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Membrane Currents (membrane + current)
Selected AbstractsInhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to EthanolALCOHOLISM, Issue 7 2008Seung-Yeon Yoo Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source] NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATPBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2003Brigitte Pelzmann The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36,37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP-dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 ,g ml,1 NADH (4,6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF,1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg,1 protein versus control: 35.4±2.57 nmol mg,1 protein, P<0.000005). These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP. British Journal of Pharmacology (2003) 139, 749,754. doi:10.1038/sj.bjp.0705300 [source] 5-hydroxytryptamine-mediated increase in glutamate uptake by the leech giant glial cellGLIA, Issue 8 2006Ingolf C. Hirth Abstract The clearance of synaptically released glutamate is one of the pivotal functions of glial cells. We have studied the role of 5-hydroxytryptamine (5-HT, 30 ,M), a neurotransmitter and neurohormone in the leech central nervous system with a versatile action spectrum, on the efficacy of glial glutamate uptake. The activity of the glutamate uptake carrier in the giant glial cell in isolated ganglia of Hirudo medicinalis was monitored by measuring the membrane current and the change in the intracellular Na+ concentration (Na+i) as induced by the glutamate carrier substrate D -aspartate (D -asp, 1 mM). 5-HT increased the D -asp-induced current (EC50 at 5 ,M) and rise in Na+i, an effect which was mimicked by the membrane-permeable cyclic nucleotide analogue dibutyryl-cyclic AMP (db-cAMP). The adenylyl cyclase inhibitor SQ 22,536 and the protein kinase A antagonist Rp-cAMP inhibited the effect of 5-HT. Blocking the G protein in the giant glial cell by injecting GDP-,-S suppressed the effect of 5-HT, but not the effect of db-cAMP, on the D -asp-induced current. Our results suggest that 5-HT enhances the glial uptake of glutamate via cAMP- and PKA-mediated pathway. © 2006 Wiley-Liss, Inc. [source] Changes in extracellular K+ concentration modulate contractility of rat and rabbit cardiac myocytes via the inward rectifier K+ current IK1THE JOURNAL OF PHYSIOLOGY, Issue 3 2004Ron Bouchard The mechanisms underlying the inotropic effect of reductions in [K+]o were studied using recordings of membrane potential, membrane current, cell shortening and [Ca2+]i in single, isolated cardiac myocytes. Three types of mammalian myocytes were chosen, based on differences in the current density and intrinsic voltage dependence of the inwardly rectifying background K+ current IK1 in each cell type. Rabbit ventricular myocytes had a relatively large IK1 with a prominent negative slope conductance whereas rabbit atrial cells expressed much smaller IK1, with little or no negative slope conductance. IK1 in rat ventricle was intermediate in both current density and slope conductance. Action potential duration is relatively short in both rabbit atrial and rat ventricular myocytes, and consequently both cell types spend much of the duty cycle at or near the resting membrane potential. Rapid increases or decreases of [K+]o elicited significantly different inotropic effects in rat and rabbit atrial and ventricular myocytes. Voltage-clamp and current-clamp experiments showed that the effects on cell shortening and [Ca2+]i following changes in [K+]o were primarily the result of the effects of alterations in IK1, which changed resting membrane potential and action potential waveform. This in turn differentially altered the balance of Ca2+ efflux via the sarcolemmal Na+,Ca2+ exchanger, Ca2+ influx via voltage-dependant Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+ release in each cell type. These results support the hypothesis that the inotropic effect of alterations of [K+]o in the heart is due to significant non-linear changes in the current,voltage relation for IK1 and the resulting modulation of the resting membrane potential and action potential waveform. [source] Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2001Carlos Cepeda Abstract Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N -methyl- d -aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders. [source] Role of Ca2+ -Activated Cl, Current in Ventricular Action Potentials of Sheep During Adrenoceptor StimulationEXPERIMENTAL PHYSIOLOGY, Issue 2 2001Arie O. Verkerk Adrenoceptor stimulation enhances repolarising and depolarising membrane currents to different extents in cardiac myocytes. We investigated the opposing effects of the repolarising Ca2+ -activated Cl, current (ICl(Ca)) and depolarising L-type Ca2+ current (ICa,L) on the action potential configuration of sheep ventricular myocytes stimulated with noradrenaline. Whole-cell current-clamp recordings revealed that noradrenaline accelerated and prolonged phase-1 repolarisation. We define the minimal potential at the end of phase-1 repolarisation as ,notch level'. Noradrenaline (1 ,M) caused the notch level to fall from 14 ± 2.6 to 7.8 ± 2.8 mV (n= 24), but left action potential duration, resting membrane potential or action potential amplitude unaffected. Whole-cell voltage-clamp recordings showed that 1 ,M noradrenaline increased both ICa,L and ICl(Ca), but it had no significant effect on the principal K+ currents. Blockage of ICl(Ca) by 0.5 mM 4,4,-diisothiocyanatostilbene-2,2,-disulphonic acid (DIDS) in both the absence and the presence of noradrenaline abolished phase-1 repolarisation. In the presence of noradrenaline, DIDS caused elevation of the plateau phase amplitude and an increase in the action potential duration. In conclusion, elevation of the plateau phase amplitude and action potential prolongation associated with an increased ICa,L upon adrenoceptor stimulation is prevented by an increased ICl(Ca) in sheep ventricular myocytes. [source] Altered functional properties of satellite glial cells in compressed spinal gangliaGLIA, Issue 15 2009Haijun Zhang Abstract The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low-back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole-cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium-sized neurons compared to non-nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state. © 2009 Wiley-Liss, Inc. [source] Block of HERG-Carried K+ Currents by the New Repolarization Delaying Agent H 345/52JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2003Gregory J. Amos M.D. Ph.D. Introduction: The aim of this study was to analyze the block of HERG-carried membrane currents caused by H 345/52, a new antiarrhythmic compound with low proarrhythmic activity, in transfected mouse fibroblasts. Methods and Results: Using the whole-cell configuration of the voltage patch clamp technique, it was demonstrated that H 345/52 concentration-dependently blocked HERG-carried currents with an IC50 of 230 nM. H 345/52 preferentially bound to the open channel with unusually rapid kinetics and was trapped by channel closure. Voltage-independent behavior of H 345/52 was observed during both square-pulse and action potential clamp protocols. In contrast, the Class III agents dofetilide (10 nM) and almokalant (250 nM) demonstrated significant membrane potential-dependent effects during square-pulse clamp protocols. When using action potential clamp protocols, voltage dependence was seen with dofetilide but not with almokalant. Mathematical simulations of human ventricular action potentials predicted that the different voltage-dependent behaviors would not produce marked variations in action potential duration prolongation patterns. Conclusion: We propose that block of IKr is the principal mechanism by which H 345/52 delays repolarization in human myocardium. The voltage independence of HERG/IKr block is unlikely to underlie the low proarrhythmic potential, and ancillary effects on other membrane currents must be considered. (J Cardiovasc Electrophysiol, Vol. 14, pp. 651-658, June 2003) [source] Changes in Left Ventricular Repolarization and Ion Channel Currents Following a Transient Rate Increase Superimposed on Bradycardia in Anesthetized DogsJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2000MICHAEL RUBART M.D. Electrical Remodeling of the Heart due to Rate. Introduction: We previously demonstrated in dogs that a transient rate increase superimposed on bradycardia causes prolongation of ventricular refractoriness that persists for hours after resumption of bradycardia. In this study, we examined changes in membrane currents that are associated with this phenomenon. Methods and Results: The whole cell, patch clamp technique was used to record transmembrane voltages and currents, respectively, in single mid-myocardial left ventricular myocytes from dogs with 1 week of complete AV block; dogs either underwent 1 hour of left ventricular pacing at 120 beats/min or did not undergo pacing. Pacing significantly heightened mean phase 1 and peak plateau amplitudes by ,6 and ,3 mV, respectively (P < 0.02). and prolonged action potential duration at 90% repolarization from 235 ± 8 msec to 278 ± 8 msec (1 Hz; P = 0.02). Rapid pacing-induced changes in transmembrane ionic currents included (1) a more pronounced cumulative inactivation of the 4-aminopyridine-sensitive transient outward K+ current, I to over the range of physiologic frequencies, resulting from a ,30% decrease in the population of quickly reactivating channels; (2) increases in peak density of L-type Ca2+ currents, Ica.I.' by 15% to 35% between +10 and +60 mV; and (3) increases in peak density of the Ca2+ -activated chloride current, ICl.Ca' by 30% to 120% between +30 and +50 mV. Conclusion: Frequency-dependent reduction in Ito combined with enhanced ICa.I. causes an increase in net inward current that may he responsible for the observed changes in ventricular repolarization. This augmentation of net cation influx is partially antagonized by an increase in outward ICa.Cl. [source] Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2009Lauri M. Louhivuori TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67,74, 2009. © 2009 Wiley-Liss, Inc [source] Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathwayJOURNAL OF PINEAL RESEARCH, Issue 3 2001Cirstin Blumenau Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1,100 ,mol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P<0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway. [source] A modelling study of locomotion-induced hyperpolarization of voltage threshold in cat lumbar motoneuronesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2002Yue Dai During fictive locomotion the excitability of adult cat lumbar motoneurones is increased by a reduction (a mean hyperpolarization of ,6.0 mV) of voltage threshold (Vth) for action potential (AP) initiation that is accompanied by only small changes in AP height and width. Further examination of the experimental data in the present study confirms that Vth lowering is present to a similar degree in both the hyperpolarized and depolarized portions of the locomotor step cycle. This indicates that Vth reduction is a modulation of motoneurone membrane currents throughout the locomotor state rather than being related to the phasic synaptic input within the locomotor cycle. Potential ionic mechanisms of this locomotor-state-dependent increase in excitability were examined using three five-compartment models of the motoneurone innervating slow, fast fatigue resistant and fast fatigable muscle fibres. Passive and active membrane conductances were set to produce input resistance, rheobase, afterhyperpolarization (AHP) and membrane time constant values similar to those measured in adult cat motoneurones in non-locomoting conditions. The parameters of 10 membrane conductances were then individually altered in an attempt to replicate the hyperpolarization of Vth that occurs in decerebrate cats during fictive locomotion. The goal was to find conductance changes that could produce a greater than 3 mV hyperpolarization of Vth with only small changes in AP height (< 3 mV) and width (< 1.2 ms). Vth reduction without large changes in AP shape could be produced either by increasing fast sodium current or by reducing delayed rectifier potassium current. The most effective Vth reductions were achieved by either increasing the conductance of fast sodium channels or by hyperpolarizing the voltage dependency of their activation. These changes were particularly effective when localized to the initial segment. Reducing the conductance of delayed rectifier channels or depolarizing their activation produced similar but smaller changes in Vth. Changes in current underlying the AHP, the persistent Na+ current, three Ca2+ currents, the ,h' mixed cation current, the ,A' potassium current and the leak current were either ineffective in reducing Vth or also produced gross changes in the AP. It is suggested that the increased excitability of motoneurones during locomotion could be readily accomplished by hyperpolarizing the voltage dependency of fast sodium channels in the axon hillock by a hitherto unknown neuromodulatory action. [source] Regulation of glucagon release in mouse ,-cells by KATP channels and inactivation of TTX-sensitive Na+ channelsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2000S. O. Göpel 1The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting ,-cells in intact mouse pancreatic islets. 2,-cells were distinguished from the ,- and ,-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. 3The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at ,45, ,47 and ,68 mV, respectively. 4Pancreatic ,-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0·1 mm) evoked a brief period of electrical activity followed by a depolarisation to a plateau of ,30 mV with no regenerative electrical activity. 5Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mm glucose, concentrations up to 2 ,m stimulated glucagon secretion to the same extent as removal of glucose. 6We conclude that electrical activity and secretion in the ,-cells is dependent on the generation of Na+ -dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the ,-cell with resultant inactivation of the ion channels participating in action potential generation. [source] Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMPTHE JOURNAL OF SEXUAL MEDICINE, Issue 4 2009Gerard P. Sergeant PhD ABSTRACT Introduction., Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca2+ level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca2+ signals regulate tone, and erection occurs as a result of inhibition of Ca2+ oscillations by NO. Aims., The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca2+ oscillations and, if so, whether these were regulated by NO. Methods., Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca2+ (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca2+. Main Outcome Measure., Cytosolic Ca2+ was measured in isolated smooth muscle cells and tissue slices. Results., Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca2+ waves that spread at a mean velocity of 65 µm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca2+ waves was associated with an inward current typical of the Ca2+ -activated Cl - currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca2+ store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP3 -mediated Ca2+ release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca2+ oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. Conclusions., These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO. Sergeant GP, Craven M, Hollywood MA, McHale NG, and Thornbury KD. Spontaneous Ca2+ waves in rabbit corpus cavernosum: Modulation by nitric oxide and cGMP. J Sex Med **;**:**,**. [source] Electrophysiological classification of P2X7 receptors in rat cultured neocortical astrogliaBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2010W Nörenberg Background and purpose:, P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. Experimental approach:, We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial,neuronal co-cultures prepared from the cerebral cortex of rats. Key results:, All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [,,,-methylene ATP and 2,-3,-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5,-O-(2-thiodiphosphate), uridine 5,-diphosphate, uridine 5,-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. Conclusions and implications:, Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury. [source] Multiple effects of mefenamic acid on K+ currents in smooth muscle cells from pig proximal urethraBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2003N Teramoto The effects of mefenamic acid on both membrane potential and K+ currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 ,M glibenclamide. In the voltage-clamp mode, mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300 nM) at ,50 mV. ATP-sensitive K+ channels (KATP channels) could be activated in the same patch by mefenamic acid and levcromakalim, with the same unitary amplitude and the similar opening gating at ,50 mV in cell-attached configuration. In outside-out recording, external application of mefenamic acid activated intracellular Ca2+ -activated IbTX-sensitive large-conductance K+ channels (BKCa channels). Mefenamic acid (30 ,M) activated spontaneous transient outward currents (STOCs). In contrast, mefenamic acid (100 ,M) increased sustained outward currents, diminishing the activity of STOCs. Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 ,M glibenclamide. In the presence of 10 mM 4-aminopyridine (4-AP), mefenamic acid only increased the outward currents. These results indicate that mefenamic acid increases the channel activities of two distinct types of K+ channels (i.e. BKCa channels and KATP channels) and decreased 4-AP-sensitive K+ channels in pig urethral myocytes. British Journal of Pharmacology (2003) 140, 1341,1350. doi:10.1038/sj.bjp.0705524 [source] Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2003Li-Ping He We have investigated the effects of loperamide on intracellular Ca2+ stores and membrane K+ channels in insulin-secreting hamster insulinoma (HIT-T15) cells. In cell-attached patch-clamp mode, loperamide (3,250 ,M) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca2+ -activated K+ channel (KCa) currents based on their single-channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single-channel currents with a conductance (32 pS) indicative of adenosine triphosphate-sensitive K+ channels (KATP channels) were also recorded, but were insensitive to loperamide. Surprisingly, the loperamide-activated currents persisted in the absence of extracellular Ca2+. Yet under these conditions, we still measured loperamide-induced Ca2+ increases. These effects are dose dependent. Loperamide had no effects in the inside-out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca2+ from intracellular stores. Carbachol (100 ,M), an agonist of muscarinic receptors, which mediates IP3 -dependent intracellular Ca2+ release, enhanced the effects of loperamide on KCa channels. Both the putative KCa currents and Ca2+ signals induced by loperamide (with ,0' [Ca2+]o) were abolished when the intracellular Ca2+ stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca2+ -ATPase inhibitor that blocks reuptake of calcium. These data indicate that loperamide in insulin-secreting , -cells evokes intracellular Ca2+ release from IP3 -gated stores and activates membrane currents that appear to be carried by KCa, rather than KATP channels. British Journal of Pharmacology (2003) 139, 351,361. doi:10.1038/sj.bjp.0705263 [source] ELECTROPHYSIOLOGICAL EFFECTS OF KETAMINE ON HUMAN ATRIAL MYOCYTES AT THERAPEUTICALLY RELEVANT CONCENTRATIONSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2008Chun-Yu Deng SUMMARY 1Ketamine is widely used for the induction of anaesthesia in high-risk patients with cardiovascular instability or severe hypovolaemia. However, the ionic mechanisms involved in the effects of ketamine at therapeutically relevant concentrations in human cardiac myocytes are unclear. The present study was designed to investigate the effects of ketamine on L-type Ca2+ (ICa), transient outward K+ (Ito), ultra-rapid delayed rectifier K+ (IKur) and inward rectifier potassium (IK1) currents, as well as on action potentials, in human isolated atrial myocytes. 2Atrial myocytes were isolated enzymatically from specimens of human atrial appendage obtained from patients undergoing coronary artery bypass grafting. The action potential and membrane currents were recorded in both current- and voltage-clamp modes using the patch-clamp technique. 3Ketamine inhibited ICa with an IC50 of 1.8 µmol/L. In addition, 10 µmol/L ketamine decreased the ICa peak current at +10 mV from 5.1 ± 0.3 to 2.1 ± 0.4 pA/pF (P < 0.01), but did not change the threshold potential, peak current potential and reverse potential. 4Ketamine had no effect on Ito, IKur or IK1, but it reversibly shortened the duration of the action potential in human atrial myocytes. 5In conclusion, ketamine, at a clinically relevant concentration, shortens the action potential duration of the human atrial myocytes, probably by inhibiting ICa. [source] | |||||||||||||