Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells

A new triple-barrelled ion-sensitive microelectrode was used to investigate the importance of bicarbonate for the regulation of intracellular Na⁺ and pH (Na_i and pH_i, respectively) of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis. Addition of CO²/HCO ₃ ^– produced an increase of the Na_i activity and an intracellular alkalinization, indicating bicarbonate accumulation in the glial cells. Changes of external pH (from 7.4 to 7.0 and 7.8) produced large and rapid shifts of pH_i and Na_i and of the membrane potential in the presence, but not in the absence, of bicarbonate. Thus, acid/base transport and Na⁺ movements across the glial membrane into and out of the cells were accelerated severalfold in CO²/HCO ₃ ^– -buffered saline as compared to a CO²/HCO ₃ ^– -free, HEPES-buffered saline. The results suggest that the electrogenic, reversible, cotransport of Na⁺ and HCO ₃ ^– in the glial cell membrane [3, 9] can produce significant changes in intraglial pH and Na activity, and can carry a significant fraction of the total Na⁺ flux across the cell membrane.     

Whole-cell recording of intracellular pH with silanized and oiled patch-type single or double-barreled microelectrodes

Conventional ion-sensitive microelectrodes cannot be used in small cells, since they create too large an electrical leak at the site of penetration. Membrane potentials can be measured in such cells with the whole-cell configuration of the patch-clamp technique, after obtaining a high-resistance seal (giga-seal) to the cell membrane. Achieving such seals with patch-type microelectrodes silanized and filled with ion-sensitive cocktails has proved very difficult. Since ion-sensitive microelectrodes offer advantages over fluorescent techniques, we have developed a method which enables whole-cell recordings of membrane potential and intracellular pH to be achieved with silanized microelectrodes. We have been able to obtain high-resistance seals with silanized tips by dipping them in mineral oil. We describe the method for both single-barrel and theta-glass double-barreled microelectrodes. Double-barreled microelectrodes can be used to measure and control membrane potential in the whole-cell patch-clamp configuration while also measuring ionic activities with the adjacent barrel. We present illustrative experiments showing intracellular pH recordings in snail neurones and rat dorsal root ganglion cells, and we suggest the method can also be applied to other liquid-sensor ion-sensitive microelectrodes.     

Comparison of simultaneous pH measurements made with 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and pH-sensitive microelectrodes in snail neurones

 We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pH_i) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H⁺ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50–7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pH_i values, 7.16±0.05 (n=10) and 7.17±0.06 (n=9) respectively in nominally CO₂/HCO₃ ^–-free saline. At both extremes of the pH range there were small discrepancies. At acidic pH_i, 6.87±0.09 (n=5), the intracellular HPTS measurement differed by –0.08±0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pH_i,7.32±0.10 (n=5), HPTS measurements produced pH values that differed by +0.07±0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77±15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 μM to 2 mM, did not block activity-dependent pH_i changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00–8.00. Received: 19 February 1998 / Received after revision and accepted: 22 April 1998     

Maximal elevation of 2,3-diphosphoglycerate concentrations in human erythrocytes: Influence on glycolytic metabolism and intracellular pH

Summary The 2,3-DPG content of human red blood cells can be elevated in vitro to a maximum of 24 Moles/g by incubating the cells in media containing inosine, pyruvate and inorganic phosphate (=IPP media). The rate of accumulation depends on the extracellular phosphate level. The concentrations of organic phosphate fractions other than 2,3-DPG also increase during the initial phase of incubation in IPP media, but rediminish thereafter. As a consequence of these changes, the total concentration of acid-soluble organic phosphates in the red cells rises from 14 to 55 Moles P/g red cells. This increase of non-penetrating anions produces a shifting of the Donnan ratio H _e ⁺/H _i ⁺ to lower values and thereby diminishes progressively the intracellular pH of the red cells during incubation in IPP media. The extent of these changes can be calculated on the basis of equations derived by van Slyke.The Donnan induced intracellular acidification causes a gradual impairment of red cell metabolism as the 2,3-DPG concentration increases, thus imposing a selflimitation to the accumulation of 2,3-DPG in the presence of IPP and leading to an inhibition of glucose consumption in IPP-pretreated cells.The findings are discussed with respect to their metabolic and biophysical basis and in view of possible implications for the regulation of red cell metabolism and function.Supported by the Deutsche Forschungsgemeinschaft.     

Effects of acidic stimuli on intracellular calcium in isolated type I cells of the neonatal rat carotid body

We have investigated the effects of acidic stimuli upon [Ca²⁺]_i in isolated carotid body type I cells from the neonatal rat using indo-1 (AM-loaded). Under normocapnic, non-hypoxic conditions (23 mM HCO₃ ^–, 5% CO₂ in air, pH_o=7.4), the mean [Ca²⁺]_i for single cells was 102±5.0 nM (SEM, n=55) with 58% of cells showing sporadic [Ca²⁺]_i fluctuations. A hypercapnic acidosis (increase in CO₂ to 10%–20% at constant HCO₃ ^–, pH_o 7.15–6.85), an isohydric hypercapnia (increase in CO₂ to 10% at constant pH_o=7.4) and an isocapnic acidosis (pH_o=7.0, constant CO₂) all increased [Ca²⁺]_i in single cells and cell clusters. The averaged [Ca²⁺]_i response to both hypercapnic acidosis and isohydric hypercapnia displayed a rapid rise followed by a secondary decline. The averaged [Ca²⁺]_i response to isocapnic acidosis displayed a slower rise and little secondary decline. The rise of [Ca²⁺]_i in response to all the above stimuli can be attributed to no single factor other than to a fall of pH_i. The hypercapnia-induced rise of [Ca²⁺]_i was almost completely abolished in Ca²⁺-free solution, suggesting a role for Ca²⁺ influx in triggering and/or sustaining the [Ca²⁺]_i response. These results are consistent with a role for type I cell [Ca²⁺]_i in mediating pH/PCO₂ chemoreception.     

The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney

The present study was performed to test the influence of sodium coupled transport of neutral substrates on intracellular pH and sodium activity in proximal tubules of the amphibian kidney. To this end, kidneys of rana esculenta have been isolated and perfused both through the portal vein (peritubular capillaries) and the aorta (luminal perfusate). The potential difference across the peritubular membrane of proximal tubule cells has been redorced with conventional (PD_pt) as well as with sodium (PD_na) and hydrogen ion (PD_h) selective microelectrodes continuously before during and after the luminal application of 10 mmol/l phenylalanine, replacing 10 mmol/l raffinose. PD_b and PD_na allowed the calculation of intracellular pH (pH_i) and sodium activity (Na_i), respectively. In the absence of phenylalanine in the tubule lumen, PD_pt approximates –57.5±2.3 mV (n=27), pH_i 7.73±0.04 (n=14, extracellular pH 7.77), and Na_i 13.3±0.9 mmol/l (n=13, extracellular sodium activity 74 mmol/l). Within 1 min the luminal application of phenylalanine leads to a depolarisation of PD_pt by +32±2 mV, as well as an increase of pH_i by 0.24±0.04 and of Na_i by 5.2±1.0 mmol/l. At 8 min from luminal application of phenylalanine, Na_i plateaus 5±1 mmol/l above control value, PD_pt increases again to a value of +12±2 mV below and pH_i decreases to a value 0.04±0.07 above their respective control values. All changes are fully reversed after removal of phenylalanine from the tubule lumen. The steady state of intracellular sodium activity might be explained by an extrusion of sodium via the sodium/potassium-ATPase, which approaches the entry across the luminal membrane, the intracellular alkalinisation is probably due to the reduced exit of bicarbonate across the peritubular cell membrane following the depolarisation of PD_pt.     

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a _Na ⁱ ) and intracellular pH (pH_i) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na₂SO₄ buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10^–4M) blocks net H⁺ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (mc). We demonstrate the these changes are accompanied by a fall ina _Na ⁱ from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pH_i from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping mc to its control value in the presence of ethoxzolamide restoreda _Na ⁱ but the pH_i remained alkaline. Furthermore, the fall ina _Na ⁱ produced by ethoxzolamide could be mimicked by voltage clamping mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na⁺ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H⁺ pump at the apical membrane.Addition of amiloride (10^–5 mol/l) or substitution of external Na⁺ by Mg²⁺ or K⁺ caused a hyperpolarization of mc and a fall ina _Na ⁱ . These effects were accompanied by an inhibition of H⁺ excretion and a fall in pH_i of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na⁺ transport inhibition on mc was prevented by imposing a voltage clamp no effects of amiloride on H⁺ excretion or pH_i were observed. Moreover, the effect of amiloride on pH_i could be reproduced in control skins by voltage clamping mc to –100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10^–5 mol/l) inhibited H⁺ excretion and decreased pH_i from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.These results indicate that an apical membrane H⁺ ATPase plays a role in regulating pH_i and the mechanism is sensitive to membrane potential.     

Concurrent measurements of the free cytosolic concentrations of H+ and Na+ ions with fluorescent indicators

We report a method for the concurrent measurement of intracellular [Na+] ([Na+ ]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower p Ka in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

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.     

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland

 To investigate the role of Cl^– in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pH_i) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl^– was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl^– on the Na⁺-K⁺-2Cl^– cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl^– by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pH_i consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO₃ ^– omission, 100 μM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pH_i was due to Cl^–/HCO₃ ^– exchange while the secondary rise resulted from activation of Na⁺/H⁺ exchange. Although replacement of Cl^– by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl^– replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl^– concentration contribute to the up-regulation of the exchanger following muscarinic stimulation. Received: 12 November 1996 / Received after revision: 11 August 1997 / Accepted: 18 August 1997     

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A

The intracellular pH (pH_i) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na⁺/H⁺ exchange and a stilbene-insensitive Cl^– /HCO₃ ^– exchange mechanism have been identified in the basolateral membrane. Removal of Na⁺ from the basolateral solution caused a decrease of pH_i by 0.50±0.09 unit (n=4). Amiloride or Na⁺-free solution at the apical side had no effect on pH_i. Cl^– removal at the basolateral side led to an increase of pH_i by 0.20±0.03 unit (n=4) whereas apical removal had no influence on pH_i. This effect was independent of Na⁺ and was insensitive to 0.2 mM 4,4-diisothiocyanatodihydrostilbene-2, 2-disulphonic acid. A basolateral Cl^–/ HCO₃ ^– exchanger is the most likely explanation for this observation. The Na⁺/H⁺ exchange mechanism in the basolateral membrane is an acid extruder, whereas the C1^–/HCO₃ ^– exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.     

Lactate/proton co-transport in skeletal muscle: regulation and importance for pH homeostasis

In skeletal muscle, intracellular pH is more alkaline than would be predicted if H⁺ were passively distributed across the sarcolemma. Therefore, the passive influx of H⁺ must be counteracted by transport processes mediating H⁺ efflux. In resting skeletal muscle, these transport processes are Na⁺/H⁺ exchange and bicarbonate-dependent systems. During periods of high energy demand, skeletal muscle produces large amounts of lactic acid. The internal accumulation of lactic acid reduces pH, which may cause fatigue. It is therefore important for muscle cells to be able to regulate pH during and after activity. A part of the accumulated lactate and H⁺ is metabolized, but a considerable fraction is released from the cell. The efflux of H⁺ and lactate might be mediated by the lactate/proton co-transport system found in almost all cell types in the body. The role of lactate/proton co-transport in pH regulation has been studied both with intact cells and with sarcolemmal vesicles. In intact cells, inhibitors of lactate/proton transport have been shown to accelerate the development of fatigue, and to delay the recovery after activity. A comparison with vesicles has demonstrated that, at low pH, and with a high lactate concentration, the capacity for H⁺ removal is higher via the lactate/proton co-transport system than via the sum of the Na⁺/H⁺ exchange and bicarbonate-dependent exchange systems. Therefore, the carrier-mediated lactate/proton efflux is of major importance for pH regulation in connection with muscle activity. The lactate/proton transport system has been shown to undergo long-term changes depending on the level of physical activity. The capacity of the system was enhanced after intense training or chronic stimulation, and reduced after denervation. It is concluded that the lactate/proton transport system is of major importance for pH regulation in skeletal muscle, and that changes in the amount of transporters are one of the many adaptations to physical activity.     

Effects of intracellular pH and Ca2+ on the activity of stretch-sensitive cation channels in leech neurons

The effects of intracellular pH and calcium on the activity of the leech mechanosensitive cation channels have been studied. These channels exhibited two activity modes denoted as spike-like (SL) and multiconductance (MC). In the absence of mechanical stimulation, acidification of the intracellular side of membrane patches from 7.2 to 6.2 reversibly increased the mean channel open time as well as the opening frequency in the SL mode. Channels in MC mode were activated by a pH_i reduction from 7.2 to 6.2, but were inhibited at pH_i 5.5. Unlike MC mode, SL mode was strongly activated by intracellular Ca²⁺. Fura-2 imaging experiments showed that intracellular calcium was induced to increase by hypotonic cell swelling. The major component of this response did not require extracellular calcium. A component of the swelling-induced calcium response was sensitive to blockers of stretch-sensitive cation channels. The results indicate that the two activity modes of mechanosensitive channels of leech neurons respond differently to changes of intracellular pH and calcium. The sensitivity of the channel to micromolar concentrations of internal free calcium, along with its permeability to this ion, is consistent with a role in the amplification of mechanically induced Ca²⁺ signals in leech neurons.     

Role of physiological HCO3 –buffer on intracellular pH and histamine release in rat peritoneal mast cells

 The purpose of this study was to examine how intracellular pH (pH_i) regulation and histamine release are affected by HCO₃ ^–in rat peritoneal mast cells. The pH_i was measured using the pH-sensitive dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pH_i of 6.88±0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67±0.015 (n=23) when we exposed the mast cells to a HEPES/HCO₃ ^–buffer equilibrated at all time with 5% CO₂ (pH 7.4). This fall in pH_i is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na⁺-independent, indicating the involvement of Na⁺-independent Cl^–/HCO₃ ^–exchange activity. Furthermore removal of external Cl^–in the presence but not in the absence of HCO₃ ^–reversed the Cl^–/HCO₃ ^–exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl^–but independent of Na⁺. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). In addition, ³⁶Cl^–uptake measurements confirm the presence of a Cl^–/HCO₃ ^–exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO₃ ^–buffer (pH_o 7.4, pH_i 6.66). Histamine release was increased, however, when pH_i was clamped to 6.66 in HCO₃ ^–-free media (pH_o 6.9). We conclude that: (1) Na⁺-independent Cl^–/HCO₃ ^–exchange determines steady-state pH_i in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO₃ ^–is not due to its effect on pH_i per se, but rather on other changes in ion transport. Received: 29 January 1998 / Received after revision and accepted: 3 April 1998     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones

1. Intracellular pH (pH(i)), Cl(-) and Na(+) levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH(i) recovery from internal acidification investigated.2. Reducing the external HCO(3) (-) concentration greatly inhibited the rate of pH(i) recovery from HCl injection.3. Reducing external Cl(-) did not inhibit pH(i) recovery, but reducing internal Cl(-), by exposing the cell to sulphate Ringer, inhibited pH(i) recovery from CO(2) application.4. During pH(i) recovery from CO(2) application the internal Cl(-) concentration decreased. The measured fall in internal Cl(-) concentration averaged about 25% of the calculated increase in internal HCO(3) (-).5. Removal of external Na inhibited the pH(i) recovery from either CO(2) application or HCl injection.6. During the pH(i) recovery from acidification there was an increase in the internal Na(+) concentration ([Na(+)](i)). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na(+)](i) that occurred during pH(i) recovery from an injection of HCl was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH(i) recovery after HCl injection were not additive.9. It is concluded that the pH(i) regulating system involves tightly linked Cl(-)-HCO(3) (-) and Na(+)-H(+) exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO(3) (-) and the movement outward of Cl(-) and H(+) ions.     

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.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Comparison of the Myotoxic Effects of Levobupivacaine,Bupivacaine, and Ropivacaine: An Electron Microscopic Study

The aim of this study was to investigate the myotoxic effects of bupivacaine, ropivacaine, and levobupivacaine which were applied intramuscularly to rat skeletal muscle. Forty Wistar-Albino rats were divided into four groups. In the study, .5% bupivacaine (Group B), .5% ropivacaine (Group R), .5% levobupivacaine (Group L), or .9% normal saline (Group SF) was applied intramuscularly to the right gastrocnemius muscle of rats. The rats in each group were sacrificed on the second day after injection. Sections of muscle samples were stained with hematoxylin–eosin for light microscopic investigation and prepared for the evaluation of ultrastructural changes in the subcellular level with transmission electron microscopy. All three local anesthetic agents caused qualitatively similar skeletal muscle damage. The most observed muscle damage was in Group B, muscle damage of Group R was less than that of Group B, and the least damage was seen in Group L quantitatively. Electron microscopic examination of each group that caused cellular damage was qualitatively similar. The most subcellular damage was observed in the group receiving bupivacaine, less was seen in the ropivacaine group, and the least was observed in the levobupivacaine group. The results indicated that bupivacaine caused more myotoxic damage than the other two agents in the skeletal muscle of rats and that levobupivacaine caused less myotoxic damage than both bupivacaine and ropivacaine at the cell and tissue levels.