Oxygen supply and ion homeostasis of the respiratory network in the in vitro perfused brainstem of adult rats

An in vitro arterially perfused medulla preparation of 3- to 8-week-old rats is described in which synchronous rhythmic activity (frequency 4.5 ± 1.7 cycles/min, burst duration 3.1 ± 1.1 s, n = 40) was recorded from hypoglossal (XII), vagal (X), or spinal (C1–2) nerves and from different classes of neurons in the region of the ventral respiratory group (VRG). Stimulation of dorsal X nerve rootlets produced a reversible blockade of rhythmic activity. Under steady-state conditions, tissue oxygen (pO₂) in the VRG (depth of 600–1600 m below the ventral surface) fell from 180 to 40 mmHg. Extracellular K⁺ activity (aK_e) in the VRG was about 0.3 mM higher, calcium concentration ([Ca]_e) did not differ, and pH (pH_e) was about 0.27 units lower than in the perfusion or superfusion solution (with an aK_e of 2.2 mM, a [Ca]_e of 1.5 mM and a pH_e of 7.4). During inspiratory XII nerve discharges, rhythmic increases of aK_e by up to 0.8 mM were detected in the VRG. Perfusion of N₂-gassed hypoxic solutions (5–10 min) resulted in a tissue anoxia of the VRG and a reversible cessation of rhythmic activity after 2–7 min. Such anoxia was accompanied by a rise of aK_e by up to 35 mM, whereas pH_e and [Ca]_e fell (from mean levels of 7.17 and of 1.5 mM, respectively) by more than 0.2 pH units and 1 mM. Similar observations were made during a 2- to 5-min arrest of the perfusion pump to simulate ischaemia, whereas significantly larger changes in aK_e, pH_e and [Ca]_e were revealed during an ischaemia period of 10 min. The results indicate that the rhythmic activity is generated by the functionally intact respiratory network of the VRG in which neurons are under aerobic conditions and ion homeostasis is not impaired. We conclude that the preparation is an appropriate in vitro model for the analysis of the cellular mechanisms for generation of respiratory rhythm and of metabolic perturbations like anoxia and ischaemia in the mature respiratory network.     

Functional relevance of anaerobic metabolism in the isolated respiratory network of newborn rats

Respiratory (C₃–C₅) activity and extracellular K⁺, pH and Ca²⁺ (aK_e, pH_e, [Ca]_e, respectively) in the ventral respiratory group (VRG) were measured in vitro. In brainstem-spinal cord preparations from 0- to 1-day-old rats, lowering of bath glucose content from 30 to 10 mM for 1 h did not affect aK_e or rhythmic activity. In preparations from 2- to 3-day-old animals, however, an aK_e rise by about 1 mM and disturbance of rhythm occurred after a delay of 50 min. Glucose-free saline resulted, after about 30 min, in reversible blockade of respiratory rhythm and an aK_e rise by more than 8 mM, whereas pH_e remained unaffected. Exposure to anoxia for 30 min after 1 h of pre-incubation in 10 mM glucose led to a progressive rise of aK_e, and a fall of [Ca]_e. The concomitant suppression of rhythm was irreversible in preparations from 2- to 3-day-old animals. Similar effects on aK_e and [Ca]_e and irreversible blockade of rhythm were revealed during anoxia in glucose-free solution, or by addition of 2–5 mM iodoacetate to oxygenated or hypoxic solutions. Iodoacetate led to a slow increase of pH_e by more than 0.2 pH units, which was accelerated by anoxia. Our findings show that normal respiratory network functions in the en bloc medulla, in particular from rats older than 1 day, depend on high bath glucose levels, necessary for effective utilization of anaerobic metabolism. Received: 18 December 1995/Accepted: 2 April 1996     

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture

The influence of changes in intra- and extracellular pH (pH_i and pH_e, respectively) on the cytosolic, free calcium concentration ([Ca²⁺]_i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pH_e, pH_i was lowered with the NH₄Cl prepulse technique, or by a transient increase in CO₂ tension, [Ca²⁺]_i invariably increased, the magnitude of the rise being proportional to pH_i. Since similar results were obtained in Ca²⁺-free solutions, the results suggest that the rise in [Ca²⁺]_i was due to calcium release from intracellular stores. The initial alkaline transient during NH₄Cl exposure was associated with a rise in [Ca²⁺]_i. However, this rise seemed to reflect influx of Ca²⁺ from the external solution. Thus, in Ca²⁺-free solution NH₄Cl exposure led to a decrease in [Ca²⁺]_i. This result and others suggest that, at constant pH_e, intracellular alkalosis reduces [Ca²⁺]_i, probably by enhancing sequestration of calcium. When cells were exposed to a CO₂ transient at reduced pH_e, Ca²⁺ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO ₃ ^- concentration was reduced at constant CO₂ tension. Unexpectedly, such results were obtained only in Ca²⁺-containing solutions. In Ca²⁺-free solutions, acidosis always raised [Ca²⁺]_i. It is suggested that a lowering of pH_e stimulates extrusion of Ca²⁺ by ATP-driven Ca²⁺/2H⁺ antiport.     

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.     

Calcium influx pathways in rat pancreatic ducts

A number of agonists increase intracellular Ca²⁺ activity, [Ca²⁺]_i, in pancreatic ducts, but the influx/efflux pathways and intracellular Ca²⁺ stores in this epithelium are unknown. The aim of the present study was to characterise the Ca²⁺ influx pathways, especially their pH sensitivity, in native pancreatic ducts stimulated by ATP and carbachol, CCH. Under control conditions both agonists led to similar changes in [Ca²⁺]_i. However, these Ca²⁺ transients, consisting of peak and plateau phases, showed different sensitivities to various experimental manoeuvres. In extracellular Ca²⁺-free solutions, the ATP-induced [Ca²⁺]_i peak decreased by 25%, but the CCH-induced peak was unaffected; both plateaus were inhibited by 90%. Flufenamate inhibited the ATP-induced peak by 35%, but not the CCH-evoked peak; the plateaus were inhibited by 75–80%. La³⁺ inhibited the ATP-induced plateau fully, but that induced by CCH by 55%. In resting ducts, an increase in extracellular pH, pH_e, by means of HEPES and HCO₃ ⁻/CO₂ buffers, increased [Ca²⁺]_i; a decrease in pH_e had the opposite effect. In stimulated ducts the pH-evoked effects on Ca²⁺ influx were more pronounced and depended on the agonist used. At pH_e 6.5 both ATP- and CCH-evoked plateaus were inhibited by about 50%. At pH 8.0 the ATP-stimulated plateau was inhibited by 27%, but that stimulated by CCH was increased by 72%. Taken together, we show that CCH stimulates Ca²⁺ release followed by Ca²⁺ influx that is moderately sensitive to flufenamate, La³⁺, depolarisation, it is inhibited by low pH, but stimulated by high pH. ATP stimulates Ca²⁺ release and probably an early Ca²⁺ influx, which is more markedly sensitive to flufenamate and La³⁺, and is both inhibited by low and high pH. Thus our study indicates that there are at least two separate Ca²⁺ influx pathways in pancreatic ducts cells. Received: 4 December 1995/Received after revision and accepted: 1 February 1996     

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.     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

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     

Untersuchungen zum zentralen chemosensiblen Mechanismus der Atmung

Summary In cats lightly anesthetized with chloralose-urethane, the respiratory response to changes in the medullary extracellular pH (pH _e ) was studied before and after cervical vagotomy. pH _e was varied by perfusion of the ventral medullary surface with mock CSF of different pH (pH_CSF) and by changes inP _ACO₂; it was calculated from the acid-base parameters of the blood and the mock CSF by means of a mathematical model described by Berndt, Berger, and Mückenhoff (1972). Results. 1. The hydrogen ion concentration in the CSF does not influence respiration at pH_CSF values below 6.5. 2. An increase of ventilation as a response to increasing extracellular hydrogen ion concentration is only obtained in a pH _e range of about 0.2 pH units, which under the conditions of the present experiments lay between pH _e 7.1 and 7.3, due to the methods of calculation employed. pH _e -values below 7.1 usually caused a decrease in ventilation. 3. Vagotomy influences the response of the tidal volume and the respiratory frequency to changes in pH _e : the tidal volume is increased after vagotomy and the slope of the pH _e -response curve ofV _T is steeper, while the frequency decreases and further diminishes with decreasing pH _e . The average response of ventilation, however, is not significantly altered after vagotomy. This confirms the hypothesis that in the case of the central chemical control of respiration the vagal activity is responsible for the partition of the total response to chemical stimuli into partial responses ofV _T andf rather than for the magnitude of the total ventilatory response.

     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

The weak bases NH3 and trimethylamine inhibit the medium and slow afterhyperpolarizations in rat CA1 pyramidal neurons

The weak bases NH₃ and trimethylamine (TMeA), applied externally, are widely used to investigate the effects of increasing intracellular pH (pH_i) on neuronal function. However, potential effects of the compounds independent from increases in pH_i are not usually considered. In whole-cell patch-clamp recordings from rat CA1 pyramidal neurons, bath application of 1–40 mM NH₄Cl or TMeA HCl reduced resting membrane potential and input resistance, inhibited the medium and slow afterhyperpolarizations (AHPs) and their respective underlying currents, mI_ahp and sI_ahp, and led to the development of depolarizing current-evoked burst firing. Examined in the presence of 1 M TTX and 5 mM TEA with 10 mM Hepes in the recording pipette, NH₃ and TMeA increased pH_i and the magnitudes of depolarization-evoked intracellular [Ca²⁺] transients, Ca²⁺-dependent depolarizing potentials, and inward Ca²⁺ currents but reduced the slow AHP and sI_ahp. When internal H⁺ buffering power was raised by including 100 mM tricine in the patch pipette, the effects of NH₃ and TMeA to increase pH_i and augment Ca²⁺ influx were attenuated whereas the reductions in the slow AHP and sI_ahp (as well as membrane potential and input resistance) were maintained. The findings indicate that increases in pH_i contribute to the increases in Ca²⁺ influx observed in the presence of NH₃ and TMeA but not to the reductions in membrane potential, input resistance or the magnitudes of AHPs. The results have implications for the interpretation of data from experiments in which pH_i is manipulated by the external application of NH₃ or TMeA.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

A comparison of the abilities of CO2/HCO 3 − , protonophores and changes in solution pH to release Ca2+ from the SR of barnacle myofibrillar bundles

Increases in solution pH from 6.5 to 7.0 to 7.5 at 0.1 M free Ca²⁺ concentration had no effect on the isometric tension of barnacle myofibrillar bundles in relaxing solutions containing 0.1–0.16 mM BAPTA. Decreases in pH in the same range were also without effect. Under the same conditions CO₂-induced Ca²⁺ release from the SR could be readily obtained by replacing the Cl^–-containing relaxing solution with one containing HCO ₃ ^– and 100% CO₂ at the same pH. At a higher free Ca²⁺ of 2.5 M, there was a contraction on increasing the pH of the Cl^–-containing solution from 7.0 to 7.5. This reponse could be abolished by 1 mM procaine suggesting that it was due to Ca²⁺ release from the SR. The protonophores monensin, gramicidin, CCCP and FCCP at concentrations of 10–100 M had no effect on resting tension at either free Ca²⁺ concentration and did not inhibit the response to 100% CO₂. It is concluded that dissipation of a possible pH gradient across the SR membrane by protonophores does not release Ca²⁺ from the SR of barnacle muscle. Since both CO₂ (by possibly lowering SR pH) and an increase in solution pH can release Ca²⁺ at 2.5 M free Ca²⁺, the existence of a Ca²⁺ release channel which is opened by a change in the trans-SR pH gradient cannot be discounted. In a separate series of experiments, the CO₂-releasable Ca²⁺ store was first depleted by exposure of bundles to 100% CO₂ in the presence of 1 mM BAPTA for 10 min and then partially reloaded by 15 s exposure to a free Ca²⁺ of 0.6 M buffered by 2 mM EGTA (total) in the Cl^– relaxing solution. The same level of reloading was obtained when the loading solution contained HCO ₃ ^–/100% CO₂; this result tends to discount inhibition of the Ca²⁺ uptake pump as a possible mechanism for CO₂-induced Ca²⁺ release. Loading at 2.6 M free Ca²⁺ for 1 min resulted in almost complete recovery of the CO₂ response to its value before depletion of the store.     

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na⁺/K⁺ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K⁺ (aK_e) and intracellular Na⁺ (aNa_i) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10⁻⁶ M) resulted in a membrane hyperpolarization and transient decreasesaK_e andaNa_i which could be blocked by ouabain (3×10⁻⁴ M). In the h.p.p. muslce a continuous rise ofaNa_i induced by elevation ofaK_e to 5.2 mM could be stopped by ADR. In addition, the intracellular K⁺ activity (aK_i), the free intracellular Ca²⁺ concentration (pCa_i) and intracellular pH (pH_i) were monitored in rat soleus muscle. During ADRaK_i increased, pH_i remained constant and intracellular Ca²⁺ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na⁺/K⁺ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. Parts of the results have been presented to the German Physiological Society (Ballanyi and Grafe 1987)     

Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling inXenopus oocytes

The relationship among intracellular pH (pH_i), –log₁₀ intracellular Ca²⁺ concentration (pCa_i) and gap junctional conductance, the participation of Ca²⁺ stores, and the role of calmodulin in channel regulation have been studied inXenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO₂. The time courses of pH_i [measured with 2,7-bis(2-carboxyethyl)-5,6-carboxylluorscein (BCECF)], (pCa_i) (measured with the membrane-associated fura-C₁₈) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with (pCa_i) than with pH_i. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) on CO₂-induced uncoupling, coupled to negative results with Ca²⁺-free external solutions, point to a low-pH_i-induced Ca²⁺ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5 h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO₂ within 72 h. The effect of CO₂ on junctional conductance was reduced by 60% in 24 h, by 76% in 48 h and by 93% in 72 h. Oocytes that had lost gating sensitivity to CO₂. partially recovered gating competency following calmodulin injection. The data suggest that lowered pH_i uncouples gap junctions by a Ca²⁺-calmodulin-mediated mechanism.     

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.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Contribution of Ca2+ inflow to quantal,phasic transmitter release from nerve terminals of frog muscle

Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca²⁺ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cd _e ²⁺ or with an intracellular, EGTA-buffered solution containing less than 0.1 M Ca _e ²⁺ . Pulse application and recording were by a perfused macro-patch-clamp electrode. The muscle outside the electrode (bath) was superfused with Ringer's solutions containing Cd _b ²⁺ to block Ca²⁺ inflow and normal (1.8mM) or elevated (10 mM) Ca _b ²⁺ . The depolarization level of the terminal during current pulses that generated maximal Ca²⁺ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca²⁺ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca²⁺ inflow is high. However, for large depolarizations (>1.5 relative units), the depression of release by block of Ca²⁺ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca²⁺ inflow. If the extra-electrode superfusion of Ca _b ²⁺ of the muscle was elevated to 10 mM and Cd _b ²⁺ was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1M Ca _e ²⁺ raised the average release paradoxically. With 0.5 mM Cd _b ²⁺ this paradoxical increase of release was, on average, 4-fold at 6 °C, and 19-fold at 16 °C. Quantal endplate currents recorded in less than 0.1 M Ca _e ²⁺ had slightly increased amplitudes, and decay time constants were prolonged by about 50%. The results are interpreted to support the Ca²⁺/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca²⁺ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca²⁺ concentration is sufficiently high, large depolarizations can elicit release independent of the presence or absence of Ca²⁺ inflow.     

On the kinetics of the potassium channel activated by acetylcholine in the S-A node of the rabbit heart

In the isolated frog spinal cord depolarization of motoneurons (MNs) induced by glutamate (GLUT) was not accompanied by measurable changes of neuronal input resistance when chemical synaptic transmission was blocked by Mn²⁺ or Mg²⁺. The GLUT depolarization was, however, paralleled by a considerable increase of K⁺ in the extracellular space.To clarify, whether the GLUT depolarization was exclusively due to a reduction of the transmembrane K⁺ gradient or whether ion conductances not detectable by measurements of neuronal input resistance were involved, membrane potential (MP) was plotted semilogarithmically versus extracellular K⁺ activity (aK_e ⁺). During experimental elevation of aK_e ⁺ the function MP/dec. aK_e ⁺ was found to agree fairly with the Nernst equation. The slope of this function was much steeper during GLUT superfusion, indicating an influx of positive ions.The elevation of aK_e ⁺ during the GLUT action can mimic postsynaptic effects by release of transmitter from presynaptic terminals synapsing with the recorded cell.In vivo preparations do not allow blockade of chemical synaptic transmission. Therefore, it is impossible to decide, whether the recorded cell is depolarized either postsynaptically by GLUT or by K⁺ release from surrouding GLUT sensitive cells.As an experimental proof of the postsynaptic GLUT action is not feasible in such preparations, the abiquitous action of GLUT in the CNS may have been overestimated.     

Imaging the intratumoral–peritumoral extracellular pH gradient of gliomas

Solid tumors have an acidic extracellular pH (pH_e) but near neutral intracellular pH (pH_i). Because acidic pH_e milieu is conducive to tumor growth and builds resistance to therapy, simultaneous mapping of pH_e inside and outside the tumor (i.e., intratumoral‐peritumoral pH_e gradient) fulfills an important need in cancer imaging. We used B iosensor I maging of R edundant D eviation in S hifts (BIRDS), which utilizes shifts of non‐exchangeable protons from macrocyclic chelates (e.g., 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrakis(methylene phosphonate) or DOTP^8?) complexed with paramagnetic thulium (Tm³⁺) ion, to generate in vivo pH_e maps in rat brains bearing 9L and RG2 tumors. Upon TmDOTP^5? infusion, MRI identified the tumor boundary by enhanced water transverse relaxation and BIRDS allowed imaging of intratumoral‐peritumoral pH_e gradients. The pH_e measured by BIRDS was compared with pH_i measured with ³¹P‐MRS. In normal tissue, pH_e was similar to pH_i, but inside the tumor pH_e was lower than pH_i. While the intratumoral pH_e was acidic for both tumor types, peritumoral pH_e varied with tumor type. The intratumoral–peritumoral pH_e gradient was much larger for 9L than RG2 tumors because in RG2 tumors acidic pH_e was found in distal peritumoral regions. The increased presence of Ki‐67 positive cells beyond the RG2 tumor border suggested that RG2 was more invasive than the 9L tumor. These results indicate that extensive acidic pH_e beyond the tumor boundary correlates with tumor cell invasion. In summary, BIRDS has sensitivity to map the in vivo intratumoral–peritumoral pH_e gradient, thereby creating preclinical applications in monitoring cancer therapeutic responses (e.g., with pH_e‐altering drugs). Copyright © 2016 John Wiley & Sons, Ltd.