The effect of short-term fluctuations in pH on NO−3 uptake and intracellular constituents in Skeletonemacostatum (Grev.) Cleve

Measurements of uptake rates, intracellular nitrogen pools, and other key intracellular constituents were made during exponential growth in Skeletonema costatum (Grev.) Cleve under varying pH levels. An understanding of the overall effects of extracellular pH on the above mentioned cellular parameters is crucial in order to ascertain the degree to which pH must be regulated and monitored in laboratory experiments with marine phytoplankton.It was found that uptake rates and intracellular pool sizes of NO^?₃ were directly influenced by the extracellular pH level, whereas, other cellular compounds remained relatively unchanged. Therefore, nitrogen uptake and intracellular nitrogen storage are dependent on key H⁺ and OH_? ion transport mechanisms that are associated with phytoplankton metabolism. These findings reiterate the fact that investigators examining nitrogen uptake and assimilatory mechanisms in marine phytoplankton must be conscious of cellular H ⁺ and OH^? fluxes that contribute to intracellular pH regulation and changes in extracellular pH levels, both of which interact to affect phytoplankton metabolic processes.     

Dynamic Volume Changes in Astrocytes Are an Intrinsic Phenomenon Mediated by Bicarbonate Ion Flux

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K⁺]_o) and pH homeostasis. Pathological brain states that result in [K⁺]_o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K⁺]_o (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO₃₋) ions as swelling was significantly decreased when the influx of HCO₃₋ was reduced. We found: 1) in HCO₃₋ free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO₃₋ efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO₃₋ mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink −7.7±0.5% of control in response to the GABA_A channel agonist muscimol. Astrocyte shrinkage from GABA_A channel activation was significantly decreased to −5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.     

A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

Astrocytes fulfill a central role in regulating K⁺ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na⁺ ions and one proton and the efflux of one K⁺ ion. Thus, intracellular K⁺ concentration ([K⁺]_i) is potentially influenced both by extracellular K⁺ concentration ([K⁺]_o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K⁺ ion movements on [K⁺]_i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K⁺]_i using the recently developed K⁺ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K⁺]_i at 133±1 mM. We first investigated the dependency of [K⁺]_i levels on [K⁺]_o. We found that [K⁺]_i followed [K⁺]_o changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K⁺ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K⁺]_i that depended on the glutamate concentration with an apparent EC₅₀ of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K⁺]_i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K⁺ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K⁺]_i regulation with excellent spatial resolution.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

Influx and efflux of inorganic carbon in Synechococcus UTEX625

The CO₂ and HCO₃^? fluxes in air-grown cells of Synechococcus UTEX 625 al pH 8-0 were measured during dark to light and light to dark transitions using a mass spectrometer and sampling of the reaction medium. The kinetic parameters for initial uptake of CO₂ and HCO₃^? were determined during the initial period of illumination. The development of the internal C_i pool was followed up to steady-state photosynthesis, which occurred when the size of the internal inorganic carbon pool remained apparently constant for a limited period. The experimental procedure confirmed that only CO₂ transport occurred with 100mmolm^?3 Na⁺ and that both CO₂ and HCO^?3 transport occurred with 25molm^?3 Na⁺. The K_1/2 values of initial CO₂ and HCO₃ uptake were 0.7 and 17.2 mmolm^?3respectively and agreed closely with the K_1/2 values of net CO₂ and HCO₃^? transport during steady-state photosynthesis, which were 0.66 and 17.1 mmolm^?3 respectively. Maximum rates of CO₂and HCO₃^? transport were 423 and 219mmolh^?1 g^?1 Chl. Maximum CO₂ efflux observed upon darkening was 118mmolh^?1 g^?1 Chl. A permeability coefficient of the cell for CO₂ of 3 × 10^?8 m s^?1 was determined from the dark CO₂ efflux assuming an internal pH of 7.2 in the dark. Following the initial CO₂ uptake in the light, the extracellular [CO₂] steadily declined when only CO₂ transport was allowed, but an increase in the extracellular [CO₂] when HCO₃^? transport was allowed to proceed suggested that an enhanced CO₂ efflux occurred as a result of the larger size of the intracellular C_i pool.     

Photosynthesis and inorganic carbon acquisition in the cyanobacterium Chlorogloeopsis sp. ATCC 27193

The ability of the morphologically complex cyanobacterium Chlorogloeopsis sp. ATCC 27193 to actively transport and accumulate inorganic carbon (C₁= CO₂+ HCO₃^?+ CO₃^2?) for photosynthetic CO₂ fixation was investigated. Mass-spectrometric assays revealed that Chlorogloeopsis cells grown under C₁ limitation rapidly took up CO₂ from the medium in a light-dependent reaction which was independent of CO₂ fixation. Ethoxyzolamide, a carbonic anhydrase (CA) inhibitor, inhibited CO₂ transport. Since electrometric and mass-spectrometric assays did not detect the presence of a periplasmic CA, it is suggested that CO₂ transport was mediated by a CA-like activity which converted CO₂ to HCO₃^? during passage across the membrane. Radiochemical assays, using H¹⁴CO₃ as substrate, showed that C₃-limited cells also had a high affinity (K_0.5 HCO₃^?= 37 μM), Na⁺-independent HCO₃^? uptake mechanism. HCO₃^?uptake was light dependent and occurred against its electrochemical potential indicating a carrier-mediated, active transport process. The rate of Na⁺-independent HCO₃^? transport was sufficient to account for the steady state rate of CO₂ fixation. Although not absolutely required. Na⁺ did specifically enhance the rate of HCO₃^? transport by up to 2-fold, but had no effect on the apparent affinity of the transport system for HCO₃^? Combined CO₂ and HCO₃^? transport resulted in C₁ accumulation as high as 25 mM and in excess of 300 times the external concentration. The C₁ pool was the source of CO₂ for photo-synthetic fixation and was generated, presumably, by the dehydration of HCO₃^? catalyzed by an intracellular CA. The collective evidence indicates that Chlorogloeopsis has a physiologically functional CO₂-concentrating mechanism which is essential for photosynthesis.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Acid-base regulation during nitrate assimilation in Hydrodictyon africanum

Abstract The acid-base balance during NO₃^? assimilation in Hydrodictyon africanum has been investigated during growth from (1) an analysis of the elemental composition of the cells, (2) the alkalinity of the ash and (3) the net H⁺ changes in the medium during growth. These investigations agree in showing that some 0.25 excess organic negative charges are generated per N assimilation from No₃^? as N-source and C02 as C-source; the excess OH^? (0.75 OH^? per NO₃^? assimilated) appears in the medium. Approximately half of the excess organic negative charge is attributable to cell wall uronates; the remainder is intracellular. All of the excess OH^? appearing in the medium must have crossed the plasmalemma (as net downhill H⁺ influx or OH^? efflux). Previous work has shown that the value of ψco is more negative than ψ_K⁺ during NO₃^? assimilation, suggesting that the active electrogenic H⁺ extrusion pump is still operative despite the net downhill H⁺ influx. The interpretation of this in terms of H⁺?NO₃^? symport which causes the entry of more H⁺ than is consumed in NO₃^? metabolism, with extrusion of the excess H⁺via the active, electrogenic H⁺ pump, was tested by measuring short-term H⁺ influx upon addition of NO^?₃. A net H⁺ influx occurs before NOa assimilation (as indicated by additional O₂ evolution in the light) has commenced, suggesting a mechanistic relation of H⁺ and NO₃^? influxes. This is consistent with the interpretation suggested above. Determinations of cytoplasmic pH showed no significant effect of NO₃^? assimilation, suggesting that cytoplasmic pH changes sufficient to change the ‘pH-regulating’ H⁺ fluxes are smaller than the errors in the determination of cytoplasmic pH.     

Effects of hyper-osmotic stress on K+ fluxes, H+ extrusion, transmembrane electric potential difference and comparison with the effects of fusicoccin

The stimulation of H⁺ extrusion by hyper-osmotic stress (0.2–0.3 M mannitol) in cultured cells of Arabidopsis thaliana (L.) Heynh. was shown to be associated with an inhibition of Cl^? efflux, whereas hypo-osmotic stress, inhibiting H⁺ extrusion, early and strongly stimulated Cl^? efflux. In this paper, we investigate the contribution of other factors [K⁺ transport and transmembrane electric potential difference (E_m)] to the hyper-osmotic-induced activation of the plasma membrane (PM) H⁺-ATPase. The effects of mannitol (MA) on K⁺ transport and on E_m were compared with those of fusicoccin (FC) since the modes of action of osmotica and of the toxin in stimulating H⁺-ATPase activity seem to differ at least in some steps. The changes in H⁺ extrusion induced by hyper- or hypo-osmotic stress were opposite and could be reversed by the application of the respective opposite stress. The effect of MA on H⁺ extrusion was dependent on the presence of K⁺ (or Rb⁺) similarly to that of FC, while Na⁺ and Li⁺, which also stimulated the FC effect, were ineffective on that of MA. The MA effect was independent of the anions (Cl^?, SO₄^2?, NO₃^?) accompanying K⁺. K⁺ net uptake and K⁺ influx were stimulated by both MA and FC. Tetraethylammonium (TEA⁺) and Cs⁺ inhibited both MA- and FC-induced H⁺ extrusion, suggesting the involvement of K⁺ channels. MA (0.2 M) induced a strong hyperpolarization of E_m both in the absence and in the presence of K⁺. The hyperpolarizing effect of MA was also found when the cells were already hyperpolarized by FC, and was rapidly reversed by removing the osmoticum from the medium. In the presence of the lipophilic cation tributylbenzylammonium (TBBA⁺), MA was no longer able to stimulate H⁺ extrusion, while FC still stimulated it. In cells pretreated with TBBA⁺, which strongly depolarized E_m, the subsequent addition of FC repolarized it, while the hyperpolarizing effect of MA was lacking. On the contrary, in cells pretreated with Erythrosine B (EB), E_m was strongly depolarized and the following addition of FC did not hyperpolarize it, while the hyperpolarizing effect of MA was still observed. These results suggest that the mechanism of MA in activating H⁺ extrusion and K⁺ uptake is different from that of FC. The rise in net K⁺ uptake seems to be driven by the activation of some hyperpolarizing system that does not seem to depend on a direct activation of PM H⁺-ATPase, but rather on the inhibition of Cl^? efflux induced by hyper-osmotic stress.     

Sodium-Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit

Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na⁺-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO₃^? plus 95% O₂/5% CO₂. Intracellular pH (pH_i) was determined from the steady-state distribution of [¹⁴C]benzoate. Recovery after acidification with NH₄Cl was rapid (t_1/2= 5 min) and was dependent on external Na⁺ (EC₅₀= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na⁺/H⁺ antiport, blocked 80% of recovery when [Na⁺] was 5 mM with IC₅₀ values of 100 nM. However, neither drug blocked recovery in normal [Na⁺]. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl^?/HCO₃^? antiports, blocked recovery of pH_i in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na⁺ channel, nor with dimethylbenzamil, an inhibitor of Na⁺/Ca²⁺ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of ²²Na⁺ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na⁺] with an EC₅₀ value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of ²²Na⁺ uptake by hexamethyleneamiloride was 60% with an IC₅₀ value of 76 nM. We conclude that both the Na⁺/H⁺ antiport and a DIDS-sensitive bicarbonate-dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na⁺/H⁺ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Effects of pH on ammonium uptake by Typha latifolia L.

The effects of solution pH on NH₄⁺ uptake kinetics and net H⁺ extrusion by Typha latifolia L. were studied during short-term (days) and long-term (weeks) exposure to pH in the range of pH 3.5–8.0. The NH₄⁺ uptake kinetics were estimated from depletion curves using a modified Michaelis-Menten model. T. latifolia was able to grow in solution culture with NH₄⁺ as the sole N source and to withstand a low medium pH for short periods (days). With prolonged exposure (weeks) to pH 3.5, however, the plants showed severe symptoms of stress and stopped growing. The solution pH affected NH₄⁺ uptake kinetics. The affinity for NH₄⁺, as quantified by the half saturation constant (K_1/2) and C_min (the NH₄⁺ concentration at which uptake ceases), decreased with pH. K_1/2 was increased from 7.1 to 19.2 mmol m^?3 and C_min from 2.0 to 5.7 mmol m^?3 by lowering the pH in steps from 8.0 to 3.5. V_max was, however, largely unaffected by pH (～22 μmol h^?1 g^?1 root dry weight). Under prolonged exposure to constant pH, growth rates were highest at PH 5.0 and 6.5. At pH 8.0 growth was slightly depressed and at pH 3.5 growth completely stopped. NH₄⁺ uptake kinetics were similar at pH 5.0, 6.5 and 8.0 whereas at pH 3.5 NH₄⁺ uptake almost completely stopped. The ratio between net H⁺ extrusion and NH₄⁺ uptake decreased significantly at low pH. The adverse effects of low pH on NH₄⁺ uptake kinetics are probably a consequence of a reduced H⁺-ATPase activity and/or an increased re-entry of H⁺ at low pH, and the associated decrease in the electrochemical gradient across the plasma membranes of the root cells.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K⁺ concentration ([K⁺]_o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pH_i) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na⁺/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na⁺, K⁺-ATPase); and (2) activation of cellular reuptake of K⁺ mediated by the Na⁺, K⁺-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pH_i is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third ‘classical’ anti-bipolar drug, ‘lithium’ increases astrocytic pH_i by a different mechanism (stimulation of the acid extruding Na⁺/H⁺ exchanger NHE1). The acid extruder fluxes, which depend upon the change in pH_i per time unit (ΔpH_i/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pH_i was determined using the fluorescent pH-sensitive indicator BCECF–AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pH_i changes/min during recovery after acid-loading with NH₃/NH₄ ⁺, NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K⁺ concentration. Energetically, K⁺ uptake is much costlier than NBCe1 activity.     

Cytosolic sodium regulation in mouse cortical astrocytes and its dependence on potassium and bicarbonate

Sodium plays a major role in different astrocytic functions, including maintenance of ion homeostasis and uptake of neurotransmitters and metabolites, which are mediated by different Na⁺-coupled transporters. In the current study, the role of an electrogenic sodium-bicarbonate cotransporter (NBCe1), a sodium-potassium-chloride transporter 1 (NKCC1) and sodium-potassium ATPase (Na⁺-K⁺-ATPase) for the maintenance of [Na⁺]_i was investigated in cultured astrocytes of wild-type (WT) and of NBCe1-deficient (NBCe1-KO) mice using the Na⁺-sensitive dye, asante sodium green-2. Our results suggest that cytosolic Na⁺ was higher in the presence of CO₂/HCO₃⁻ (15 mM) than CO₂/HCO₃⁻-free, HEPES-buffered solution in WT, but not in NBCe1-KO astrocytes (12 mM). Surprisingly, there was a strong dependence of cytosolic [Na⁺] on the extracellular [HCO₃⁻] attributable to NBCe1 activity. Pharmacological blockage of NKCC1 with bumetanide led to a robust drop in cytosolic Na⁺ in both WT and NBCe1-KO astrocytes by up to 6 mM. There was a strong dependence of the cytosolic [Na⁺] on the extracellular [K⁺]. Inhibition of the Na⁺-K⁺-ATPase led to larger increase in cytosolic Na⁺, both in the absence of K⁺ as compared with the presence of ouabain and in NBCe1-KO astrocytes as compared with WT astrocytes. Our results show that cytosolic Na⁺ in mouse cortical astrocytes can vary considerably and depends greatly on the concentrations of HCO₃⁻ and K⁺, attributable to the activity of the Na⁺-K⁺-ATPase, of NBCe1 and NKCC1.