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PubMed1993

Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements.

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K(+)-dependent, amiloride-sensitive K+/H+ exchange system.

Amiloride↗

PubMed1993

Interstitial PCO2 and pH in rat hippocampal slices measured by means of a novel fast CO2/H(+)-sensitive microelectrode based on a PVC-gelled membrane.

We describe here the construction and properties of a double-barrelled microelectrode (tip diameter 4-10 microns) which permits simultaneous measurements of PCO2 and pH, and which has a 90% response time of only one or a few seconds for a step change in PCO2. The fast response of the CO2-sensitive barrel is due to (i) the use of a PVC-gelled (tridodecylamine-containing) membrane solution which enables the construction of extremely short (> or = 4 microns), yet mechanically stable, membrane columns, and (ii) the presence of carbonic anhydrase in the filling solution. Recordings made in the pyramidal layer of area CA1 in rat hippocampal slices showed that the deviation in the acid direction of the basal interstitial pH (pH0) from that of the perfusion solution was attributable to a higher PCO2 level within the tissue. Most of the late acid shift evoked by stimulation of the Schaffer collaterals (5- to 20-s trains at 10 Hz) could also be explained on the basis of an accumulation of interstitial CO2 at a constant HCO3- concentration. This conclusion was supported by the finding that inhibition of extracellular carbonic anhydrase activity by 10 microM benzolamide completely abolished the activity-induced fall in pH0, but not the increase in PCO2. The initial stimulus-induced alkalosis was accompanied by a slight decrease in PCO2 only, implying a parallel increase in the interstitial HCO3- concentration. Benzolamide produced a dramatic enhancement of the early alkaline shift as well as of the simultaneous fall in PCO2. The latter effect of the drug unmasks a cellular CO2 sink that is induced by neuronal activity.

Animals↗

PubMed1992

Microelectrode determination of oxyntic cell pH in intact frog gastric mucosa. Effect of histamine.

Intracellular pH (pHi) of acid-secreting cells was measured in intact gastric fundus mucosa of Rana esculenta with double-barrelled pH microelectrodes. Tissues were mounted, serosal side up, between two half chambers and individual cells were impaled after microsurgical removal of the serosal muscle layer. Transepithelial potential difference (Vt) and resistance (Rt) as well as serosal cell membrane potential (Vs) and pHi were continuously recorded at rest (0.1 mmol/l cimetidine) or during stimulation (0.5 mmol/l histamine). During chamber perfusion with HCO3-/CO2-buffered Ringer solution of pHo = 7.36, Vt and Rt were -21.7, SD +/- 6.0 mV and 229 +/- 83 omega cm2 (n = 17) while Vs and pHi averaged -57.3 +/- 6.9 mV and 7.4 +/- 0.11 (n = 25). The latter value is considerably more alkaline than all recent pHi measurements obtained with microspectrofluorometric techniques on isolated cells, glands or intact tissue. The difference may in part be explained by use of HCO3(-)-free solutions in most of the previous studies because we observed that such solutions decrease pHi to 6.89 +/- 0.18 (n = 4). Again, in contrast to recent literature, application of histamine in HCO3-/CO2-buffered solution led to further transient alkalinization by 0.12 +/- 0.05 pH unit (n = 8). Since in accidental punctures of the gastric gland lumen we noticed that H+ secretion only began approximately 5 min after histamine application, we conclude that the histamine-induced initial alkalinization does not reflect stimulation of the H+/K+ ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1977

[Animal experimental investigations of the electrophysiologic parameters of 'fast fibers' of extraocular muscles by means of glass microelectrodes (author's transl)].

The author reports on animal experimental investigations of the electrophysiologic parameters of 'fast fibers' of extraocular muscles in rabbits in vivo by means of standardized glass microelectrode. The submitted results of investigation enlarge our knowledge of the electrophysiologic parameters of 'fast fibers'. The statistically analyzed results on musclefiber action potential parameters, on musclefiber membrane resting potentials, strengthduration curves and the data on the electro-mechanical latency period of 'fast fibers' are illustrated by figures. The most important conclusions for basic research on 'fast fibers', for clinical ophthalmo-electromyography and for the duality concept of eyemovement control are given.

Action Potentials↗

PubMed1977

The use of microelectrodes for measurement of local H+ activity in the cortical subarachnoidal space of cats.

pH microelectrodes with pointed tip (Hinke-type) were constructed for the continuous measurement of the local pH in the perivascular space of pial arteries in the feline cerebral cortex. The sensitive tip had a length of 20-60 mu and a base diameter of 10-25 mu. As reference electrode, a micropipette (tip diameter 2 mu), filled with 150 mM KCl was used. Calibration curves were linear and showed a sensitivity of 54.5-57.5 mV/pH unit at 38 degrees C. Advantages of such electrodes are the easy penetration of the subarachnoid membrane, the long life span, the quick response, and a minimal drift. The electrodes were tested in vivo during hyper- and hypoventilation and during local perivascular injection of mock spinal fluid at varying pH. A close correlation was observed between the change in perivascular pH and the corresponding change in pial arterial diameter.

Animals↗

PubMed1988

Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: studies with Cl- -sensitive microelectrode.

The mechanism of uphill Cl- accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl- -selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential (Vm) was -39.2 +/- 0.4 mV and intracellular Cl- activity (AiCl) was 34.6 +/- 0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na+-free solution, AiCl decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level of AiCl in 5 min. In the presence of furosemide (1 mmol/l), Cl- uptake induced by Na+-readmission was inhibited by 44%. Superfusion with a K+-free solution gradually decreased AiCl until it was close to the equilibrium level after 75 min; superfusion with a high-K+ (29.5 mmol/l) solution increased AiCl significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K+-free to high-K+ and from high-K+ to K+-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (-7 mmol/l) in AiCl. These changes in AiCl were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl- uptake after cessation of acetylcholine (ACh 1 mumol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na+-K+-Cl- cotransport.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1982

Postdenervation changes of intracellular potassium and sodium measured by ion selective microelectrodes in rat soleus and extensor digitorum longus muscle fibres.

The intracellular concentration of free [K+]i and [Na+]i in innervated and denervated rat extensor digitorum longus (EDL) and soleus (SOL) muscles was measured by double-barrel glass microelectrodes filled with liquid ion exchanger. In both muscles, postdenervation fall of resting membrane potential was accompanied by a decrease of [K+]i and increase of [Na+]i. The relative permeability, PNa/PK of the muscle fibre membrane increased three times in EDL and 1.5 times in SOL respectively by the third day of denervation and then dropped within 14 days to the values which were only slightly but significantly lower than the control ones.

Animals↗

PubMed1984

Free calcium in sheep cardiac tissue and frog skeletal muscle measured with Ca2+-selective microelectrodes.

Microelectrodes filled with neutral carrier selective to Ca2+ were used to measure the free intracellular Ca2+ concentration [( Ca2+]i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca2+]i at rest in normal physiological saline (20-22 degrees C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca2+]o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca2+]i. Elevation of [Ca2+]o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca2+]i. Exposure to Na+-free solution (Li+-substituted) gave rise to elevation of [Ca2+]i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca2+]i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.

Animals↗

PubMed1983

Microelectrode measurement of intracellular chloride activity in smooth muscle cells of guinea-pig ureter.

The intracellular Cl activity (aiCl) of smooth muscle cells in the guinea-pig ureter was measured using double-barrelled Cl-sensitive microelectrodes. The mean aiCl in normal Krebs solution was 51.1 mM, equivalent to an ECl of -18.6 mV, with a mean Em of -48.7 mV. Thus aiCl was three times higher than predicted from a passive distribution, aiCl exceeded the extracellular activity by nearly 10 mM in 10%-Cl solution. Complete removal of extracellular Cl (Cl0) caused a decline in aiCl to an apparent level of 3.3 mM with a mean time constant of 6.7 min. Cl ions were reaccumulated against their electrochemical gradient on readmission of Cl0 with a mean time constant of 6.6 min. Both the reaccumulation and loss of Cl ions on changing Cl0 were slowed about three-fold by the presence of the anion exchange inhibitor DIDS. It is therefore concluded that most of the transmembrane Cl movements are mediated by a reversible anion exchange carrier and that PCl is very low. These results are similar to those obtained in vas deferens and may be a general feature of smooth muscle cells.

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

PubMed1977

A technique for recording local blood flow and neuronal activity with a single microelectrode.

A technique is presented for recording both local blood flow, by means of hydrogen clearance method, and neuronal activity of the cat's cortex using a single microelectrode. The requirements of electrodes for recording cerebral blood flow are different from those for recording extracellular spikes. Only electrodes within a small range of impedance (measured at 10 Hz) are suitable to record both. A bridge method is used for the recordings. The bridge signal is split in order to amplify cell activities and H2 responses separately. Three examples demonstrate the method and the importance of appropriately selected electrodes.

Animals↗

PubMed1976

Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method.

When a strand of the rabbit sinoatrial node tissue was shortened by ligation, the spatial decay of electrotonic potential decreased and the input impedance increased. In a piece of the tissue 0.2-0.3 mm in diameter apparently uniform current spread was obtained. Action potentials recorded from three different sites in this small piece occurred simultaneously and were superimposable. In voltage clamp experiments using the double microelectrode method, the membrane potential was usually held at -30 to -40 mV, where no net current flowed. When membrane potential was suddenly changed from the holding potential, the sign and the time course of the ionic current varied with membrane potential. Hyperpolarization gave an inward current which increased with time. Depolarization gave a transient inward current followed by sustained outward current, and repolarization gave an outward current tail which exponentially subsided with a time constant of 0.37 s. The membrane time constant was 12.0 ms. When the specific membrane capacitance was assumed to be 1 muF/cm2, the specific membrane resistance at the resting potential was 12 Komega cm2. The peak of the transient inward current on depolarization was 1.3 X 10(-5) A/cm2.

Action Potentials↗

PubMed1977

The influence of carbon dioxide, bicarbonate and other buffers on the potential of antimony microelectrodes.

Antimony microelectrodes were calibrated at 37 degrees C in phosphate buffers, in different bicarbonate solutions at various CO2-partial pressures and in buffers like TRIS1, TES2, MES3 and malonic acid. By use of the latter buffers (with exception of malonic acid) the most reliable calibration curves were obtained ("normal values"). The usual calibration in 67 mmol/l standard phosphate buffers turned out to be unacceptable because the obtained mV-values were too high (negative) in comparison to all other buffers. Different calibration curves resulted from the use of pure bicarbonate solution whether the pH-values were changed by variation of pCO2 or of the bicarbonate concentration. Low bicarbonate concentrations in combination with low pCO2 gave mV-values which were too low relative to the other buffers. Both the increase of pCO2 as well as of the bicarbonate concentration caused a shift of the potential of the antimony electrodes toward "normal values". In solutions containing other buffers the influence of bicarbonate and pCO2 became negligible with increasing buffer concentration. Decreasing oxygen partial pressure was found to cause an increase of the potential of the antimony electrodes. The influence of liquid junction potentials at the reference electrode is discussed.

Antimony↗

PubMed1977

Time course of anoxia-induced K+ concentration changes in the cochlea measured with K+ specific microelectrodes.

The endocochlear potential (EP), potassium concentration in the endolymph (Ke+) and in the perilymph (Kp+) were measured in guinea-pigs during anoxia of different duration. Specific K+ double-barrel microelectrodes with liquid ion exchanger were used. The resting K+ concentration in the endolymph was 146.8 +/- 9.2 mM and in the perilymph 3.2 +/- 0.5 mM. The following time course of events was observed in the cochlea during anoxia: 40-50 s after the arrest of ventilation the K+ concentration decreased by 0.1-0.2 mM in the scala vestibuli, which was time related to a rapid fall of EP to negative values. Perilymphatic K+ started to increase in both scalae with a latency of 2-2.5 min, reaching a concentration of about 14 mM 60 min after the arrest of ventilation. The endolymphatic K+ began to decrease after a latency of 2.5-3 min, and 60 min after the arrest of ventilation an 80% concentration (average 112 mM K+) was reached as compared to the initial value. From the comparison of K+ concentration changes with the experimental values of the negative EP, it may be assumed that the negative EP is mainly generated by the K+ gradient between the perilymph and endolymph.

Animals↗

PubMedDate not supplied

Microelectrode recording of responses in visually observed neurons of the cat motor cortex.

With a contact optical system it is possible to carry out intravital studies of neurons and other structures, stained with vital dyes in reflected light in a specially prepared specimen of the cat cerebral cortex. The high-quality characteristics of the optical system used have made combined morphological and intracellular electrophysiological investigations of these neurons possible. The nature of intravital morphological changes in cortical neurons was established in response to their puncture by microelectrodes with tips with different external diameters and configurations; certain morpho-functional correlations were found in the response of pyramidal neurons to disturbance of their temperature regime.

Animals↗

PubMed1982

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin.

Microelectrodes were used to investigate the effect of 0.5 mM mucosal lanthanum (La3+) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potential Vsco (mean value = -65.4 +/- 3.2 mV, inside negative) hyperpolarized to -108.7 +/- 2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potential Vo or the inner membrane potential Vi. The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La3+ to the outer medium increased the short-circuit current to 190% of the control value. Vsco concomitantly changed to -28 +/- 3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La3+ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La3+ on short-circuit current has been observed. It is ascribed to the penetration of La3+ into the sodium channels.

Animals↗

PubMed1978

Intracellular gradients of ion activities in the epithelial cells of the Necturus gallbladder recorded with ion-selective microelectrodes.

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.

Animals↗

PubMed1983

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion.

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.

Animals↗

PubMed1983

Influx mechanisms for Na+ and Cl- across the brush border membrane of leaky epithelia: a model and microelectrode study.

This paper presents a numerical model for the movement of Na+, K+, Cl-, H+ and HCO3- in a leaky epithelium. The model describes the active transport of Na+ and K+ at the serosal membrane and electrodiffusive permeation across the mucosal, serosal and junctional pathways. The model accounts for H+ and HCO3- production in the cell. The influx of Na+ and Cl- is assumed to occur mainly via Na/H and Cl/HCO3 exchange. The behavior of the cell, with this influx mechanism, is compared to a cell with an obligatory neutral coupled influx of Na+ and Cl-. All parameters are obtained from the literature, primarily from studies utilizing the Necturus gallbladder. The analysis shows (i) that it is virtually impossible in steady-state experiments to distinguish between cells with Na/H - HCO3/Cl transport and cells with Na/Cl transport mechanisms. (ii) That nonsteady-state experiments can decide whether Na/H - HCO3/Cl or Na/Cl transport mechanisms mediate the influx of salt. A comparison between studies with ion-selective microelectrodes and the model predictions indicates that the influx of Na+ and Cl- is mediated by Na/H - HCO3/Cl transport when the external solutions contain CO2 and HCO3. (iii) The model also explains the diuretic effects of furosemide and carbonic anhydrase inhibitor, as well as the stimulatory effects on salt transport of elevated levels of HCO3- at a constant pH. (iv) The model fails to explain some experiments performed in HCO3/Co2-free media and some experiments using inhibitors.

Animals↗