Construction of K+- and Na+-sensitive theta-microelectrodes with fine tips: an easy method with high yield

A new method is described to prepare theta-microelectrodes with tips up to 0.15 m diameter controlled under scanning electron microscope. K⁺- and Na⁺-sensitive resins were tested.Method features are the following: i) hard drying of the glass, ii) rehydration of one channel and weak wetting of the other with a three-methylchlorosilane solution before pulling, iii) simultaneous presence of water and silane in the two channels during pulling, iv) gradual silanization from the tip to the shank.Selective and conventional channels did not affect each other and no displacements of resins were observed. The change of potential difference of the selective channel was more than –50 mV/decade. Apical membrane potentials and cell Na⁺ and K⁺ activities of the epithelial cells of rabbit gall-bladder (cell diameter: 5–10 m) were measured with these theta-microelectrodes and with single-barrel microelectrodes of similar tip size: results obtained were not significantly different.     

Comparison of the effects of potassium and pH on the calibre of cerebral veins and arteries

The vasomotor responses of individual pial veins and arteries on the convexity of the cerebral cortex to perivascular microinjection of mock cerebrospinal fluid (CSF) containing various concentrations of potassium (K⁺) and of various pH (achieved by altering the bicarbonate, HCO ₃ ^– concentration) have been examined in cats anaesthetised with -chloralose.Microapplication of CSF containing 0 mM HCO ₃ ^– (pH 4.80) effected significant increases in calibre of pial veins and arteries of 9.3±2.4% and 38.2±4% respectively (mean calibre change ±SE), whereas CSF containing 22 mM HCO ₃ ^– (pH 7.45) which constricted pial arteries significantly (–18.5±2.9%) minimally altered venous calibre (–4.3 ±2.2%).Microapplication of CSF containing 0 mM potassium resulted in a significant reduction in pial arterial calibre (–11.4±2.8%) but failed to alter pial venous calibre (–0.3 ±0.6%). Perivascular microapplication of CSF containing moderately elevated potassium concentrations (10 mM) which effected marked, significant increases in pial arterial calibre (49.3±3.9%) did not significantly alter the calibre of the pial veins (mean response –1.6±2.4%). The perivascular administration of CSF containing a high concentration of potassium (40 mM) resulted in the significant constriction of both pial veins (–13.5±0.9%) and pial arteries (–47.2±6.3%). The magnitude of the response was significantly smaller in the pial veins.The relative insensitivity to K⁺ and pH of the pial veins as compared to pial arteries suggests that alteration in the chemical composition of the perivascular fluid are of lesser importance in the control of cerebrovascular capacitance than for the regulation of cerebrovascular resistance.     

Perivascular pH and pial arterial diameter during bicuculline induced seizures in cats

The aim of the present study was to correlate locally at the same pial artery the vascular reaction with the perivascular pH during the initial phase of functional hyperemia. As a model of functional hyperemia, bicuculline (3 mg/kg i.v.) induced seizure was taken. Normally, a strong increase of blood pressure occurs together with the start of seizure. Since a discrimination between metabolically induced and pressure dependent vascular reactions is not possible under such conditions, the cats (anesthetized with 40–50 mg/kg chloralose) received in addition 3 mg/kg phentolamine and 10 mg/kg pentobarbital. Under these conditions a significant increase of blood pressure started only 50 s after the onset of seizure. Perivascular pH was recorded using spear type pH microelectrodes in the subarachnoid space surrounding a pial artery. The diameter of the respective artery was measured continuously. After onset of seizure an immediate, increasing perivascular acidosis developed which was accompanied by an increase in pial arterial diameter. The maximal decrease of pH was 0.29 units and occurred 30 s after the start of seizure. These data show that a decrease in perivascular pH can be one factor mediating functional hyperemia in the brain.Preliminary reports of these investigations were presented at the spring meeting of the German Physiological Society 1979, (Pflügers Arch. Suppl.379, R 11, 1979) and at the 36^th Annual Meeting of the Federation of American Societies for Experimental Biology, Dallas, Texas, 1979Supported by the Deutsche Forschungsgemeinschaft     

Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit

In intracellular electrolyte solutions a Ca²⁺-selective microelectrode based on the synthetic electrically neutral carrier N,N,N,N-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca²⁺ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa=9.2 in Ca²⁺ buffers containing an intracellular background of K⁺ (125 mM). Selectivity studies in mixed solutions show a preference of Ca²⁺ over Na⁺ of 6·10⁵, over K⁺ of 1.6·10⁶, and over Mg²⁺ of 5·10⁶. The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca²⁺ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa=7 and pCa=8 are reproducible within 24.7±0.4 mV (SD,n=8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca²⁺ electrode response is not disturbed by the contact of a cytosolic milieu.     

Intracellular pH in diluting segment of frog kidney

Chronic exposure to high potassium (K⁺ adaptation) stimulates H⁺ net secretion in the diluting segment of the frog kidney. In order to investigate the cellular mechanism of the H⁺ secretory process intracellular pH (pH_i) measurements were performed in cells of the diluting segment of the isolated doubly-perfused kidney of K⁺ adaptedRana esculenta. pH_i changes were monitored by pH-sensitive microelectrodes while the tubule lumen was rapidly perfused with various solutions. With control solutions (extracellular pH=7.80) pH_i averaged 7.60±0.05. Luminal application of furosemide (5 · 10^–5 mol/l) or reduction of luminal Cl^– (from 104 mmol/l to 9 mmol/l) hyperpolarized the cell membrane potentials but pH_i was not altered. Reduction of luminal Na⁺ (from 98 mmol/l to 3 mmol/l) depolarized the cell membrane potentials but pH_i remained constant. Complete removal of luminal Na⁺, however, led to a significant decrease of pH_i from 7.61±0.08 to 7.18±0.08. Luminal application of amiloride (1 · 10^–3 mol/l) also decreased pH_i significantly (pH_i=0.15±0.02).The results indicate that an amiloride-sensitive H⁺ extrusion mechanism exists in the luminal cell membrane of the K⁺ adapted frog diluting segment. The data are consistent with Na⁺/H⁺ exchange which maintains a constant pH_i even at extreme experimental conditions.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)     

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

Summary The endocochlear potential (EP), potassium concentration in the endolymph (K _e ⁺ ) and in the perilymph (K _p ⁺ ) were measured in guinea-pigs during anoxia of different duration. Specific K⁺ doublebarrel 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 ±0.5mM.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.     

K+ recirculation in A6 cells at increased Na+ transport rates

Homocellular regulation of K⁺ at increased transcellular Na⁺ transport implies an increase in K⁺ exit to match the intracellular K⁺ load. Increased K⁺ conductance, g_K, was suggested to account for this gain. We tested whether such a mechanism is operational in A6 monolayers. Na⁺ transport was increased from 5.1±1.0 A/cm² to 20.7±1.3 A/cm² by preincubation with 0.1 mol/l dexamethasone for 24 h. Basolateral K⁺ conductances were derived from transference numbers of K⁺, t _K, and basolateral membrane conductances, g_b, using conventional microelectrodes and circuit analysis with application of amiloride. Activation of Na⁺ transport induced an increase in g_b from 0.333±0.067 mS/ cm² to 1.160±0.196 mS/cm² and t _K was reduced to 0.22±0.01 from a value of 0.70±0.05 in untreated control tissues. As a result, g_K remained virtually unchanged at increased Na⁺ transport rates. The increase in g_b after dexamethasone was due to activation of a conductive leak pathway presumably for Cl^–. Increased K⁺ efflux, I _K, was a consequence of the larger driving force for K⁺ exit due to depolarization at an elevated Na⁺ transport rate. The relationship between calculated K⁺ fluxes and Na⁺ transport rate, measured as the I _sc, is described by the linear function I _K=0.624×I _Na–0.079, which conforms with a stoichiometry 23 for the fluxes of K⁺ and Na⁺ in the Na⁺/K⁺-ATPase pathway. Our data show that homocellular regulation of K⁺ in A6 cells is not due to up-regulation of g _K .     

Structure and mechanics of growing arterial microvessels from hypertrophied urinary bladder in the rat

Rat bladder hypertrophy, induced by a partial ligation of the urethra, was used to study the accompanying changes of microvascular smooth muscle mechanics, pharmacology and morphology. A segment of a microarterial vessel to the bladder was taken from a defined anatomical location and studied in a wire myograph in vitro at the length for maximal isometric force development (L _max). After 10 days of ligation, bladder hypertrophy resulted in a microvascular growth response compared to non-operated controls which was characterized by (i) an increase of the calculated diameter at L _max from 134±5 m to 222±19 m; (ii) an increase of the media thickness from 22.4±1.9 m to 32.2±3.0 m; (iii) an increase of the active tension from 1.42±0.28 mN/mm to 3.06±0.33 mN/mm; (iv) no change of the wall/lumen ratio (from 0.83±0.10 to 0.79±0.15). Normalized length/force relations (active, passive and total) did not differ significantly between microarteries from control and hypertrophic bladders. Microvascular smooth muscle growth was also associated with a decreased sensitivity to K⁺-induced depolarization and an increased sensitivity to ₁-adrenergic stimulation. No differences were noted regarding the Ca²⁺ sensitivity of force during K⁺-induced depolarization. The results suggest that microvascular growth (1) is immediately and positively influenced by the organ growth; (2) results in a functional resetting of the microvascular segments towards larger diameters without gross morphological or mechanical alterations; and (3) is accompanied by pharmacological alterations of the smooth muscle reactivity.     

Activation of luminal Na+/H+ exchange in distal nephron of frog kidney

Increased chronic intake of K⁺ induced H⁺ and K⁺ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed)Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pH_1u) was measured continuously with pH-sensitive microelectrodes while aldosterone (3·10^–7 to 3·10^–6 mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (V _te) from 8.5±1.1 mV to 4.0±0.6 mV pH_1u dropped from 7.73±0.02 to a new steady-state value of 7.17±0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10^–3 mol/l amiloride reversed luminal acidification to a pH_1u of 7.68±0.04; at the same timeV _te recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone — after interaction with cytoplasmic receptors — activates the luminal, amiloride-inhibitable Na⁺/H⁺ exchanger. This mechanism could explain enhanced H⁺ secretion found in the K⁺ adapted animal.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

Adenosine response on pial arteries,influence of CO2 and blood pressure

The television image-splitting technique was used to study the influence of arterialpCO₂ and blood pressure on the dilatatory response of pial arterioles to topically applied adenosine in chloralose anaesthetised cats. At normocapnia (pCO₂35 mm Hg) 10^–5 adenosine caused pial arteriole dilatation of 29.2 ±2.7% (S.E.M.). This was significantly reduced to 14.5±1.6% (P<0.001) atpCO₂ 25 mm Hg and to 8.5±1.6% (P<0.001) atpCO₂ 48 mm Hg. Lowering the blood pressure to 65–85 mm Hg had no significant effect on the adenosine response, but raising the blood pressure to 140–160 mm Hg significantly reduced the adenosine response to 22.1±1.8% (P<0.05). The response was independent of vessel size except at hypertension where vessels<150 m were significantly more reactive than the larger vessels (P<0.01). These results indicate that adenosine induced vasodilatation of pial arterioles shows little change in the face of alterations in vessel tone induced by altering blood pressure, but is markedly decreased by the combination of changing perivascular pH and vascular resistance through moderate changes in arterialpCO₂. The importance of these results in assessing the role of adenosine as a cerebral vasodilator is discussed.This study was supported by the Medical Research Council of Great Britain. D. P. J. Boisvert was supported by a Medical Research Council of Canada Fellowship     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation

The presence of an H⁺/K⁺-ATPase and its contribution to the regulation of intracellular pH (pH_i) was investigated in Caco-2 cells. The H⁺/K⁺-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H⁺/K⁺-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pH_i, measured in HCO ₃ ^– -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pH_i following a NH ₄ ⁺ /NH₃ pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na⁺. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H⁺/K⁺-ATPase and a Na⁺/H⁺ exchanger in regulating pH_i. Recovery rates at pH_i 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na⁺/H⁺ exchange to the recovery. Recovery of pH_i was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H⁺-ATPase, since bafilomycin A₁ had no effect on pH_i recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH_i, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na⁺/H⁺ exchanger to pH_i regulation is decreased.     

Sodium movement into and out of corneal endothelium

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na⁺-selective and conventional KCl-filled microelectrodes. The membrane potential (V _m) was –30.4±0.8 mV (mean ±SEM, n = 55) and the intracellular [Na⁺]_i (calculated from the Na⁺-selective electrode potential, V_Na) was 13.7 ±1.9 mM (mean±SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both V _m and V_Na to increase with a very similar time course. Final V _m was –6.3±0.6 mV (mean ±SEM, n = 15), and the final [Na⁺]_i was 114±6.9 mM (mean ± SEM, n = 5). The parallel increase in V _m and rise in [Na⁺]_i suggest that a component of the ouabain-induced depolarisation of the cell (increase in V _m) is due to Na⁺ entry into the cell down its concentration gradient. The lateral and basal location of the Na⁺/K⁺-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the 1 subunit of Na⁺/K⁺-ATPase. The absence of a net Na⁺ flux across these cells combined with the basolateral location of the ATPase suggest that Na⁺ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).     

Erythrocyte sodium content and transport in borderline and mild hypertension

Summary Sodium content and transport of red blood cells were examined in 98 male blood donors. Regarding their blood pressure they were classified into the following groups: (a) 57 normotensives, (RR<140/90 mm Hg); (b) 24 borderline hypertensives (140/90RR<160/95 mm Hg); and (c) 17 hypertensives (RR>160/100 mm Hg). Compared with the normotensives the borderline hypertensives have significantly reduced red cell sodium content. The ouabain-resistant net Na⁺ uptake and the relative Na⁺ uptake, as a measure of the Na⁺/K⁺ pump, were significantly increased. With rising blood pressures the measured values turn to normal, so that no difference exists between the normotensive and hypertensive groups. It is supposed that in the initial or even prehypertensive state a considerable enhancement of the pump activity occurs, simultaneously accompanied by less marked increases in sodium influx, leading to a reduced intracellular sodium content. In the course of hypertension, possibly caused by the formation of a pump inhibitor, the sodium content of red cells turns to normal or supernormal values.Abbreviations BMI body mass index - BHT borderline hypertensive - Ca _ion ²⁺ ionized plasma calcium - HT hypertensive - k relative OR net Na⁺ uptake - [Na⁺]_i, [K⁺]_i intracellular sodium and potassium content in RBCs - NT normotensive - OR ouabain-resistant - RBCs red blood cells - Na OR net Na⁺ uptake     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.     

pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V _m), whole-cell conductances (g _c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V _m, g _c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V _m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g _c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P _o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄ ⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄ ⁺/NH₃ removal. In the presence of NH₄ ⁺/NH₃ V _m depolarized by 16±2 mV (n=66) and g _c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄ ⁺/NH₃. The effect of NH₄ ⁺/NH₃ on V _m and g _c was markedly increased when the pH of the NH₄ ⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄ ⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.     

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat

Sodium transport correlates with varying Na⁺-K⁺-ATPase activity rates along the nephron. Whether differences in Na⁺-K⁺-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na⁺-K⁺-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na⁺-K⁺-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43±1.99 versus 2.3±0.20, respectively, P<0.01) while Na⁺-K⁺-ATPase activity was higher in mTAL (3,402±83 vs 711±158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10^–6 M decreased phosphorylation in PCT to 125±10% (P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na⁺-K⁺-ATPase. Both calcineurin inhibitor FK-506 10^–6 M and phorbol 12-myristate 13-acetate (PMA) 10^–6 M increased the degree of Na⁺-K⁺-ATPase phosphorylation (P<0.05) and inhibited Na⁺-K⁺-ATPase activity to 657±152 and 1,448±347 pmol/mm tubule per hour, respectively, in mTAL (P<0.01). Increase in [Na⁺]_i to 30, 50 and 70 mM resulted in no changes in Na⁺-K⁺-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na⁺]_i were paralleled by decreased phosphorylation (from 120±7 to 160±15% of controls, P<0.05) and increased Na⁺-K⁺-ATPase activity (from 850±139 to 1,874±203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10^–6 M decreased both Na⁺-K⁺-ATPase dephosphorylation to 41.85±9.58% (P<0.05) and Na⁺-K⁺-ATPase activity to 2,405±176 pmol/mm tubule per hour in mTAL (P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.