ABSTRACT
The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.
Subject(s)
Cations/metabolism , Chlorides/physiology , Receptors, Nicotinic/metabolism , Superior Cervical Ganglion/metabolism , Synapses/metabolism , Acetylcholine/pharmacology , Animals , Bungarotoxins/pharmacology , Cells, Cultured , Chlorides/metabolism , Chlorides/pharmacology , Electrophysiology , Ganglia/drug effects , Ganglia/metabolism , Ganglia/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurons/drug effects , Neurons/physiology , Patch-Clamp Techniques , Rats , Receptors, Nicotinic/physiology , Substrate Specificity , Superior Cervical Ganglion/physiology , Synapses/physiologyABSTRACT
BACKGROUND: The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). METHODS: Chloride secretion was stimulated via cAMP by forskolin (FSK, 10(-5) M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10(-4) M), and via protein kinase C by the phorbol ester PMA (5 x 10(-9) M). Stimulated Cl- secretion was quantified as short circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. RESULTS: Loperamide (5 x 10(-5) M) inhibited I(SC) stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10(-5) M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10(-4) M) inhibited I(SC) induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5 x 10(-5) M), on the other hand, inhibited only PMA-stimulated I(SC),but had no effect on FSK or CCh-induced I(SC) CONCLUSIONS: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.
Subject(s)
Antidiarrheals/pharmacology , Chlorides/physiology , Colon/cytology , Enterocytes/drug effects , Enterocytes/metabolism , HT29 Cells/drug effects , HT29 Cells/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Loperamide/pharmacology , Potassium Channels/drug effects , Calcium Channel Blockers/pharmacology , Calmodulin/antagonists & inhibitors , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Colforsin/pharmacology , Drug Evaluation, Preclinical , Humans , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Signal Transduction/drug effects , Time Factors , Trifluoperazine/pharmacology , Verapamil/pharmacologyABSTRACT
BACKGROUND & AIMS: When rice is incorporated into oral rehydration therapy for patients with secretory diarrhea, clinical outcomes improve. We have shown that a factor purified from boiled rice (RF) blocks the secretory response of intestinal crypt cells to adenosine 3',5'-cyclic monophosphate (cAMP). Now we report that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the cellular target for this rice inhibitor. METHODS: We used RF, the same previously described extract prepared from boiled rice, to assess chloride channel activation in vitro, measuring (1) cell volume regulation of guinea pig intestinal crypt epithelial cell suspensions using standard Coulter counter technology, (2) transepithelial chloride current in monolayers of T84 cells mounted in Ussing chambers, and (3) whole-cell and single-channel currents using the patch-clamp technique in cells transfected to express CFTR. RESULTS: RF inhibited activation by cAMP of CFTR chloride channels in all experimental preparations; RF did not block volume-stimulated Cl- secretion, suggesting that its effect might be specific for CFTR chloride channels. RF inhibited transepithelial cAMP-stimulated Cl- current in T84 cells and inhibited forskolin (i.e., cAMP)-induced current in cells transfected with CFTR. Excised patch and single-channel patch-clamp recordings supported the view that the response was a direct effect on CFTR rather than on cAMP signal transduction. CONCLUSIONS: RF exerts a specific inhibitory effect on CFTR chloride channels, blocking activation from the luminal surface of the cell and reversing established activation. Many major diarrheal states are based on cAMP-induced CFTR activation, leading to excessive gut secretion; our findings could have clinical relevance.
Subject(s)
Chlorides/metabolism , Cooking , Intestinal Mucosa/metabolism , Oryza , Animals , CHO Cells , Cell Line/drug effects , Cell Line/metabolism , Chlorides/physiology , Cricetinae , Cyclic AMP/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Electric Conductivity , Guinea Pigs , Humans , Intestines/cytology , Oryza/chemistry , Patch-Clamp Techniques , Plant Extracts/pharmacology , TransfectionABSTRACT
Influx of D-aspartate (D-Asp), L-glutamate (L-Glu), and serine (Ser) across the brush-border membrane of the intact mucosa from rabbit ileum has been examined. L-Glu influx is chloride independent and completely sodium dependent. D-Asp and L-Glu share a transport system with a maximum transport rate of 1 micromol. cm-2. h-1 and an apparent affinity constant (K1/2) of approximately 0.3 mM. The function of this transport system is pH insensitive between pH 5.65 and 8.2, and bipolar amino acids do not affect the way in which the transport system handles D-Asp and L-Glu. The characteristics of this transport system match those of system X-AG. L-Glu and Ser share a transporter for which the inhibitor constant (Ki) of L-Glu against Ser decreases from 54 to 10 mM when pH is reduced from 7.2 to 5.65, while the maximum rate of transport remains unaffected at approximately 10 micromol. cm-2. h-1. The Ki values (5 mM) of Ser against L-Glu influx and the L-Glu-sensitive contribution to Ser influx (0.8 micromol. cm-2. h-1 at 1 mM Ser) are the same at both pH values. The L-Glu-sensitive transport of Ser together with the contribution of system bo,+ account for approximately 50% of Ser influx at pH 7.2. The remaining 50% can be ascribed to system B. Transport of Ser by system B is reduced by >95% at pH 5.65. At pH 7. 2 Ki of Ser against transport of leucine (Leu) by system B is 18 mM and Ki of Leu against transport of Ser is 1.7 mM. The low-affinity transport of L-Glu and the L-Glu-sensitive transport of Ser are performed by an equivalent of system ASC. Supplementary experiments using the jejunum confirm the validity of these results for a major portion of the rabbit small intestine.
Subject(s)
Aspartic Acid/metabolism , Glutamic Acid/metabolism , Hydrogen/metabolism , Ileum/metabolism , Serine/metabolism , Amino Acid Transport Systems , Animals , Biological Transport/physiology , Carrier Proteins/metabolism , Chlorides/physiology , Female , Hydrogen-Ion Concentration , Jejunum/metabolism , Kinetics , Microvilli/metabolism , Rabbits , Sodium/physiologyABSTRACT
Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations =3 mM. In summary, the present data suggest that Ip does not reflect charge transfer related to Pi-uptake, but a Pi-mediated modulation of GCl.
Subject(s)
Carrier Proteins/biosynthesis , Carrier Proteins/physiology , Chloride Channels/metabolism , Ion Channel Gating/physiology , Oocytes/metabolism , Phosphates/metabolism , Symporters , Animals , Biological Transport/genetics , Carrier Proteins/genetics , Chlorides/physiology , Extracellular Space/chemistry , Gene Expression/drug effects , Microinjections , Nitrobenzoates/pharmacology , RNA, Complementary/pharmacology , Sodium/physiology , Sodium-Phosphate Cotransporter Proteins , XenopusABSTRACT
The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.
Subject(s)
Brain Injuries/pathology , Calcium/physiology , Chlorides/physiology , Dendrites/pathology , Sodium/physiology , Animals , Bridged Bicyclo Compounds/pharmacology , Calcium/analysis , Cells, Cultured , Chlorides/analysis , Coculture Techniques , Cytosol/chemistry , Dendrites/drug effects , Dendrites/ultrastructure , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Kainic Acid/pharmacology , Mice , Microscopy, Fluorescence , N-Methylaspartate/pharmacology , Quinoxalines/pharmacology , Receptors, Glutamate/physiology , Sodium/analysis , Veratridine/pharmacologyABSTRACT
Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.
Subject(s)
Chloride Channels/genetics , Nicotiana/genetics , Plants, Toxic , Amino Acid Sequence , Animals , Base Sequence , Chlorides/physiology , Cloning, Molecular , DNA Primers/chemistry , DNA, Complementary/genetics , Ion Channel Gating/physiology , Membrane Potentials , Molecular Sequence Data , Plant Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Xenopus laevisABSTRACT
We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/metabolism , Gene Transfer Techniques , Glycoconjugates/metabolism , Trachea/metabolism , Adenoviridae/genetics , Base Sequence , Chlorides/physiology , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , DNA, Complementary/metabolism , Electric Conductivity , Epithelium/metabolism , Epithelium/pathology , Genetic Vectors , Humans , Molecular Probes/genetics , Molecular Sequence Data , Protein Kinase C/metabolism , Trachea/pathologyABSTRACT
GABAergic inhibition synchronizes oscillatory activity in the thalamocortical system. To understand better the role of this neurotransmitter in generation of thalamocortical rhythmicity, the postnatal development of GABAergic function mediated through activation of GABAA receptors was studied in thalamus and cortex. GABA-evoked chloride currents were recorded in dissociated rat cortical and thalamic neurons during postnatal development. Kinetic fits of GABA concentration/response relationships revealed developmental and regional alterations in the potency of GABA. Early in postnatal development (p5-p8), both thalamic and cortical neurons exhibited reduced potency of GABA (27-31 microM KD). Potency increased by p18-p25 in thalamic and cortical neurons (19-22 microM KD), to a level maintained in adult thalamic neurons. Adult cortical neurons exhibited reduced potency of GABA (40 microM KD). Benzodiazepine modulation of GABAA currents was also studied. Kinetic analyses of benzodiazepine augmentation of GABAA currents were best fitted assuming two effective sites with different affinities for clonazepam. The high-affinity site (KD of 0.05-0.27 nM) showed little variation with development in cortical neurons, contributing about 16-23% potentiation at all postnatal ages. Developing thalamic neurons (p5-p25) showed similar potency and efficacy of the high-affinity benzodiazepine site to cortical neurons. High-affinity benzodiazepine effects disappeared in adult thalamic neurons. A lower-affinity benzodiazepine site (25-50 nM KD) was greater in efficacy in cortical neurons compared to thalamic neurons at all ages, with efficacy ranging from 50% to 110% in cortex and from 20% to 60% in thalamus. Knowledge of developmental and regional alterations in GABAA receptor function may aid in understanding mechanisms involved in generation and control of normal and pathological thalamocortical rhythms.
Subject(s)
Aging/metabolism , Neurons/metabolism , Receptors, GABA/metabolism , Somatosensory Cortex/metabolism , Thalamus/metabolism , Animals , Animals, Newborn , Cell Separation , Chlorides/physiology , Clonazepam/pharmacology , Electrophysiology , Neurons/physiology , Rats , Somatosensory Cortex/cytology , Somatosensory Cortex/physiology , Thalamus/cytology , Thalamus/physiologyABSTRACT
The properties of serotonin (5-HT)-activated Cl- receptor/ion channel complexes in neurons of the CNS of the medicinal leech were analyzed. These channels mediate the postsynaptic response at the serotoninergic Retzius P-cell as well as Retzius-Retzius cell synapses. 5-HT-induced Cl- currents were activated by fast superfusion of transmitter on cells dissociated from embryonic leeches including histochemically identified Retzius cells. Whole-cell currents elicited by partial superfusion of the membrane and summated single-channel currents of outside-out patches showed times to peak of 18 +/- 4 msec and 10 +/- 5 msec, respectively, and desensitized with time constants of 28 +/- 3 msec and 20 +/- 11 msec. Persistence of single-channel openings in the outside-out configuration as well as lack of effect of dialysis of the whole cell with AMP-PNP or GDP-beta-S indicated that 5-HT directly gates the Cl- channels without involving second messenger cascades. In outside-out patches, two single-channel conductances of 13 pS and 32 pS were identified. While the 13 pS conductance desensitized, the 32 pS conductance activated within several tens of msec and showed no desensitization. We postulate that two subtypes of channels are coactivated by 5-HT and that the activation of the fast desensitizing channel could be responsible for the fast decaying component of the post-synaptic response. The slow conductance explains the second slower decay time constant of the postsynaptic response and could account for the tonic component sometimes observed at Retzius P-cell synapses.
Subject(s)
Chlorides/physiology , Leeches/physiology , Neurons/physiology , Serotonin/pharmacology , Synapses/physiology , Animals , Chloride Channels/drug effects , Chloride Channels/physiology , Electric Conductivity , Ion Channel Gating , Kinetics , Leeches/embryologyABSTRACT
The effects of plant diterpenes, horminone (HMN) and taxodione (TXN), on the GABAA receptor-operated Cl-current (IGABA) were investigated in voltage-clamped and internally perfused neurones dissociated from frog dorsal root ganglia. Both diterpenes depressed IGABA in a concentration-dependent manner similar to that of picrotoxin. Concentrations required to elicit 50% depression of the IGABA were 10(-6) M for picrotoxin, 10(-5) M for HMN and 10(-4) M for TXN. Blocking and restoration kinetics of the IGABA with HMN were also similar to those of picrotoxin. Time constants for both the blockade and restoration of the IGABA with TXN were more than five times greater than those with HMN.
Subject(s)
Abietanes , Chlorides/physiology , Diterpenes/pharmacology , Neurons, Afferent/physiology , Receptors, GABA/physiology , Animals , Electric Conductivity , Ganglia, Spinal/cytology , Kinetics , Neurons, Afferent/drug effects , Osmolar Concentration , Picrotoxin/pharmacology , Plant Extracts/pharmacology , Rana catesbeiana , Receptors, GABA/classification , gamma-Aminobutyric Acid/pharmacologyABSTRACT
The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pHi), high-energy metabolites, and lactate was investigated using 31P and 1H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO3-/CO2 in the presence of Na+ led to a sustained split of the inorganic phosphate (Pi) peak so that the pHi indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO3-. The pH in the compartment with a higher pHi value returned to 7.29 +/- 0.04 by 10.5 min of superfusion in a HCO3(-)-free medium, whereas the pHi in an acidic compartment was reduced to 7.02. In the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or the absence of external Cl-, removal of HCO3- caused alkalinization without split of the Pi peak. Both treatments reduced the rate of pHi normalization following alkalinization. Simultaneous omission of external HCO3- and Na+ did not inhibit alkalinization of the pHi following CO2 exit. All these data show that the acid loading mechanism at neutral pHi is mediated by an Na(+)-independent anion transport. During severe hypoxia, pHi dropped from 7.29 +/- 0.05 to 6.13 +/- 0.16 and from 7.33 +/- 0.03 to 6.67 +/- 0.05 in the absence and presence of HCO3-, respectively, in Na(+)-containing medium. Lactate accumulated to 18.7 +/- 2.8 and 19.6 +/- 1.5 mmol/kg under the respective conditions. In the HCO3(-)-free medium supplemented with 1 mM amiloride, the pHi fell only to 6.94 +/- 0.08 despite the lactate concentration of 18.9 +/- 2.4 mmol/kg.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Bicarbonates/metabolism , Cerebral Cortex/metabolism , Chlorides/physiology , Extracellular Space/metabolism , Intracellular Membranes/metabolism , Magnetic Resonance Spectroscopy , Animals , Guinea Pigs , Hydrogen , Hydrogen-Ion Concentration , Hypoxia/metabolism , In Vitro Techniques , Phosphorus , Reference ValuesABSTRACT
Slow, rhythmic membrane potential (Vm) fluctuations occur spontaneously in cortical neurons of urethane-anesthetized rats, and likely underlie EEG activity in the same low-frequency (1-4 Hz or delta) range. Nucleus basalis (NB) stimulation elicits neocortical activation, simultaneously modifying Vm and EEG fluctuations, by way of cortical muscarinic ACh receptors (Metherate et al., 1992). To investigate the nature of spontaneous fluctuations and their modification by NB stimulation, we have obtained intracellular recordings from auditory cortex using the whole-cell recording technique in vivo. Spontaneous Vm fluctuations appeared to contain three components whose polarity and time course resembled the EPSP, putative Cl(-)-mediated IPSP, and putative K(+)-mediated, long-lasting IPSP elicited by thalamic stimulation. The spontaneous, long-lasting hyperpolarization, whose rhythmic occurrence appeared to set the slow-wave rhythm, was associated with an increased conductance that could shunt the thalamocortical EPSP. We hypothesized that spontaneous Vm fluctuations arise from intermixed rapid depolarizations, rapid Cl(-)-mediated hyperpolarizations, and long-lasting, K(+)-mediated hyperpolarizations. NB-mediated cortical activation might then result from muscarinic suppression of K+ permeability, allowing the rapid depolarizations and Cl- fluxes to continue uninterrupted. Tests of this hypothesis showed that (1) intracellular blockade of K+ channels by rapid diffusion of Cs+ from the recording pipette resulted in suppression of spontaneous, long-lasting hyperpolarizations, mimicking the effect of NB stimulation, and reducing shunting of the thalamocortical EPSP; (2) effects of Cs+ and NB stimulation suggested overlapping, or converging, mechanisms of action; however, there were important differential effects on the spontaneous, long-lasting hyperpolarizations and the K(+)-mediated IPSP; and (3) modifying Cl- fluxes with intracellular picrotoxin or high intracellular Cl- concentrations resulted in spontaneous and NB-elicited large-amplitude depolarizations. We conclude that spontaneous, long-lasting hyperpolarizations are K+ fluxes, but are not "spontaneous" K(+)-mediated IPSPs. Since NB-mediated reduction of spontaneous hyperpolarizations implies muscarinic suppression of a K+ conductance, the spontaneous hyperpolarizations more likely result from the calcium-activated K+ current, IK(Ca). Finally, Cl- fluxes form an important component of activated Vm fluctuations that acts to restrain excessive depolarization.
Subject(s)
Auditory Cortex/physiology , Substantia Innominata/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Cesium/pharmacology , Chlorides/physiology , Electric Stimulation , Electrophysiology , Evoked Potentials/physiology , Picrotoxin/pharmacology , Potassium/pharmacology , Potassium/physiology , Rats , Synapses/physiology , Thalamus/physiologyABSTRACT
Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.
Subject(s)
Chlorides/physiology , Membrane Proteins/genetics , Muscles/physiology , Amino Acid Sequence , Animals , Base Sequence , Chloride Channels , Cloning, Molecular , Gene Expression , Membrane Proteins/physiology , Molecular Sequence Data , Muscle Development , Oocytes/physiology , RNA, Messenger/genetics , Rats , Sequence Alignment , Xenopus laevisABSTRACT
1. The physiological and functional features of time-dependent anomalous rectification activated by hyperpolarization and the current which underlies it, Ih, were examined in guinea-pig and cat thalamocortical relay neurones using in vitro intracellular recording techniques in thalamic slices. 2. Hyperpolarization of the membrane from rest with a constant-current pulse resulted in time-dependent rectification, expressed as a depolarizing sag of the membrane potential back towards rest. Under voltage clamp conditions, hyperpolarizing steps to membrane potentials negative to approximately -60 mV were associated with the activation of a slow inward current, Ih, which showed no inactivation with time. 3. The activation curve of the conductance underlying Ih was obtained through analysis of tail currents and ranged from -60 to -90 mV, with half-activation occurring at -75 mV. The time course of activation of Ih was well fitted by a single-exponential function and was strongly voltage dependent, with time constants ranging from greater than 1-2 s at threshold to an average of 229 ms at -95 mV. The time course of de-activation was also described by a single-exponential function, was voltage dependent, and the time constant ranged from an average of 1000 ms at -80 mV to 347 ms at -55 mV. 4. Raising [K+]o from 2.5 to 7.5 mM enhanced, while decreasing [Na+]o from 153 to 26 mM reduced, the amplitude of Ih. In addition, reduction of [Na+]o slowed the rate of Ih activation. These results indicate that Ih is carried by both Na+ and K+ ions, which is consistent with the extrapolated reversal potential of -43 mV. Replacement of Cl- in the bathing medium with isethionate shifted the chloride equilibrium potential positive by approximately 30-70 mV, evoked an inward shift of the holding current at -50 mV, and resulted in a marked reduction of instantaneous currents as well as Ih, suggesting a non-specific blocking action of impermeable anions. 5. Local (2-10 mM in micropipette) or bath (1-2 mM) applications of Cs+ abolished Ih over the whole voltage range tested (-60 to -110 mV), with no consistent effects on instantaneous currents. Barium (1 mM, local; 0.3-0.5 mM, bath) evoked a steady inward current, reduced the amplitude of instantaneous currents, and had only weak suppressive effects on Ih. 6. Block of Ih with local application of Cs+ resulted in a hyperpolarization of the membrane from the resting level, a decrease in apparent membrane conductance, and a block of the slow after-hyperpolarization that appears upon termination of depolarizing membrane responses, indicating that Ih contributes substantially to the resting and active membrane properties of thalamocortical relay neurones.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Ion Channels/physiology , Neurons/physiology , Thalamus/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Cations/pharmacology , Cats , Chlorides/physiology , Female , Guinea Pigs , In Vitro Techniques , Male , Membrane Potentials/physiology , Potassium/physiology , Sodium/physiology , Thalamic Nuclei/physiology , Time FactorsABSTRACT
Potassium salts, often in the form of KCl, have had variable hypotensive effects in experimental animals and man. The chloride anion has recently been implicated as the hypertensive agent in salt-sensitive hypertension. We therefore hypothesized that non-chloride salts of potassium would have superior hypotensive effects to KCl. In randomized, double-blind fashion, we administered placebo, KCl (75 mmol/day) or potassium citrate (75 mmol/day) to 24 normotensive adult males. In these subjects, neither KCl nor potassium citrate affected blood pressure, despite urinary verification of potassium supplement intake. Thus, in these experimental circumstances, neither potassium nor its conjugate anion were of importance in lowering blood pressure.
Subject(s)
Blood Pressure/drug effects , Potassium/pharmacology , Adult , Analysis of Variance , Blood Pressure/physiology , Chlorides/physiology , Citrates/pharmacology , Citric Acid , Double-Blind Method , Humans , Male , Potassium Chloride/pharmacologyABSTRACT
The involvement of sodium and chloride ions in the process of alpha-melanocyte-stimulating hormone (a-MSH) release from hypothalamic neurons was investigated using perifused rat hypothalamic slices. Three different stimuli were found to increase a-MSH release from hypothalamic slices: high K+ concentration (50 mM), veratridine (50 microM), and the Na+/K(+)-ATPase inhibitor ouabain (1 mM). Spontaneous or K(+)-evoked a-MSH release was insensitive to the specific Na+ channel blocker tetrodotoxin (TTX; 1.5 microM) and to the blocker of K+ channels tetraethylammonium (TEA; 30 mM) or 4-aminopyridine (4-AP; 4 mM). In contrast, blockage of ouabain-sensitive Na+/K(+)-ATPase increased the resting level of a-MSH and caused a dramatic potentiation of K(+)-evoked a-MSH release. The Na+ channel activator veratridine (50 microM) triggered a-MSH release. This stimulatory effect was blocked by TTX and prolonged by TEA application, indicating the occurrence of voltage-sensitive Na+ and K+ channels on a-MSH neurons. Replacement of Na+ by impermeant choline ions from 95 to 60 mM did not alter K(+)-evoked a-MSH release. Conversely, dramatic reduction of the external Na+ concentration to 16 mM caused a robust increase of a-MSH secretion from hypothalamic neurons, likely through activation of the Na+/Ca2+ exchange system. These data indicate that the depolarizing effect of K+ results from direct activation of voltage-operated Ca2+ channels. The lack of effect of TEA on basal a-MSH release prompted us to investigate the possible involvement of chloride ions in the regulation of the spontaneous activity of a-MSH neurons. Substitution of Cl- for impermeant acetate ions did not affect basal or K(+)-evoked a-MSH release.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Hypothalamus/drug effects , Ion Channel Gating/drug effects , Ion Channels/physiology , alpha-MSH/metabolism , 4-Aminopyridine/pharmacology , Animals , Bicuculline/pharmacology , Calcium/physiology , Chlorides/physiology , Hypothalamus/metabolism , Ion Channels/drug effects , Male , Membrane Potentials/drug effects , Muscimol/pharmacology , Ouabain/pharmacology , Potassium/pharmacology , Rats , Rats, Inbred Strains , Secretory Rate/drug effects , Sodium/physiology , Tetraethylammonium , Tetraethylammonium Compounds/pharmacology , Tetrodotoxin/pharmacology , Veratridine/pharmacologyABSTRACT
While the molecular mechanism underlying triethyllead (TEL) neurotoxicity is unknown, we hypothesize that triethyllead mediates an accelerated Cl-/OH exchange across neuronal membranes leading to prolonged depolarization and neuronal cell injury. As a test of this hypothesis we have investigated the effect of external ion modulation on triethyllead neurotoxicity in cerebellar granule cell culture. Cultures were prepared from neonatal rats and used 10-20 days in vitro. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and trypan blue exclusion. A slow, dose-dependent (1-30 microM TEL) release of LDH occurred after a variable latent period dependent upon [TEL]. External replacement of [Cl-]e by Na isothionate dramatically shifted the dose response curve to the left reflecting an accelerated stimulation of LDH release, while replacement of extracellular [Na+]e with equimolar choline chloride had a minimal protective effect. Similarly, high [Mg2+]e or low [Ca2+]e did not protect or potentiate TEL cytotoxicity. The low [Cl-]e accelerated TEL cytotoxicity was dependent on medium pH: alkaline pH potentiated the cytotoxicity. Low [Cl-]e had no significant effect on culture ATP over 5 hrs. ATP reduction was markedly stimulated by TEL in low Cl- medium in contrast to the minimal decline in [ATP] in the control medium. The reduction of ATP in the low [Cl-]e medium occurred prior to LDH or trypan blue staining release confirming that such reduction in [ATP] was not secondary to cell damage. Substituting K sulfate or Na sulfate for the Cl(-)-free medium revealed marked loss of ATP without LDH release in control and TEL supplemented cultures. These observations provide supporting evidence for the role of an abnormal Cl- flux in mediating TEL-induced neurotoxic injury. Specifically, the membrane depolarization is proportional to the gradient imposed by Cl- efflux/OH influx, stimulated by low [Cl-]e. The rapid loss in ATP appeared early, was not a secondary reflection of neuronal damage but a result of a combination of increased ion flux at the plasma membrane, stimulation of Na+/K+ ATPase and direct TEL-induced inhibition of mitochondrial oxidative phosphorylation.
Subject(s)
Cerebellum/drug effects , Ions , Organometallic Compounds/toxicity , Adenosine Triphosphate/analysis , Animals , Calcium/physiology , Cell Survival/drug effects , Cells, Cultured , Cerebellum/cytology , Cerebellum/metabolism , Chlorides/physiology , L-Lactate Dehydrogenase/metabolism , Magnesium/physiology , Nerve Tissue Proteins/analysis , Potassium Cyanide/pharmacology , Rats , Rats, Inbred Strains , Sodium/physiologyABSTRACT
The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.
Subject(s)
Chemoreceptor Cells/physiology , Ion Channels/physiology , Nephropidae/physiology , Neurons/physiology , Animals , Calcium Channels/physiology , Chlorides/physiology , In Vitro Techniques , Potassium Channels/physiologyABSTRACT
1. Electrical and pharmacologic properties of glycine-induced currents were investigated in single hypothalamic neurons acutely isolated from young and adult rats by the use of a "concentration-clamp" technique, which allows both internal perfusion and rapid application of an external solution under single-electrode voltage-clamp. 2. The glycine-induced current reversed at the Cl- equilibrium potential (ECl), and a 10-fold decrease of extracellular Cl- with a large impermeable anion resulted in a 53 mV shift of the glycine reversal potential (EGly). 3. Glycine-induced Cl- currents (ICl) increased sigmoidally in a concentration-dependent manner with a Kd of 9 X 10(-5) M at a Hill coefficient of 1.8. Current inactivation occurred completely at all concentrations within 10 s. EGly remained unchanged during continuous application of glycine, suggesting that the inactivation process is because of desensitization. 4. The glycine-induced conductance exhibited a striking voltage dependency at membrane potentials more negative than -50 mV and reached a steady state value when hyperpolarized beyond -110 mV. 5. Both the activation and inactivation phases of glycine-induced ICl are described by double exponential (fast and slow components) functions with the concentrations used. All four time constants decreased with increasing glycine concentration. 6. The slow time constant of the current decay induced by glycine increased with depolarization and decreased with hyperpolarization, indicating that the rate of desensitization is considerably voltage dependent. The fast decay showed little voltage dependency. 7. Recovery of the glycine response after complete desensitization consisted of two components. 8. The blockade of the glycine response by strychnine and picrotoxin was noncompetitive.