current deflection accompanied by a decrease of conductance. It is concluded that the direct blockade of membrane K+ channels by THB underlies its inhibition of intracellular message-mediated outward currents.     

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP > GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.     

Somatostatin increases a voltage-insensitive K+ conductance in rat CA1 hippocampal neurons

Somatostatin (SST) is a neuropeptide involved in several central processes. In hippocampus, SST hyperpolarizes CA1 pyramidal neurons and augments the K+ M current (IM). However, the limited involvement of IM at resting potential in these cells suggests that the peptide also may modulate another channel to hyperpolarize hippocampal pyramidal neurons (HPNs). We studied the effect of SST on noninactivating conductances of rat CA1 HPNs in a slice preparation. Using MK886, a specific inhibitor of the enzymatic pathway that leads to the augmentation of IM by SST, we have uncovered and characterized a second conductance activated by the peptide. SST did not affect IM when applied with MK886 or the amplitudes of the slow Ca2+-dependent K+ afterhyperpolarization-current and the cationic Q current but still caused an outward current, indicating that SST acts upon another conductance. In the presence of MK886, SST elicited an outward current that reversed around -100 mV and that displayed a linear current-voltage relationship. Reversal potentials obtained in different external K+ concentrations are consistent with a conductance carried solely by K+ ions. The slope of the current-voltage relationship increased proportionately with the extracellular K+ concentration and remained linear. This suggests that SST opens a voltage-insensitive leak current (IK(L)) in HPNs not an inwardly rectifying K+ current as reported in other neuron types. A low concentration of extracellular Ba2+ (150 M) only slightly decreased the SST-induced effect in a voltage-independent manner, whereas a high concentration of Ba2+ (2 mM) completely blocked it. Extracellular Cs+ (2 mM) did not affect the outward SST current but inhibited the inward component. We conclude that SST inhibits HPNs by activating two different K+ conductances: the voltage-insensitive IK(L) and the voltage-dependent IM. The hyperpolarizing effect of SST at resting membrane potential appears to be mainly carried by IK(L), whereas IM dominates at slightly depolarized potentials.     

The number of postsynaptic currents necessary to produce locomotor-related cyclic information in neurons in the neonatal rat spinal cord

To understand better how synaptic signaling contributes to network activity, we analyzed the potential contribution of putative unitary postsynaptic currents (PSCs) to locomotor-related information received by spinal interneurons in neonatal rats. The average cyclic modulation of the whole-cell current in 13 neurons was quantified as the difference between the current integral (charge) during the first and second halves of the cyclic locomotor network output. Between 7.6 and 303 average unitary PSCs per second were needed to produce the cyclic modulation. This number is so low that very few (1-5) of the synapses contributing to the cyclic information need to be active simultaneously. This suggests that individual presynaptic cells in a central locomotor network can have a powerful influence on other neurons.     

Zinc and flunitrazepam modulation of GABA-mediated currents in rat suprachiasmatic neurons

We know that upper body obesity is associated with metabolic complications, but we don't know how regional body fat distribution influences postprandial lipemia in obese adults. Thus, this study explored the respective effects of android or gynoid types of obesity and fasting triglyceridemia on postprandial lipid metabolism and especially triglyceride-rich lipoproteins. Twenty-four obese and 6 lean normotriglyceridemic women (control), age 24-57 yr, were enrolled. Among obese women with an android phenotype, 9 exhibited normal plasma triglyceride levels (mean: 1.38 mmol/L) (NTAO), and 7 displayed a frank hypertriglyceridemia (mean: 2.40 mmol/L) (HTAO). The 8 patients with a gynoid phenotype had normal triglyceride levels (mean: 1.00 mmol/L) (GO). All were given a mixed test meal providing 40 g triglycerides. Serum and incremental chylomicron triglycerides 0-7 h areas under the curve (AUCs) as well as triglyceride levels in apoB-48-containing triglyceride-rich lipoprotein (TRLs) or chylomicrons were significantly higher in HTAOs and NTAOs than in GOs and controls postprandially. The size of chylomicron particles was bigger in controls and GOs than in HTAOs and NTAOs postprandially. Android obese subjects showed abnormally elevated fasting apoB-48 and apoB-100 triglyceride-rich lipoprotein (TRL) levels. Most abnormalities that were found correlated to plasma levels of insulin and apoC-III. In conclusion, an abnormal postprandial lipid pattern is a trait of abdominal obesity even without fasting hypertriglyceridemia.     

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat

Trigeminal primary neuronal cell bodies were labeled by retrograde transport of Fluoro-gold (FG) from the nasal mucosa of rats. The trigeminal ganglion containing the labeled cell bodies were processed for double stain for calretinin- and tachykinin-immunoreactivities (CR- and TK-irs). Except for a few contralateral cells, all the cells that innervated the nasal mucosa (NM cells) were confined to the ophthalmo-maxillary division of the trigeminal ganglion ipsilateral to the FG application. In the dorsal two-thirds of the ganglion, NM cells formed a cluster in the rostromedial part of ophthalmo-maxillary division (the rostromedial cluster). In the ventral third, the number of cells in the rostromedial cluster markedly decreased. Instead, numerous NM cells were found in the caudolateral part of the ophthalmo-maxillary division (the caudoventrolateral cluster). CR- and TK-irs were detected in 18% and 54% of overall population of NM cells, respectively. Virtually all of CR-immunoreactive (-ir) NM cells coexpressed TK. Although the proportion of TK-ir cells, irrespective of CR-ir, was similar for both clusters, CR-ir cells were more frequent in the caudoventrolateral cluster than in the rostromedial cluster. In the dorsal 1/3 of the ganglion where all the NM cells belonged to the rostromedial cluster, only 8.4% exhibited CR-ir. On the other hand, as much as 30.1% of NM cells expressed CR-ir in the ventral 1/3 where most NM cells were found in the caudoventrolateral cluster. Trigeminal cell bodies innervating the cornea and conjunctivum were located in the rostromedial part of the ophthalmo-maxillary division.(ABSTRACT TRUNCATED AT 250 WORDS)     

RGS8 accelerates G-protein-mediated modulation of K+ currents

Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signalling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. Here we report the isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, and the determination of its function. We show that RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and that it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerated not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. We conclude that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors.     

KCR1, a membrane protein that facilitates functional expression of non-inactivating K+ currents associates with rat EAG voltage-dependent K+ channels

Cerebellar granule neurons possess a non-inactivating K+ current, which controls resting membrane potentials and modulates the firing rate by means of muscarinic agonists. kcr1 was cloned from the cerebellar cDNA library by suppression cloning. KCR1 is a novel protein with 12 putative transmembrane domains and enhances the functional expression of the cerebellar non-inactivating K+ current in Xenopus oocytes. KCR1 also accelerates the activation of rat EAG K+ channels expressed in Xenopus oocytes or in COS-7 cells. Far-Western blotting revealed that KCR1 and EAG proteins interacted with each other by means of their C-terminal regions. These results suggest that KCR1 is the regulatory component of non-inactivating K+ channels.