Ion-selective Microelectrode Technique Research Articles

Intracellular chloride activity increases in guinea pig ventricular muscle during simulated ischemia.

We investigated the effects of simulated ischemia on intracellular Cl- activity ([Cl-]i) in isolated guinea pig ventricular papillary muscles using ion-selective microelectrode techniques. Simulated ischemia in ventricular muscles was produced by stopping the flow of superfusion and immersing preparations in mineral oil as previously described [B. Vanheel, L. Leybaert, A. De Hemptinne, and I. Leusen. Am. J. Physiol. 257 (Cell Physiol. 26): C365-C379, 1989; Z. F. Lai, J. Liu, and K. Nishi. Jpn. J. Pharmacol. 72: 161-174, 1996]. When preparations were exposed to paraffin oil for 15 min, [Cl-]i markedly increased and the peak magnitude of [Cl-]i reached 55.3 +/- 2.5 mM from 18.7 +/- 3.5 mM, whereas membrane potentials (Vm) depolarized from -82.5 +/- 1.1 to -54.7 +/- 2.4 mV (n = 6 muscles from 6 animals). SITS (0.5 mM), a known blocker of the Cl-/HCO-3 exchanger, suppressed the ischemia-induced depolarization of Vm and delayed the onset of the ischemia-induced increase in [Cl-]i but did not suppress the magnitude of the increase of [Cl-]i. Under Cl--free conditions created by replacing Cl- with equimolar gluconate, the increase in [Cl-]i during ischemia was transient and suppressed by >60% compared with that in normal-Cl- conditions (peak value was 20. 3 +/- 1.7 mM, n = 6 muscles from 6 animals). The present results provide direct evidence that [Cl-]i in ventricular muscle increases in ischemic conditions in quiescent guinea pig ventricular muscle, suggesting that activation of the Cl-/HCO-3 exchanger by ischemia would partially contribute to the elevation of [Cl-]i during the initial stage of ischemia.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine.

Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J. Neurophysiol. 78: 2582-2591, 1997. Ion-selective (H+ and K+) microelectrode techniques as well as conventional extra- and intracellular recordings were used to study the ionic mechanisms of propagating spontaneous GABAergic events (SGEs) in rat hippocampal slices exposed to 4-aminopyridine (4-AP, 50-100 mu M). All experiments were made in the presence of antagonists of ionotropic glutamate receptors [10 mu M 6-nitro-7-sulphamoylbenzoquinoxaline-2,3-dione (NBQX) and 40 mu M -2-amino-5-phosphonopentanoic acid (AP5)]. The SGEs were composed of a negative-going change in field potential with a temporally coincident increase (0.7 +/- 0.3 mM; mean +/- SE) in extracellular K+ ([K+]o) and an alkaline transient (0.01-0.08 units) in extracellular pH (pHo) in stratum radiatum of the area CA1. Simultaneous intracellular recordings showed a triphasic hyperpolarization-depolarization-late hyperpolarization response in pyramidal cells. Application of pentobarbital sodium (PB, 100 mu M) decreased the interval between SGEs from a mean value of 35 to approximately 20 s and shortened the period of refractoriness of stimulus-evoked propagating events. This was accompanied by an increase in the amplitude of the field potential response of the [K+]o and the pHo shifts and of the depolarizing phase of the pyramidal-cell response. The SGEs were completely blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist, picrotoxin (PiTX; 100 mu M). The amplitudes of the negative-going field potential and of the depolarizing phase of the pyramidal-cell response as well as the ionic shifts associated with SGEs were strongly suppressed in the nominal absence of CO2/HCO-3. There was a five-fold increase in the interevent interval, and propagating SGEs could not be evoked by stimuli given at intervals shorter than approximately 2-3 min. Exposure to inhibitors of carbonic anhydrase, benzolamide (BA; 10 micro M) or ethoxyzolamide (EZA; 50 mu M) fully blocked the alkaline pHo transients and turned them into acid shifts. The poorly membrane-permeant BA had no discernible effect on the other components of the SGEs, but application of EZA had effects reminiscent to those of CO2/HCO-3-free medium. Addition of the GABAA receptor-permeant weak-acid anion, formate (20 mM) reestablished the SGEs that were first suppressed by exposure to the CO2/HCO-3-free medium. No SGEs were seen in the presence of a similar concentration of the GABAA receptor-impermeant anion propionate. Unlike the alkaline transients associated with HCO-3-driven SGEs, those supported by formate were not blocked by BA. The present data suggest that an inward current carried by bicarbonate is necessary for the generation of SGEs and that the GABAA receptor-mediated excitatory coupling among GABAergic interneurons is essentially dependent on the availability of intracellular bicarbonate.

Effects of Stilbene Derivatives SITS and DIDS on Development of Intracellular Acidosis during Ischemia in Isolated Guinea Pig Ventricular Papillary Muscle In Vitro

Using ion-selective microelectrode techniques, we investigated the effects of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are known as Cl−-HCO3− exchange blockers, on action potentials and intracellular pH (pHi) in guinea pig ventricular papillary muscles subjected to simulated ischemia. Simulated ischemia was produced by stopping the flow of superfusing solution and then covering the preparations with mineral oil. Simulated ischemia induced a progressive decrease in the maximum upstroke rate and resting membrane potentials, shortened action potential duration, and resulted in cessation of action potentials within 10–12 min after the onset of simulated ischemia. The pHi-measurements revealed progressive intracellular acidosis during the period of simulated ischemia. SITS (0.5 mM) or DIDS (0.1 mM) delayed the onset of ischemia-induced deterioration of action potentials and prolonged the time to cessation of action potentials. SITS or DIDS (0.1–0.5 mM) induced an increase in pH in HCO3−-buffered solution and suppressed the development of intracellular acidosis during ischemia. Under the external CP-free condition, the time to cessation of action potentials caused by ischemia was significantly delayed, and the development of intracellular acidosis during ischemia was attenuated. The present results indicate that activation of the Cl−-HCO3− exchange system would be involved, in part, in the development of intracellular acidosis during cardiac ischemia.

Protection of isolated rat heart against the Ca 2+ paradox. Are gap junction channels involved?

Enzyme release from isolated Langendorff-perfused rat hearts was studied under various protective conditions against the Ca2+ paradox. In addition sarcosolic free cation concentrations and the membrane potential were measured employing ion-selective microelectrode techniques during Ca(2+)-free perfusion. Low temperature (18 degrees C), low pH (6.5 or 6.1), and polyethylene glycol (9%) during Ca(2+)-free perfusion all protected isolated hearts against the Ca2+ paradox. Protection could only be afforded if the protective agent was continuously present from the beginning of the Ca(2+)-free perfusion period. A 10 min "normal" Ca(2+)-free pre-perfusion was sufficient to abolish the protective potency of the subsequent perfusion phase in the presence of the protective agent. The gap junction channel blocker heptanol (2 mmol/l) markedly decreased enzyme release during re-perfusion, but did not afford protection. Sarcosolic free cation concentrations were measured during Ca(2+)-free acidic perfusion. [Na+]i was markedly increased to about 44 mmol/l without predisposing to cell damage under these conditions. A marked reduction of cell damage was also afforded under conditions of hypoxia during Ca(2+)- and substrate-free perfusion. Acidosis (pHe = 6.5) under these conditions prevented a lethal increase of [Ca2+]i (2 mumol/l) and partially preserved a negative membrane potential. It is concluded that the predisposition to the Ca2+ paradox is produced by a permeabilisation of gap junction channels at low [Ca2+]e and that upon re-elevation of [Ca2+]e a serious Ca2+ influx proceeds through these leaks.

Vasopressin V1-Receptor Stimulation Produces a Positive Inotropic Response without Affecting pHi in Guinea Pig Papillary Muscles

Effects of arginine-vasopressin (AVP) on the contractile force, action potential (AP) and intracellular pH (pHj) were studied in isolated guinea pig papillary muscles using conventional and ion-selective microelectrode techniques. AVP increased the developed tension and the resting tension, and these responses were attenuated by the V1-receptor antagonist OPC-21268 (1-{1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-Quinolinone). However, AVP failed to affect AP configuration or pH^ These results suggest that AVP produces a positive inotropy by mechanism(s) other than intracellular alkalinization.

Proximal tubular cell alkalinization induced by Na(+)-glucose cotransport.

To study the ionic mechanisms of crosstalk between luminal and peritubular membranes induced by Na(+)-glucose cotransport in the proximal tubular cells, we applied the ion-selective microelectrode techniques and measured intracellular ion activities of Na+ (Nai) and H+ (pHi), as well as peritubular membrane potential (EM) in doubly-perfused bullfrog kidney. In control conditions with peritubular HCO3- (15 mM), luminal perfusion with 10 mM D-glucose induced a 12 mV depolarization of peritubular membrane (glucose-induced depolarization) with a transient increase in Nai by 3 mM and an elevation in pHi by 0.1. In the absence of peritubular HCO3- (HEPES-Ringer at constant pH), the luminal glucose produced a sustained increase in Nai and suppression of pHi elevation without affecting glucose-induced depolarization. Peritubular perfusion with DIDS (10(-4) M) abolished the change in pHi induced by luminal glucose. Furthermore, the luminal glucose increased the transport number for K+ (gK/gm) in the peritubular membrane by 20%. These results suggest that 1) glucose-induced depolarization suppresses peritubular HCO3- exit, followed by an elevation in pHi, 2) the luminal glucose increases K+ conductance of the peritubular membrane, probably by the elevation in pHi, and 3) this increased K+ conductance repolarizes the peritubular membrane, which can support the driving force for Na(+)-glucose entry across the luminal membrane. Thus, we conclude that the crosstalk between peritubular and luminal membranes by Na(+)-glucose cotransport is mainly modulated by changes in cell pH and membrane potential in the proximal tubule.

Acid-induced two-step depolarization in the peritubular membrane of bullfrog kidney proximal tubules.

Using ion-selective microelectrode technique, we investigated acid-induced changes of peritubular membrane potential (EM) and intracellular activity of Na+ and H+ ((Na)i and pHi) in the proximal tubule of perfused bullfrog kidney in vivo. When peritubular pH was reduced at constant PCO2, EM was depolarized in two steps: i.e., an initial sharp fall followed by an additional deeper fall. This was termed as the acid-induced two-step depolarization. During this change, pHi was decreased gradually from 7.4 to 6.9 in response to 1/10 HCO3- reduction (pH from 7.7 to 6.7), whereas (Na)i was increased after a transient decrease. This result supports the peritubular rheogenic HCO3- exit coupled to Na+ movement during the initial depolarization period. Complete removal of peritubular HCO3- at constant pH produced a less marked initial depolarization with a transient rise of pHi, followed by a partial repolarization without appreciable change in (Na)i. Peritubular SITS administration inhibited these depolarization responses to the acid perfusion. The above findings suggest that 1) the initial part of the two-step EM depolarization produced by low HCO3- or HCO(3-)-free perfusion resulted mainly from the peritubular rheogenic exist of HCO3-, 2) the magnitude of initial depolarization would also be affected by pHi, and 3) the subsequent delayed changes of EM were mainly determined by the pHi-sensitive K+ conductance of the peritubular membrane.

Utilization of the Vibrating Probe and Ion-Selective Microelectrode Techniques to Investigate Electrophysiological Responses to Wounding in Pea Roots

This paper examines the ionic composition of wound-induced electrical currents in higher plant tissue, using two non-injurious electrophysiological techniques. By simultaneous recording of K + , H + , and Ca2+ ion fluxes with extracellular ion-selective microelectrodes, we have determined that a Ca2+ influx (2-4 fzA cm-2), a small H+ influx (017 /zA cm2) and a large K+ efflux (16 ft A cm2) occur immediately after wounding in roots of Pisum sativum L. var. Greenfeast. Using an extracellular vibrating probe at the wound site, net ion currents of 26 fiA cm-2 were measured 5 min after wounding. In a more concentrated bathing medium (1/4 rather than 1/16 strength Hoagland's solution), net ion currents of 59 ftA cm 2 were measured, and these would appear to be the largest extracellular currents that have been measured in plants. We made a quantitative comparison of the summed ion fluxes with the net ion currents and this revealed that ion fluxes, in addition to those measured here, occur after wounding.

Transmembrane action potentials and intracellular potassium activity of baboon cardiac tissues

Preparations of baboon cardiac tissue were studied using standard microelectrode and double barrelled potassium ion selective microelectrode techniques. Transmembrane action potentials were recorded from Purkinje fibres and from atrial and ventricular muscle cells. Purkinje fibres were impaled frequently on the ventricular septum but were sparse on the endocardium of the free wall. Purkinje fibres had longer action potential durations than ventricular muscle cells, and endocardial muscle cells had longer action potential durations than epicardial muscle cells. Tissue bundles resembling the false tendons of the canine heart were found in the ventricles. Most of these false tendons consisted of ventricular muscle as indicated by transmembrane action potentials. On rare occasions, false tendon preparations were studied in which the cells had action potentials similar to those of canine Purkinje fibres. In these preparations, normal automaticity occurred from maximum diastolic potentials greater than or equal to -85 mV. Intracellular potassium activity in the baboon ventricular muscle cells was 96(3) mmol.litre-1 (mean(SEM), n = 32), which is lower than that reported for canine tissues but higher than that reported for feline or rabbit tissues. It is concluded that, although the cellular electrophysiology of primate and canine cardiac tissues is superficially similar, consistent and perhaps important differences do occur.

Extracellular K + accumulation during penicillin-induced epileptogenesis in the CA 3 region of immature rat hippocampus

Ion-selective microelectrode techniques were used to study extracellular K + changes associated with penicillin-induced epileptogenesis in the CA 3 region of hippocampal slices. Recordings were made in slices taken from rats 9–16 days of age, which have a pronounced capacty to undergo prolonged synchronized afterdischarges. Direct comparisons were made to slices from hippocampus from more mature rats, 30–35 days old, which are much less prone to seizure-like events. The amplitude and time course of the K + transients varied across the CA 3 laminae. K + signals were largest close to stratum pyramidale in stratum oriens (the infrapyramidal zone). Recordings from this site showed extracellular K + accumulation to be unusually large in immature hippocampus. The ceiling [K +] o level recorded during seizure-like events ranged from 14.4 to 20.2 mM and averaged 16.9 mM. The peak amplitude of extracellular K + transients following an epileptiform burst in slices from immature rats averaged 4.31 mM while the mean of similar recordings from mature rats was 0.97 mM. Detailed laminar distribution studies in developing hippocampus revealed that the K + signals were large in the proximal two-thirds of the basilar dendrites and proximal half of the apical dendrites. K + accumulation in stratum pyramidale was comparatively small even though at its very edge in stratum oriens large K + transients were always recorded. The latter was also true in recordings from mature hippocampus. Other dendritic signals in mature tissue were comparatively small. Laminar analysis was performed of the field potentials recorded by the reference barrel of the K + electrodes. Negative field potential for the epileptiform burst and subsequent slow potential correlated in space with sites of K + accumulation in both immature and mature hippocampal slices. Interictal and prolonged ictal-like discharges, recorded in developing hippocampus, arose from the same baseline [K +] o. However, since [K +] o is excessively high during the course of these epileptiform events it most likely has a role in the unusual propensity of immature hippocampus for seizures.

Extracellular K+ accumulation during penicillin-induced epileptogenesis in the CA3 region of immature rat hippocampus.

Ion-selective microelectrode techniques were used to study extracellular K+ changes associated with penicillin-induced epileptogenesis in the CA3 region of hippocampal slices. Recordings were made in slices taken from rats 9-16 days of age, which have a pronounced capacity to undergo prolonged synchronized afterdischarges. Direct comparisons were made to slices from hippocampus from more mature rats, 30-35 days old, which are much less prone to seizure-like events. The amplitude and time course of the K+ transients varied across the CA3 laminae. K+ signals were largest close to stratum pyramidale in stratum oriens (the infrapyramidal zone). Recordings from this site showed extracellular K+ accumulation to be unusually large in immature hippocampus. The ceiling [K+]o level recorded during seizure-like events ranged from 14.4 to 20.2 mM and averaged 16.9 mM. The peak amplitude of extracellular K+ transients following an epileptiform burst in slices from immature rats averaged 4.31 mM while the mean of similar recordings from mature rats was 0.97 mM. Detailed laminar distribution studies in developing hippocampus revealed that the K+ signals were large in the proximal two-thirds of the basilar dendrites and proximal half of the apical dendrites. K+ accumulation in stratum pyramidale was comparatively small even though at its very edge in stratum oriens large K+ transients were always recorded. The latter was also true in recordings from mature hippocampus. Other dendritic signals in mature tissue were comparatively small. Laminar analysis was performed of the field potentials recorded by the reference barrel of the K+ electrodes. Negative field potential for the epileptiform burst and subsequent slow potential correlated in space with sites of K+ accumulation in both immature and mature hippocampal slices. Interictal and prolonged ictal-like discharges, recorded in developing hippocampus, arose from the same baseline [K+]o. However, since [K+]o is excessively high during the course of these epileptiform events it most likely has a role in the unusual propensity of immature hippocampus for seizures.

Intracellular K+ and Na+ activities under hypoxia, acidosis, and no glucose in dog hearts.

To gain a better understanding of the ionic mechanisms responsible for the electrophysiological disturbances occurring during myocardial ischemia, transmembrane potentials and intracellular potassium (aiK) and sodium (aiNa) ion activities were measured under individual and combined conditions of hypoxia (Po2 less than 50 mmHg), respiratory acidosis (pH 6.6), and no glucose in isolated epicardial ventricular muscle preparations of the canine heart using conventional and ion-selective microelectrode techniques. After 30 min superfusion with hypoxic, acidic, and glucose-free solution under 2-Hz stimulation, resting membrane potential (RMP) was significantly reduced from -85.5 +/- 0.6 to -69.5 +/- 1.0 mV, accompanied by decreases in action potential amplitude, maximum upstroke velocity of phase O, and action potential duration. aiK was significantly decreased from 101.0 +/- 5.6 to 79.5 +/- 5.8 mM, whereas aiNa was not significantly altered by the combined condition. RMP and aiK were moderately decreased by hypoxia alone, slightly decreased by acidosis, and hardly affected by the glucose-free condition. The extent of depolarization was well correlated with the decrease in aiK. These results suggest that Na+-K+ pump inhibition may not be a major cause of K+ efflux from myocardial cells under the hypoxic, acidic, and glucose-free condition and that hypoxia is the most important factor affecting aiK and RMP among these conditions.

Diffusion coefficients of neurotransmitters and their metabolites in brain extracellular fluid space

Diffusion coefficients of catecholamine neurotransmitters, their metabolites and related species were measured in brain extracellular fluid using in vivo voltammetric techniques. Nanoliter volumes of the species were pressure-ejected into the rat caudate nucleus and their concentration profiles were determined at nearby voltammetric detector electrodes. Thorough testing was carried out to show that the present methodology gave results which agreed with brain diffusion coefficients measured previously by ion-selective microelectrode techniques. All of the species which are anionic at pH 7.4 have brain diffusion coefficients about one-third of their solution counterparts in accord with earlier studies of diffusion in tortuous media. However, the brain diffusion coefficients of all the cation species are about three-times slower than those of the anions. This phenomenon is believed to be caused by ion binding with the polyanionic glycosaminoglycans and related species in brain tissue. In vitro model experiments lend support to this interpretation. This new information on biogenic amines and their metabolites provides meaningful predictions of the spatio-temporal concentration distribution of these species in the extracellular fluid.

On the mechanism of lysophosphatidylcholine-induced depolarization of cat ventricular myocardium.

Lysophosphatidylcholine, a putative biochemical mediator of ischemia-induced arrhythmias, reduces the resting potential of ventricular muscle. To elucidate possible mechanisms of lysophosphatidylcholine-induced depolarization, we investigated the effects of lysophosphatidylcholine on the electrophysiological properties of cat ventricular muscle, using potassium ion-selective electrodes and conventional microelectrode, current-, and voltage-clamp techniques. Lysophosphatidylcholine (50 microM) decreased the sensitivity of the resting potential to changes in extracellular potassium concentration. Hyperpolarization of lysophosphatidylcholine-depolarized fibers by current-clamp methods failed to reveal two stable levels of resting potential. Depolarizing concentrations of lysophosphatidylcholine did not reduce the potassium equilibrium potential, as determined from the reversal potential of the time-dependent potassium current and measurements of intracellular potassium activity using potassium ion-selective electrodes. Lysophosphatidylcholine induced a depolarizing shift of the reversal potential for steady state current, and did not induce the formation of a negative slope region in the steady state current-voltage or background current-voltage relationships. Lysophosphatidylcholine induced an inward shift and linearization of the background current-voltage relationship negative to -30 mV, and the lysophosphatidylcholine-sensitive component of the background current was an inward rectifier with a reversal potential approximately equal to the potassium equilibrium potential. Lysophosphatidylcholine also reduced the amplitudes of the time-dependent potassium current, slow inward current, and the potassium accumulation and depletion currents. These results indicate that lysophosphatidylcholine-induced depolarization is due, in part, to reduced potassium conductance at voltages near the normal resting potential, and that lysophosphatidylcholine may act as a nonspecific depressant of membrane channels.

Case of Rhinolith: With Remarks

Ion flux kinetics associated with blue light (BL) treatment of two wild types (WTs) and the phot1, phot2 and phot1/phot2 mutants of Arabidopsis were studied by using the MIFE noninvasive ion-selective microelectrode technique. BL induced significant changes in activity of H+ and Ca2+ transporters within the first 10 min of BL onset, peaking between 3 and 5 min. In all WT plants and in phot2 mutants, BL induced immediate Ca2+ influx. In phot1 and phot1/phot2 mutants, net Ca2+ flux remained steady. It is suggested that PHOT1 regulates Ca2+ uptake into the cytoplasm from the apoplast. Changes in ion fluxes were measured from cotyledons of intact seedlings and from the cut top of the hypocotyl of decapitated seedlings. Thus the photoreceptors mediating BL-induced Ca2+ and H+ fluxes are present in the rest of the decapitated seedling and probably in the cotyledons as well. The H+ and Ca2+ flux responses to BL appear not to be linked because, (i) when changes were observed for both ions, Ca2+ flux changed almost immediately, whereas H+ flux lagged by about 1.5 min; (ii) in the Wassilewskija ecotype, changes in H+ fluxes were small. Finally, wave-like changes in Ca2+ and H+ concentrations were observed along the cotyledon–hook axis regardless of its orientation to the light.