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PubMed1987

Changes in the intracellular free calcium concentration of Aplysia and leech neurones measured with calcium-sensitive microelectrodes.

The intracellular free Ca concentration was measured in invertebrate neurones using single-barrelled and double-barrelled neutral-carrier microelectrodes. The electrodes were calibrated in solutions containing different Ca concentrations between 1 mM and 0.01 microM. The electrode responses were also tested at different ionic strengths and at varying Na concentrations. The electrodes responded with 25-30 mV per 10-fold change in Ca concentration between 1 mM and 1 microM and with 10-25 mV between 1 and 0.1 microM Ca. The intracellular free Ca concentration was measured to be between 0.1 and 0.7 microM in the neurones. The changes of intracellular Ca in identified voltage-clamped neurones of Aplysia californica were recorded during iontophoretic injections of Ca2+ or EGTA. The decrease of intracellular Ca following EGTA injection was correlated with the suppression of the Ca-dependent K current and with the reduction of Ca-induced inactivation of voltage-dependent Ca current. In identified neurones of the leech Hirudo medicinalis a reversible increase of intracellular Ca2+ was recorded after inhibition of the Na-K pump, either by addition of ouabain (0.5 mM) or by lowering the external K concentration (0.2 mM). This rise in intracellular Ca2+ did not occur, and was even reversed, in the absence of external Na, suggesting the existence of Na-Ca exchange across the leech neuronal membrane.

Animals↗

PubMed1987

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey.

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally HCO3(-)-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10-20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1-2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to HCO3(-)-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in HCO3(-)-buffered media was inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid and was slowed after exposure to Cl(-)-free media. Following inhibition of acid extrusion by amiloride, transition to HCO3- media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+-H+ exchange and an independent HCO3(-)-dependent mechanism. Evidence for HCO3(-)-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

A novel concentric double-barrelled calcium-selective microelectrode for small cells.

A novel concentric design of double-barrelled Ca2+-selective microelectrode, with an inner pipette tip that protrudes beyond an outer one, has recently been developed and is described. This configuration of pipettes was produced from concentric capillaries in one step using a horizontal pipette puller. For the tip of the inner barrel to protrude, Corning 1724 aluminosilicate glass was selected, as it has a higher melting point than the 1723 glass which is used for the outer barrel. To reduce electrode resistance the inner capillary was best made with a triangular shape. It was preferentially silanized in a dry box by injection of methyltrichlorosilane into only the inner barrel. The Ca2+ neutral carrier-based liquid membrane (ETH 1001) was back-filled from the tip to the shank of the inner pipette and above this CaCl2 solution was added. KCl, which contained EGTA and was buffered to pCa 7, was used to fill the reference barrel. These Ca2+ electrodes showed linear response with slope approximately equal to 30 mV for changes in Ca2+ concentration between 10(-3) and 10(-7) M in the presence of constant [K+]. They offer a number of advantages including a low noise level achieved by the presence of the external concentric KCl electrode, and a simple mechanical structure that allows applications to a variety of small cells.

Animals↗

PubMed1981

An improved liquid ion exchanger for chloride ion-selective microelectrodes.

A new chloride liquid ion exchanger, Corning 477913, suitable for fabricating ion-selective microelectrodes (ISE) is described. It differs from the standard chloride exchanger. Corning 477315, in that the concentration of organophilic ligand is increased fivefold. The properties of ISE (tip diameter less than 1 micrometer) made from the new material are superior to those of ISE made from the standard material. The yield of acceptable ISE was greater (77 vs. 34%); the ISE resistance was decreased 5.3-fold; the average slope was improved (-52.8 vs. -45.5 mV); and the selectivity over some interfering anions was increased. Measurement of intracellular chloride activity in rabbit ventricle with ISE made from the new exchanger gave results similar to those reported previously with the standard exchanger. This suggests that 477913 is satisfactory for use in biological tissue. The new formulation should be particularly advantageous for measurements requiring ISE with very fine tips and in fabrication of multibarrel ISE.

Anions↗

PubMed1985

Submicron tip breakage and silanization control improve ion-selective microelectrodes.

Probable causes of failure of otherwise well-constructed liquid ion-exchanger (LIE) micro-electrodes of average tip size less than 0.15 micron were examined. The problem could be attributed to two major variables, both localized at the tip: partial tip occlusion during fabrication prevents the generation of an electromotive force (small or absent slope and/or selectivity, high resistance); or poor hydrophobicity of the tip permits water to displace the resin from the tip (small or absent slope and/or selectivity and low electrode resistance). Controlled dry tip breakage on paper coated with glassine to final tip sizes well below 0.5 micron (confirmed by scanning electron microscopy) improves the yield of usable electrodes severalfold. Adequate silanization of the tip and consequent retention of resin at the tip can be predicted from the contact angles observed at the glass-LIE-backfilling solution interface. Satisfactory silanization can be achieved despite high ambient humidity. No evidence of shunting of Na+-LIE microelectrodes by the glass wall was seen. In the isolated perfused proximal tubule of Ambystoma tigrinum, the mean intracellular Na+ activity recorded by broken-tip electrodes (13.7 +/- 1.9 meq, n = 4) was similar to that recorded by intact electrodes (15.5 +/- 1.1 meq, n = 31).

Adsorption↗

PubMed1986

Segregation of gastric Na and Cl transport: a vibrating probe and microelectrode study.

The short-circuit current (Isc) of resting Necturus gastric mucosa (approximately 20 microA/cm2) can be attributed to the algebraic sum of the net Cl- secretion and amiloride-inhibitable net Na+ absorption. We have attempted to identify the cell types [surface epithelial cells (SCs) or oxyntic cells (OCs)] responsible for the transport of these ions in Necturus gastric mucosa using microelectrodes (ME) and a vibrating probe (VP). Mucosae were mounted horizontally in an open-topped Plexiglas chamber either serosal side up for basolateral ME impalements of OCs or mucosal side up for apical impalements of SCs and VP measurements. Cell impalements were made under open-circuit conditions, and VP measurements were performed under short-circuit conditions. Impalements of OCs indicate that neither the ratio of their apical to basolateral cell membrane resistances (Ra/Rb = 1.3 +/- 0.2) nor their cell membrane potentials were affected by 10(-6) M mucosal amiloride. In contrast, impalements of SCs indicate that amiloride increased their Ra/Rb from 3.5 +/- 0.2 to 15.6 +/- 1.8 and hyperpolarized both cell membrane potentials by greater than 20 mV. VP measurements showed that the amiloride-induced change in the current from SCs (5.6 microA/cm2) accounted for the amiloride-induced change in the Isc (5.5 microA/cm2). A non-zero current (4.4 +/- 1.0 microA/cm2) measured over SCs in the presence of amiloride was due to contamination from current arising from the gastric crypts that contain the OCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1987

Na microelectrode study of pathways of Na entry into Amphiuma intestinal absorptive cells.

To define the pathways of Na+ entry into intestinal villus cells, intracellular Na+ activity (aiNa) was measured in Amphiuma duodenum using conventional and Na-sensitive microelectrodes. Replacement of Na+ in the luminal medium reduced aiNa rapidly; replacement of Na+ in the serosal medium caused a slow decline of aiNa. Hence, mucosal and serosal membranes are both permeable to Na+. Ouabain addition to the serosal medium caused aiNa to increase over 4 h. When Na+ was present only in the mucosal medium and Na+ transport was inhibited with ouabain, aiNa increased over 4 h. With galactose or valine (20 mM) in the mucosal medium aiNa was greater at 2 h relative to paired control tissues. The gain in aiNa was unaffected by replacement of luminal medium Cl- with gluconate or exposure to 1 mM furosemide or amiloride. Amiloride, at 1 mM, was detected by the Na-sensitive neutral carrier. Over a wide range of Na+ concentrations in the luminal medium the rate of Na+ entry across the mucosal membrane correlated strongly (r = 0.95) with the electrochemical gradient for Na+ across the luminal membrane. It is concluded that aiNa of urodele intestinal cells is maintained at a low level by the operation of a Na+-K+ pump. Na+ entry across the luminal membrane occurs by diffusion and by the cotransport with sugars and amino acids. Luminal NaCl cotransport and Na+-H+ exchange do not appear to contribute to Na entry to a measurable extent but it is possible that these transport processes operate at a slow rate, but were inhibited secondary to inhibition of the Na-K pump.

Absorption↗

PubMed1989

Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes.

Trans sarcolemmal Ca movements in rabbit and rat ventricular muscle were compared using extracellular double-barreled Ca-selective microelectrodes. In rabbit ventricle, steady-state twitches were associated with transient extracellular Ca (Cao) depletions, indicative of Ca uptake during the twitch. In contrast, steady-state twitches in rat ventricle were associated with net cellular Ca extrusion. Rest periods in rabbit ventricle lead to a net loss of cell Ca and resumption of stimulation induces a net uptake of Ca by the cells. Conversely, in rat ventricle rest periods lead to cellular Ca gain and resumption of stimulation induces a net Ca loss from the cells. Thus stimulation is associated with net Ca gain in rabbit ventricle and net Ca loss in rat ventricle. These observations provide an explanation for some of the functional differences between rat and rabbit ventricle (e.g., negative force-frequency staircase and rest potentiation in rat vs. positive staircase and rest decay in rabbit). Resting intracellular Na activity (alpha iNa) was 12.7 +/- 0.6 mM in rat and 7.2 +/- 0.5 mM in rabbit ventricle. This alpha iNa in rat ventricle is sufficiently high that Ca entry via Na-Ca exchange is thermodynamically favored at the resting membrane potential. This may explain why rest potentiation is observed in rat ventricle. In contrast, the lower alpha iNa in rabbit ventricle would favor Ca extrusion via Na-Ca exchange at rest (and consequent rest decay). In rat ventricle, the increase of intracellular [Ca] ([Ca]i) associated with contraction, coupled with the short action potential duration, strongly favor Ca extrusion via Na-Ca exchange and explain the observed Cao accumulation observed during twitches in rat. The high plateau of the rabbit ventricular action potential tends to prevent Ca extrusion via Na-Ca exchange during the contraction and explains the Cao depletions observed in rabbit. It is concluded that the higher alpha iNa and shorter action potential duration in rat vs. rabbit ventricle can explain many of the functional differences observed in these tissues.

Animals↗

PubMed1990

An ultracompliant glass microelectrode for intracellular recording.

We describe the simple fabrication of a glass microelectrode that was designed for the stable impalement of contracting isolated cardiac preparations. The electrodes can be made with the aid of a standard pipette puller. The design uses a long-shanked glass electrode that is stepped below the shoulder at 70 degrees to the shaft and again near the tip. The electrodes are of marginally higher resistance than equivalent length straight electrodes (34.9 vs. 26.0 M omega). The stepped design imparts a low stiffness to the electrode in the vertical and horizontal planes, and the mechanical basis for this performance is described and discussed with regard to measured behavior. The duration of impalements with stepped electrodes is significantly longer than for straight electrodes and can usually be maintained for indefinite periods with minimal damage to small, highly contractile muscle preparations.

Animals↗

PubMed1992

Calibration of ion-selective microelectrodes.

Measurements of intracellular and extracellular ion activities with ion-selective microelectrodes generally involve calibration of the electrodes in solutions of known composition and fitting of the calibration data with a theoretical expression. The Nicolsky equation is frequently used to describe the variation of electrode potential with primary ion activity in the presence of a constant amount of interference. In this report, we review the estimation of primary ion activities in calibration solutions and discuss the practical use of the Nicolsky equation. We describe a specialized computer program, developed in this laboratory, for routine input and editing of calibration data, fitting of data with the Nicolsky equation, and calculation of experimental ion activities from the fitted curve. Earlier versions of this program have proven helpful in several investigations in this and in other laboratories.

Calibration↗

PubMed1993

Microelectrode measurements of pericellular PO2 in erythropoietin-producing human hepatoma cell cultures.

On the basis of Fick's law of gas diffusion, it has been proposed that cells in conventional monolayer cultures may be severely hypoxic. Because knowledge of the cellular O2 availability is important for the interpretation of biochemical and toxicological cell culture work, microelectrode measurements of the pericellular PO2 were carried out using the erythropoietin (Epo)-producing human hepatoma cell lines Hep G2 and Hep 3B as an in vitro model. In confluent hepatoma cultures grown in polystyrene dishes and incubated in air with 5% CO2, the pericellular steady-state PO2 was < 1 mmHg. The rates of the production of immunoreactive Epo and lactate were high due to a misproportion between O2 supply and O2 requirements. Epo production decreased when shaken instead of static cultures were studied, or when the O2 concentration in the gas atmosphere was increased gradually up to 95%. In cultures grown on gas-permeable supports, pericellular and gas PO2 values were very similar, with increased Epo production at lowered PO2. In agreement with mathematical models, our experimental data make PO2 measurements desirable for studies of O2-dependent biological functions in cell cultures.

Carcinoma, Hepatocellular↗

PubMed2006

Glucose-induced release of nitric oxide from mouse pancreatic islets as detected with nitric oxide-selective glass microelectrodes.

Nitric oxide (NO) is believed to play an important role in pancreatic islet physiology and pathophysiology. Research in this area has been hampered, however, by the use of indirect methods to measure islet NO. To investigate the role of NO in islet function, we positioned NO-sensitive, recessed-tip microelectrodes in close proximity to individual islets and monitored oxidation current to detect subnanomolar NO in the bath. NO release from islets consisted of a series of rapid bursts lasting several seconds and/or slow oscillations with a period of approximately 100-300 s. Average baseline NO near the islets in 2.8 mM glucose was 524+/-59 nM (n=12). Raising glucose from 2.8 to 11.1 mM augmented NO release by 429+/-133 nM (n=12, P<0.05), an effect blocked by the NO synthase inhibitor L-NAME (n=3). We also observed that glucose-stimulated increases in NO release were contemporaneous with changes in NAD(P)H and O2 but occurred well before increases in calcium associated with glucose-stimulated insulin secretion. In summary, we demonstrate that NO release from islets is oscillatory and rapidly augmented by glucose, suggesting that NO release occurs early following an increase in glucose metabolism and may contribute to the stimulated insulin secretion triggered by suprathreshold glucose.

Animals↗

PubMed1989

A microelectrode study of responses to secretagogues by epithelial cells on villus and crypt of rat small intestine.

The cellular origin of the response to secretagogues in small bowel epithelium was investigated by recording the effect of 5-hydroxytryptamine (5-HT, 10(-4) mol/l), acetylcholine (10(-4) mol/l), and prostaglandin E2 (PGE2, 10(-5) or 10(-4) mol/l) on apical membrane potentials (Va) of crypt and villus cells of rat ileum and jejunum in vitro using intracellular microelectrodes. Experiments were performed under visual control; addition of secretagogues and other manipulations were carried out during single impalements. Under basal conditions, apical membrane potential differences were consistently higher in jejunum than ileum (-72 +/- 1 vs. -47 +/- 2 mV, respectively, for villus impalements; -61 +/- 2 mV vs. -57 +/- 1 mV, respectively, for crypt impalements), and in jejunum villous membrane potentials exceeded those in the crypt. In the ileum, this crypt-villus gradient was reversed. The three secretagogues increased transmural potential difference and transiently reduced Va in cells in both crypt and villus regions by 8 mV or more. Fractional apical membrane resistance (FR) declined in ileum by approximately 30% in response to 5-HT and PGE2, whereas little change in FR was observed in jejunal recordings. PGE2 was ineffective in crypt and villus when Cl- was replaced by gluconate in both the luminal and serosal perfusates but depolarized the apical membrane in both regions after serosal restoration of Cl- from -76 +/- 4 to -56 +/- 8 mV in villus and from -58 +/- 4 to -45 +/- 6 mV in crypt. Rapid luminal Cl- substitution depolarized Va on the villus from -77 +/- 2 to -74 +/- 3 mV, but this effect was enhanced in the presence of PGE2, reducing Va from -65 +/- 8 to -43 +/- 12 mV. Prior to PGE2 addition, Va was -81 +/- 4 mV for this group of experiments. FR rose in the nominal absence of luminal Cl- from 0.69 +/- 0.09 to 0.77 +/- 0.06. It is concluded that because a Cl(-)-dependent depolarization of apical membrane potentials occurs in villi and crypts, net secretion in the small bowel is probably not confined to the crypts and may also occur from villous epithelium.

Acetylcholine↗

PubMed1991

Defibrillation shocks increase myocardial pacing threshold: an intracellular microelectrode study.

Defibrillation is known to cause inability to pace the heart acutely, but the mechanism is unknown. This study used microelectrode techniques to directly evaluate the effect of defibrillation shocks on the pacing threshold and membrane potentials from superfused guinea pig papillary muscles. Failure of pacing stimuli to induce action potentials (pacing failure) followed shocks of 50-200 V/cm, with pacing failure duration correlated with shock intensity. Increasing pacing strength from one to three times diastolic threshold decreased the incidence and duration of pacing failure. Decreased extracellular calcium concentration and verapamil added to the superfusate increased the duration of pacing failure. Membrane potential depolarization occurred after shock, but pacing failure did not correlate with depolarization magnitude. We conclude that defibrillation shocks directly cause shock intensity-dependent increase of myocardial pacing threshold. The pacing threshold of the myocardium can be increased after defibrillation shock independent of hypoxia or shock-induced depolarization and may involve membrane changes in calcium handling.

Action Potentials↗

PubMed1976

Comparison of microelectrode, DMO, and methylamine methods for measuring intracellular pH.

The intracellular pH (pHi) of giant barnacle muscle fibers was measured with glass microelectrodes and also calculated from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) and methylamine (MA). Simultaneously applying any two of these methods to muscle fibers of the same barnacle, we found the pH measured with an intracellular electrode (pH-Elec) to be about 0.06 higher than the DMO-derived pH (pH-DMO), and pH-DMO to be about 0.10 higher than the MA-derived pH (p-ma). in studies on the pHi of squid giant axons, we found that pH-Elec (7.35) and pH-DMO (7.36) were not significantly different. In the barnacle experiments, DMO required about 30 min to reach a steady-state distribution, while MA required more than 5 h. The deviations of pH-DMO and pH-MA from pH-Elec for the barnacle can be explained by a) an error in the assumed intracellular pKa' of DMO or MA, b) membrane permeability to the ionic form of DMO or MA, or c) intracellular compartmentalization. Included is a detailed study of the apparent dissociation constant of DMO as affected by temperature, and ionic strength and composition.

Animals↗

PubMed1983

Intracellular microelectrode characterization of the rabbit cortical collecting duct.

Cortical collecting ducts of the rabbit were perfused in vitro and the intracellular potential (Vbl) was measured with KCl-filled microelectrodes. The ratio of apical to basolateral membrane resistance (Ra/Rbl) was estimated from the voltage divider ratio using cable analysis. In control tubules Vbl averaged--84.0 +/- 2.5 mV and Ra/Rbl was 0.83 +/- 0.11. Pretreatment of the rabbits with mineralocorticoid caused Vbl to hyperpolarize to--105.8 +/- 3.1 mV and Ra/Rbl to decrease slightly to 0.62 +/- 0.10. A 10-fold increase of the luminal [K+] caused a 40.6 +/- 3.1 mV depolarization of Vbl in control tubules and a 33.0 +/- 4.2 mV depolarization in tubules from DOCA-pretreated rabbits. Concurrently, Ra/Rbl decreased in both groups, consistent with the existence of a conductive K+ channel at the apical cell membrane. This apical K+ channel was not sensitive to amiloride but was blocked by Ba2+. Conductive movement of Na+ across the apical membrane was also apparent in that Ra/Rbl increased with amiloride from 0.61 +/- 0.10 to 1.45 +/- 0.28. A 10-fold increase in the bath [K+] caused a 28.6 +/- 3.8 and a 49.4 +/- 4.4 mV depolarization of Vbl in tubules obtained from control and DOCA-pretreated rabbits, respectively. In both groups Ra/Rbl increased, suggesting that the basolateral cell membrane also contains a conductive K+ channel. Taken together the results support a model in which the transepithelial reabsorption of Na+ and the transepithelial secretion of K+ are driven by the Na+-K+-ATPase located in the basolateral cell membrane, with passive movement of these ions occurring through separate conductive pathways in the apical cell membrane.

Amiloride↗

PubMed1984

Microelectrode assessment of chloride-conductive properties of cortical collecting duct.

The chloride-conductive properties of the isolated rabbit cortical collecting duct were assessed with microelectrode techniques. The transepithelial, apical, and basolateral membrane potential differences, Vte, Va, and Vb, respectively, were monitored continuously along with periodic measurements of the transepithelial conductance, Gte, and fractional resistance, fRa (ratio of apical to apical plus basolateral membrane resistance). Active transport was eliminated in all experiments by luminal addition of 50 microM amiloride in HCO3-free solutions. Upon reducing the chloride activity in the bath (gluconate replacement), there was a marked depolarization of Vb and decrease in Gte and fRa, demonstrating a major dependence of the basolateral membrane conductance on the bath chloride activity. However, a significant K+ conductance at that barrier was also apparent since raising the bath K+ concentration caused an increase in Gte and fRa and depolarization of Vb. Lowering the chloride activity of the perfusate caused a consistent decrease of Gte but not of fRa, effects consistent with a high C1- conductance of the tight junction and little, if any, apical membrane C1- conductance. By use of the C1- -dependent conductances, the C1- permeabilities at equilibrium were estimated to be near 1.0 X 10(-5) cm X s-1 for the tight junction, PtiC1, and 5 X 10(-5) cm X s-1 for the basolateral cell membrane, PbC1. It is concluded that the paracellular pathway provides a major route for transepithelial C1- transport. Furthermore, since the isotopically measured C1- permeability is severalfold greater than PtiC1, a significant transcellular flux of C1- must exist, implicating a neutral exchange mechanism at the apical cell membrane in series with the high basolateral membrane C1- conductance.

Amiloride↗

PubMed1986

Microelectrode study of intracellular pH in frog skin: dependence on serosal chloride.

Replacement of external chloride has been known to reduce Na+ transport across whole frog skin. However, the sidedness and mechanism of the phenomenon have been unclear. In the present study, transepithelial current (IT), transepithelial resistance (RT), and basolateral membrane potential measured both with reference micropipettes (psi sc) and pH-selective microelectrodes (EscH) were monitored in isolated epithelial sheets from frog skin; removal of the underlying dermis facilitates ionic exchange across the basolateral membranes. The intracellular hydronium ion activity (acH) was 58 +/- 4 nM (means +/- SE) when the extracellular hydronium activity was 25 +/- 1 nM under base-line conditions. This measurement is equivalent to an intracellular pH (pHc) of 7.24 +/- 0.03 at an extracellular pH of 7.60 +/- 0.01, in reasonable agreement with estimates obtained by 31P- and 19F-nuclear magnetic resonance (NMR) analyses of frog skin. Complete replacement of mucosal Cl- by gluconate had variable effects on tissue current and resistance from preparation to preparation. The same ionic substitution on the serosal side uniformly produced a prompt reversible decrease in IT, increase in RT, and a substantial membrane depolarization of the short-circuited skins. In most of the preparations, the depolarization was preceded by a small hyperpolarization of 0.5-3.5 mV. The replacement of serosal Cl- also produced a fall in intracellular hydronium ion activity of 33 +/- 10 nM. The present date are consistent with the concept that serosal replacement of Cl- alkalinizes the cells by either favoring HCO3- entry or blocking HCO3- exit through a Cl- HCO3 antiport at the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗