Changes in extracellular potassium and calcium in rat cerebellar cortex related to local inhibition of the sodium pump

Extracellular K⁺, Ca²⁺, and Na⁺ ([K⁺]_e, [Ca²⁺]_e, [Na⁺]_e) were recorded with ion selective microelectrodes in the cerebellar cortex of urethane-anesthetized rats. Superfusion of the cerebellum with artificial cerebrospinal fluid containing K-strophanthidin (10^–6–10^–4 mol/l) or other cardioactive steroids, known to be inhibitors of the sodium/potassium pump, had the following effects: elevation of resting [K⁺]₃, reduction of poststimulus K⁺-undershoots, decrease of resting [Ca²⁺]_e and [Na⁺]_e. For instance, at 3×10^–5 mol/l K-strophanthidin within the superfusion solution (the unknown intracerebellar concentration being certainly much smaller), [K⁺]_e was elevated up to 130% and [Ca²⁺]_e reduced to 70% of their resting values. Iontophoretic K⁺-pulses were enhanced in amplitude at the same time. Control experiments with iontophoretic TMA application demonstrated that the glycoside effects were not due (or in higher concentrations only partly due) to shrinkage of the extracellular fluid volume. When tetrodotoxin (10^–7 mol/l) or Mn²⁺ (1–3 mmol/l) were additionally superfused, K-strophanthidin effects were qualitatively similar, though quantitatively smaller. This indicates that part of the effects were indirect via neuronal activity evoked by the blockade of the sodium pump. The experiments show that reduction of sodium pump activity in cerebellar cortex has rapid and serious consequences on the distribution of potassium and calcium in the extracellular space, resulting in an alteration of neuronal circuit excitability.     

Anoxic disturbance of the isolated respiratory network of neonatal rats

Tissue oxygen (PO₂), K⁺ (aK_e), pH (pH_e) and Ca²⁺ ([Ca²⁺]_e) were measured in the region of the ventral respiratory group (VRG) in the in vitro brainstem-spinal cord preparation of neonatal rats. During tissue anoxia, elicited by superfusion of N₂-gassed solutions, an initial increase in the frequency of respiratory activity, lasting between 2 and 12 min, turned into a frequency depression. During anoxia periods of up to 60 min, respiratory activity persisted in solutions containing CO₂/bicarbonate, whereas a complete blockade was observed after 15–25 min in N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid- (Hepes)-buffered salines. After such anoxic apnea, respiratory rhythmicity could be reactivated by superfusion of hypoxic, CO₂/bicarbonate-buffered solutions. In both types of hypoxic solutions, aK_e increased by maximally 1.5mM, whereas an initial increase of pH_e by up to 0.05 pH units turned, after 2–4 min, into an acidification which could exceed 0.5 pH units. In contrast, [Ca²⁺]_e remained unaffected by anoxia. Addition of 2–5 mM cyanide (CN^-) to oxygenated Hepes-buffered saline evoked an increase in PO₂ in the VRG from 100 to more than 300 mmHg. The effects of CN^- on respiratory activity, aK_e and pH_e were almost identical to those during anoxia. In oxygenated, CO₂/bicarbonatefree solutions of different pH, however, an increase in pH_e in the VRG led to a decrease in respiratory frequency, whereas a fall of pH_e produced a frequency acceleration. A rise of aK_e in the VRG by more than 2 mM as induced by superfusion of a 7 mM K⁺ solution led to a sustained increase of respiratory frequency. The results indicate that blockade of aerobic metabolism does not severely perturb K⁺ and Ca²⁺ homeostasis and that the biphasic response to anoxia is not directly related to the observed changes in PO₂, aK_e, pH_e, or [Ca²⁺]_e. In the respiratory network of neonatal mammals, CO₂ might provide a stimulus for long-term maintenance of respiratory activity under oxygen depletion.     

Cell membrane potential: a signal to control intracellular pH and transepithelial hydrogen ion secretion in frog kidney

The dependence of intracellular pH (pH_i) and transepithelial H⁺ secretion on the cell membrane potential (V _m) was tested applying pH-sensitive and conventional microelectrodes in giant cells fused from single epithelial cells of the diluting segment and in intact tubules of the frog kidney. An increase of extracellular K⁺ concentration from 3 to 15 mmol/l decreasedV _m from –49±4 to –29±1 mV while pH_i increased from 7.44±0.04 to 7.61±0.06. Addition of 1 mmol/l Ba²⁺ depolarizedV _m from –45±3 to –32±2 mV, paralleled by an increase of pH_i from 7.46±0.04 to 7.58±0.03. Application of 0.05 mmol/l furosemide hyperpolarizedV _m from –48±3 to –53±3 mV and decreased pH_i from 7.47±0.05 to 7.42±0.05. In the intact diluting segment of the isolated-perfused frog kidney an increase of peritubular K⁺ concentration from 3 to 15 mmol/l increased the luminal pH from 7.23±0.08 to 7.41±0.08. Addition of Ba²⁺ to the peritubular perfusate also increased luminal pH from 7.35±0.07 to 7.46±0.07. Addition of furosemide decreased luminal pH from 7.32±0.03 to 7.24±0.05. We conclude: cell depolarization reduces the driving force for the rheogenic HCO ₃ ^– exit step across the basolateral cell membrane. HCO ₃ ^– accumulates in the cytoplasm and pH_i increases. An alkaline pH_i inactivates the luminal Na⁺/H⁺ exchanger. This diminishes transepithelial H⁺ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pH_i serves as signal transducer between cell voltage and Na⁺/H⁺ exchange.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Relationship between plasma potassium and ventilation during successive periods of exercise in men

Summary During and after two successive incremental cycle ergometer tests (tests A and B), plasma potassium concentration ([K⁺]_p), plasma pH (pH_p), plasma partial pressure of carbon dioxide, blood lactate concentration ([Lac^–]_b) and ventilation (V_E) were measured. While there was a good correlation between the increase in [K⁺]_p and V_E or pH_p, respectively, in test A, in test B a close correlation was found only between the increase in V_E and [K⁺]_p (r>0.9 for nearly all single cases; r was 0.84 and 0.89 for all (pooled) cases in tests A and B, respectively; the correlation coefficients between changes in pH_p and V_E in tests A and B were r=0.74 and r=0.28, respectively, and r=0.89 and r=0.10 between the changes in [Lac^–]_b and V_E in tests A and B). The close relationship for individuals between V_E and [K⁺]_p in tests A and B supported the hypothesis that the extracellular increase in [K⁺] may contribute to the ventilatory drive during exercise. The comparison of the results of tests A and B further indicated that the relationship between pH_p and V_E was dependent on the experimental design, and that pH_p and V_E changes are unlikely to be cause and effect.The study was carried out in the Centre of Physiology, Department of Sports- and Exercise Physiology, Medical School, W-3000 Hannover, Federal Republic of Germany     

Measurements of potassium changes in the cat carotid body under hypoxia and hypercapnia

With the aid of potassium-sensitive microclectrodes reinforced by bitumen (tip diameter, 1.5 m), extracellular potassium activity ([K⁺]_e) and DC potential were measured in the cat's carotid body. Under normoxic and normocapnic conditions, potassium values of 1–16 mM (mean value 7.2 mM, standard deviation 3.8 mM) and DC potential values of –11 mV to +13 mV were recorded. With hypoxia, [K⁺]_e increased by between 1 mM and 9 mM; DC potential was reduced by between 0.5 and 3 mV. With hypercapnia, [K⁺]_e decreased by between 1 mM and 5 mM: changes in DC potential were variable. The results suggest that, during hypoxia, potassium influences the nervous structures in the carotid body whereas this influence is absent during hypercapnia.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

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.     

Red cell hemoglobin,hydrogen ion and electrolyte concentrations during exercise in trained and untrained subjects

Summary Red cell concentrations of hemoglobin (MCHC), H⁺, Na⁺, K⁺, Mg⁺⁺, Cl^– were measured in femoral venous blood of six untrained (UT), six endurance trained (TR) and three semitrained (ST) subjects during graded increasing work (4, 8, 12, 18 and 24 mkp/s, 10–15 min on each step) on a bicycle ergometer. Before exercise no significant differences were detected for the measured variables when comparing UT and TR. During exercise MCHC, [Na⁺], [K⁺] and [Mg⁺⁺] remained constant indicating lack of water shift into the erythrocytes in spite of a marked acidosis (lowest pH_Blood value 7.225). This lack resulted from an elevated extracellular osmolality. [H⁺]_Ery and [Cl^–]_Ery maximally increased by 2.0×10^–8 eq/kg H₂O and 10 meq/l, respectively. The change was markedly greater in UT than in TR at equal load. However, if [H⁺]_Ery and [Cl^–]_Ery were related to pH of whole blood, differences between groups almost disappeared and the ions were distributed as predictable from in vitro experiments (Fitzsimmons and Sendroy, 1961). Behaviour of H⁺ and Cl^– may be of importance for oxygen dissociation under in vivo conditions.Supported by Bundesinstitut für Sportwissenschaften, Köln     

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)     

Extracellular potassium and blood flow in the post-ischemic rat brain

The concentration of extracellular potassium, [K⁺]_e, was measured in parietal cortex and basal ganglia of rats during and after ten minutes of complete cerebral ischemia. The post-ischemic normalization of [K⁺]_e was considerably delayed in parietal cortex compared to basal ganglia, but in both regions, [K⁺]_e reached its normal concentration within 4 min of the end of the ischemia. Also, in both regions blood flow was elevated at the time of maximal [K⁺]₃ decrease. Our findings suggest that the normalization of [K⁺]_e and cerebrovascular resistance after ischemia are related by positive feed-back, possibly via the stimulation of Na⁺–K⁺-ATPase.Presented in part at the meeting of the Scandinavian Physiological Society, Odense, Denmark, November 1978 (Hansen et al. 1979), and at the Symposium on Cerebral Metabolism and Neural Function, US National Institute of Neurological and Communative Disorders and Stroke, Bethesda, Maryland, May 1979 (Gjedde et al. 1980a)     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

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

Intracellular pH (pH_i) 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 (V _t) and resistance (R _t) as well as serosal cell membrane potential (V _s) and pH_i were continuously recorded at rest (0.1 mmol/l cimetidine) or during stimulation (0.5 mmol/l histamine). During chamber perfusion with HCO3 ₃ ^– /CO₂-buffered Ringer solution of pH_o=7.36, V _t and R _t were –21.7, SD±6.0 mV and 229±83 cm²(n=17) while V _s and pH_i averaged –7.3±6.9 mV and 7.4±0.11 (n=25). The latter value is considerably more alkaline than all recent pH_i measurements obtained with microspectrofluorometric techniques on isolated cells, glands or intact tissue. The difference may in part be explained by use of HCO ₃ ^– -free solutions in most of the previous studies because we observed that such solutions decrease pH_i to 6.89±0.18 (n=4). Again, in contrast to recent literature, application of histamine in HCO ₃ ^– /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. Alternatively, it may result from histamine-induced activation or inactivation of other ion transporters, one possibility being activation of basolateral Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchangers.     

pH regulation of voltage-dependent K+ channels in canine pulmonary arterial smooth muscle cells

 Voltage-dependent K⁺ currents (K_v) may play a role in hypoxic pulmonary vaso constriction. The effects of changes in extracellular pH (pH_o) and intracellular pH (pH_i) on K_v currents in smooth muscle cells isolated from canine pulmonary artery were studied using the amphotericin B perforated-patch technique for whole-cell recording. Under these conditions, cellular mechanisms for pH_i regulation remain intact, and the effects of pH_o were examined by directly changing the pH of external solutions and changes in pH_i were produced by external application of weak extracellular acids and bases and the cation/H⁺ ionophore, nigericin. Ca²⁺-free external solutions were used to isolate whole-cell K_v currents from contaminating Ca²⁺-activated K⁺ currents. Extracellular acidification (pH_o = 6.4–7.0) reduced K_v currents, produced a positive voltage shift in steady-state activation and reduced maximum K_v conductance (-g _K). Extracellular alkalinization (pH_o = 8.0–8.4) increased K_v currents, produced a small negative voltage shift in steady-state activation, and increased -g _K. Intracellular acidification produced by exposure of cells to external sodium butyrate (20 mM) or nigericin (5 μg/ml) increased K_v currents, produced a negative voltage shift in steady-state activation, and increased -g _K. Intracellular alkalinization produced by exposure of cells to external trimethylamine (20 mM) reduced K_v currents, produced a small positive voltage shift in steady-state activation and reduced -g _K. These results suggest that the effects of pH_o and pH_i on K_v currents are distinctly different, but are consistent with reported effects of pH_o and pH_i on hypoxic pulmonary vasoconstriction, suggesting that such modulation may be mediated in part by pH-induced alterations in K_v channel activity. Received: 1 November 1996 / Received after revision: 19 December 1996 / Accepted: 3 January 1997     

Intracellular pH in depolarized cardiac Purkinje strands

In isolated sheep cardiac Purkinje strands the effect of membrane depolarization on intracellular pH (pH_i) and on pH_i changes produced by addition and withdrawal of NH ₄ ⁺ and CO₂/HCO ₃ ^– was investigated. pH_i was continuously measured with double-barreled glass microelectrodes. Repetitive stimulation at high rate resulted in a moderate intracellular acidification (approximately 0.03 pH unit after a 3 Hz train of 2 min), whereafter pH_i returned toward its pre-stimulus level. Prolonged depolarization, evoked either by current injection or by superfusion with high K⁺ solutions, was accompanied by a small acid shift. In the depolarized cell, addition of NH ₄ ⁺ to the superfusate caused intracellular alkalinization followed by re-acidification which was slower than at normal membrane potential. Following intracellular acidification caused by withdrawal of NH ₄ ⁺ , pH_i recovery also was slightly slower than in the normally polarized cell. In the depolarized fiber, removal and readdition of CO₂/HCO ₃ ^– produced the expected intracellular alkalinization and acidification respectively. Recovery from CO₂-induced acidosis was slowed somewhat in high K⁺ (low Na⁺) superfused fibers, not in current depolarized fibers. In the depolarized cell, steady state pH_i in CO₂/HCO ₃ ^– containing and in CO₂/HCO ₃ ^– free solution tended to become identical. These experiments support the hypothesis that in the normally polarized Purkinje fiber passive shuttle movement of NH ₄ ⁺ /NH₃ and CO₂/HCO ₃ ^– occurs and could perhaps at least be partly responsible for the lower steady state pH_i as compared to that reached in NH ₄ ⁺ -free and CO₂/HCO ₃ ^– -free solutions respectively.     

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.     

Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex

Voltage-gated Ca²⁺-current was identified in single isolated cells of the zona glomerulosa of adrenal cortex and its properties were studied by the tight-seal whole cell recording technique. The Ca²⁺-channel current was dissected from the net current by dialyzing the cells with CsCl. The identified Ca²⁺-current was found to be activated by a relatively small depolarization only when the cells were held at a large negative holding potential, but it was inactivated within 10–30 ms. The time course of activation and inactivation was voltage-dependent and became faster when the amplitude of depolarization was increased. The transmembrane potential of the glomerulosa cells was highly sensitive to [K⁺]_e, the slope of the potential change per tenfold change in [K⁺]_e being 48 mV. An increase in [K⁺]_e from 4.7 to 10 mM induce a membrane depolarization by 15 mV, which was sufficient to cause the membrane to reach the threshold potential (–60 mV) for activation of the Ca²⁺-current at physiological concentration of [Ca²⁺]_e (2.5 mM –CaCl₂). The observed properties of the Ca²⁺-current and K⁺-dependence of the membrane potential may give reasonable explanation for the mechanism of Ca²⁺-uptake and consequent aldosterone secretion induced by a small increase in [K⁺]_e, which is known to be one of the major stimulations for aldosterone secretion.     

Assessment of evidence for K+-H+ exchange in isolated type-1 cells of neonatal rat carotid body

 Intracellular pH (pH_i) was measured in enzymically isolated, neonatal rat carotid body type-1 cells, using the fluorophore carboxy-SNARF-1 (AM-loaded), and using the nigericin technique for in situ fluorescence calibration (nigericin is a membrane-soluble K⁺-H⁺ exchanger). In CO₂/HCO₃ ^–-free media, inhibiting Na⁺-H⁺ exchange produced a prompt fall of pH_i (background acid-loading), the rate of which was reduced by raising the extracellular K⁺ concentration, [K⁺]_o. pH_i recovery from an intracellular acid or alkali load was also sensitive to changes of [K⁺]_o. These results are similar to those of Wilding et al. (J Gen Physiol 100:593–608, 1992), who proposed the existence of an acid-loading, K⁺-H⁺ exchanger (KHE) in the type-1 cell. However, when nigericin was not used for post-experimental calibration, and the superfusion system was flushed exhaustively with strong detergent, alcohol and distilled water, then background acid-loading was attenuated, and the K⁺ _o sensitivity of pH_i insignificant. Background loading was increased again, and K⁺ _o sensitivity restored, when cells were monitored in a superfusion system which had previously been exposed to a single nigericin-calibration protocol (followed by a short system wash with strong detergent and distilled water). We conclude that the previously reported expression of KHE in carotid body type-1 cells is an artefact caused by nigericin contamination. We have therefore quantified the pH_i dependence of background loading in uncontaminated type-1 cells. We consider the possible implications of our work for reports of KHE in other cell types. Received: 3 March 1997 / Received after revision: 1 April 1997 / Accepted: 2 April 1997     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.