Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

Milacemide (MLM, CP 1552 S, 2-N-pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose-effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)-ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)-ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)-ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)-ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)-ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K+)-ATPase.     

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells

Resting membrane potential (RMP), K⁺ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11–22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11–13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at ∼–70 mV and the other relatively less-polarized at ∼–40 mV. Ca²⁺_o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a ∼Nernstian relationship between RMP and [K⁺]_o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca²⁺_o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K⁺_o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU^–, tetanus toxin⁺ cells. Differentiating neurons were estimated to contain increased K⁺_i relative to less-polarized cells, coinciding with the developmental expression of Cs⁺/Ba²⁺-sensitive and Ca²⁺-dependent K⁺ channels. Both K⁺ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca²⁺_o-dependent and tetrodotoxin-insensitive to Na⁺_o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K⁺ channels and voltage-dependent Na⁺ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.     

Gap junctions equalize intracellular Na+ concentration in astrocytes

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na⁺ concentration ([Na⁺]_i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na⁺ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na⁺]_i in neighboring astrocytes was very similar (12.0 ± 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na⁺]_i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and α-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na⁺ load induced by removal of extracellular K⁺, indicating that octanol's effects on baseline [Na⁺]_i were not due to inhibition of Na⁺, K⁺-ATPase activity. Under control conditions, increasing [K⁺]_o from 3 to 8 mM caused similar decreases in [Na⁺]_i in groups of astrocytes, presumably by stimulating Na⁺, K⁺-ATPase. During octanol application, [K⁺]_o-induced [Na⁺]_i decreases were amplified in cells with increased baseline [Na⁺]_i, and reduced in cells with decreased baseline [Na⁺]_i. This suggests that baseline [Na⁺]_i in astrocytes “sets” the responsiveness of Na⁺, K⁺-ATPase to increases in [K⁺]_o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na⁺]_i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na⁺ ions to equalize baseline [Na⁺]_i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion. GLIA 20:299–307, 1997. © 1997 Wiley-Liss, Inc.     

Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na+/K+ ATPase

In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na⁺/K⁺ ATPase to maintain ionic gradients. Considering that impairment of Na⁺/K⁺ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na⁺/K⁺ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current- and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1–3 μM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 μM) or strophanthidin (30 μM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1–10 μM) and strophanthidin (1–10 μM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na⁺/K⁺ ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na⁺/K⁺ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na⁺/K⁺ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.     

Role of Voltage-gated Ca2+ Channels and Intracellular Ca2+ in Rat Sympathetic Neuron Survival and Function Promoted by High K+ and Cyclic AMP in the Presence or Absence of NGF

We have examined how NGF-dependent rat sympathetic neurons maintain Ca²⁺ homeostasis when challenged with high K⁺ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K⁺ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca²⁺ channels within 2 days. Although resting [Ca²⁺]_i was elevated by 1.6-fold, this was 50% less than the rise in [Ca²⁺]_i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca²⁺]_i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca²⁺ current, suggesting that voltage-sensitive Ca²⁺ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K⁺ medium with no significant rise in resting [Ca²⁺]_i, although neurites did not grow. Neither Ca²⁺ channel density nor resting [Ca²⁺]_i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca²⁺. However, the dihydropyridine antagonist nitrendipine blocked both high K⁺- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca²⁺]_i above resting levels to survive with either high K⁺ or CPTcAMP, but dihydropyridine-sensitive Ca²⁺ channel activity may be essential for their survival promoting actions.     

Swimming training prevents pentylenetetrazol-induced inhibition of Na+, K+-ATPase activity, seizures, and oxidative stress

Purpose: In the present study we decided to investigate whether physical exercise protects against the electrographic, oxidative, and neurochemical alterations induced by subthreshold to severe convulsive doses of pentyltetrazole (PTZ). Methods: The effect of swimming training (6 weeks) on convulsive behavior induced by PTZ (30, 45, and 60 mg/kg, i.p.) was measured and different electrographic electroencephalography (EEG) frequencies obtained from freely moving rats. After EEG recordings, reactive oxygen species (ROS) generation, nonprotein sulfhydryl (NPS), protein carbonyl, thiobarbituric acid‐reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), Na⁺, K⁺‐ATPase activity, and glutamate uptake were measured in the cerebral cortex of rats. Results: We showed that physical training increased latency and attenuated the duration of generalized seizures induced by administration of PTZ (45 mg/kg). EEG recordings showed that physical exercise decreased the spike amplitude after PTZ administration (all doses). Pearson’s correlation analysis revealed that protection of physical training against PTZ‐induced seizures strongly correlated with NPS content, Na⁺, K⁺‐ATPase activity, and glutamate‐uptake maintenance. Physical training also increased SOD activity, NPS content, attenuated ROS generation per se, and was effective against inhibition of Na⁺, K⁺‐ATPase activity induced by a subthreshold convulsive dose of PTZ (30 mg/kg). In addition, physical training protected against 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) oxidation, TBARS and protein carbonyl increase, decrease of NPS content, inhibition of SOD and catalase, and inhibition glutamate uptake induced by PTZ. Conclusions: These data suggest that effective protection of selected targets for free radical damage, such as Na⁺, K⁺‐ATPase, elicited by physical training protects against the increase of neuronal excitability and oxidative damage induced by PTZ.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

Na+—Ca2+ Exchange Currents in Cortical Neurons: Concomitant Forward and Reverse Operation and Effect of Glutamate

Na⁺-Ca²⁺ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na⁺_o-Ca²⁺_i) exchange was evoked at -40 mV by switching external 140 mM Li⁺ to 140 mM Na⁺. The voltage dependence of this current was consistent with that predicted for 3Na⁺:1Ca²⁺ exchange. As expected, the current depended on internal Ca²⁺, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na⁺ from 3 to 20 mM or switching the external solution from 140 mM Li⁺ to 30 mM Na⁺ activated outward currents, consistent with reverse (Na⁺,-Ca²⁺_o) exchange. An external Ca²⁺-sensitive current was also identified as associated with reverse Na⁺-Ca²⁺ exchange based on its internal Na⁺ dependence and sensitivity to XIP. Combined application of external Na⁺ and Ca²⁺ in the absence of internal Na⁺ triggered a 3.3–fold larger inward current than the current activated in the presence of 3 mM internal Na⁺, raising the intriguing possibility that Na⁺-Ca²⁺ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3–5 min of exposure to 100–200 μM glutamate, the forward exchanger current was significantly increased even when external Na⁺ was reduced to 100 mM, and the external Ca²⁺-activated reverse exchanger current was eliminated.     

Effects of Glutamate,N-Methyl-d-Aspartate,High Potassium,and Hypoxia on Unit Discharges in CA1 Area of Hippocampal Slices of DBA and C57 Mice

Summary We studied effects of l -glutamate, N-methyl-d -aspartate (NMDA), high K⁺, and hypoxia on spontaneous unit discharges in stratum pyramidale of CA1 region of hippocampal slices in DBA and C57 mice aged 3–4 and 5–6 weeks. Application of l -glutamate (0.5–2.0 mM), NMDA (5–20 μM), high K⁺ (8.5 mM), and a brief period of hypoxia (1 min) to the perfused artificial cerebrospinal fluid (ACSF) all produced different degrees of spontaneous high-frequency discharges from CA1 area of hippocampal slices of both DBA and C57 mice. Two types of responses recorded extracellularly occurred after these manipulations: high-frequency repetitive single spikes and bursts of multiple population spikes. The rate and type of responses from CA1 region of hippocampal slices after these manipulations were different and depended on the strain and age of mice and the nature of manipulations. In general, hippocampal slices from audiogenic seizure-susceptible DBA mice were more sensitive than those from audiogenic seizure-resistant C57 mice, and hippocampal slices from younger animals were more susceptible than those from older ones. Thus, DBA mice aged 3–4 weeks of age were most susceptible and C57 mice aged 5–6 weeks were least susceptible to all these pharmacological, ionic, and hypoxic manipulations. Bursts of multiple population spikes were the most common responses in DBA mice and in younger animals, and repetitive single spikes were the predominant responses in C57 mice and in older animals. In all groups of animals, the average spontaneous discharge rate was highest after l -glutamate perfusion, next highest after NMDA, and lowest after high K⁺ and hypoxia. The latency of the appearance of spontaneous epileptiform activity from CA1 region of hippocampal slices was long (>2 min) after NMDA perfusion and short (<1 min) after l -glutamate, high K⁺ and hypoxia. The duration of the increased spontaneous discharges was short (?1 min) after l -glutamate perfusion, long (>3 min) after high K⁺ and hypoxia, and between short and long after NMDA perfusion. These results suggest that age and strain of animal and nature of stimulus precipitate different patterns of epileptiform activity in CNS.     

Relationship between L-glutamate-regulated intracellular Na+ dynamics and ATP hydrolysis in astrocytes

Summary. Glutamate uptake into astrocytes and the resulting increase in intracellular Na⁺ (Na⁺_i) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na⁺/K⁺-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg²⁺ (Mg²⁺_i), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg²⁺_i (i.e. decreased ATP), which was reversible after glutamate washout. Na⁺_i and ATP changes were then directly compared by simultaneous Na⁺_i and Mg²⁺ imaging. Glutamate increased both parameters with different rates and blocking the Na⁺/K⁺-ATPase during the glutamate-evoked Na⁺_i response, resulted in a drop of Mg²⁺_i levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na⁺ homeostasis and ATP levels in astrocytes.     

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra-and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current-and voltage-clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage-activated, delayed K⁺ outward current and the Ca²⁺ inward current were reduced by intracellular spermine in a dose- and voltage-dependent manner. The block of the K⁺ current can be described by a voltage-dependent reaction, where one spermine molecule binds to one channel. The binding constant K_b, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R-15, 223/M and 0.64 for cell L-11, and 137/M and 0.42 for cell L-3. Apparently, more than one spermine cation is needed to block one Ca²⁺ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non-integral values between 1.34 and 2.22. The binding constant K_b and the effective valency zδ had values of 265/M and 0.64 for cell R-15, 821M and 0.56 for cell L-4, and 263/M and 0.51 for cell L-6. Intracellular spermine also blocked the Ca²⁺-activated K⁺ current induced by ionophoretic Ca²⁺-injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage-dependent membrane conductances.     

AMPA/kainate receptor activation blocks K+ currents via internal Na+ increase in mouse cultured stellate astrocytes

Cultured mouse cortical astrocytes of the stellate type were studied by using the patch-clamp technique in whole-cell configuration. The astrocytes express at least two types of outwardly rectifying K⁺ channels which mediate a transient and a sustained current. Activation of AMPA receptors by kainate leads to a substantial blockade of both types of K⁺ currents. The blockade is absent when Na⁺ is withdrawn from the external medium, suggesting that it is caused by constant Na⁺ influx through AMPA receptors. The presence of high Na⁺ solutions in the pipette induces a blockade of both K⁺ currents which is very similar to the blockade induced by kainate, supporting thus the view that the mechanism of the blockade of K⁺ channels by kainate involves Na⁺ increases in the submembrane area. The blockade occurs between 20 and 40 mM [Na⁺]_i, which is within the physiological range of [Na⁺]_i in astrocytes. The data may suggest that the blockade of K⁺ channels by high [Na⁺]_i conditions could provide a mechanism to prevent K⁺ leakage from the astrocytes into the extracellular space during periods of intense neuronal activity. GLIA 20:38-50, 1997. © 1997 Wiley-Liss, Inc.     

Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH₄⁺/NH₃. We have studied the entry of NH₄⁺/NH₃ into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mm . This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 μm ) and by removal of extracellular Cl^–. These results strongly suggest that ammonium enters the cell by cotransport of NH₄⁺ with Cl^–. Removal of extracellular Na⁺ did not abolish the NH₄⁺-induced acidification. The NH₄⁺-induced pH change was unaffected when nearly all K⁺ conductance was blocked with 5 mm Ba²⁺ showing that NH₄⁺ did not enter through Ba²⁺-sensitive ion channels. Application of 2 mm NH₄⁺ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH₃, we suggest that when NH₄⁺ entered the cells, NH₃ left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mm NH₄⁺ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl^–. We conclude that NH₄⁺ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH₄⁺(K⁺)-Cl^– cotransporter described in kidney cells.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Control of the Na+, K+-ATPase under normal and pathological conditions

The Na⁺, K⁺-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (K _D) but no change in the maximum binding capacity (B _max) for [³H]ouabain binding to the cerebromicrovascular Na⁺, K⁺-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [³H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na⁺, K⁺-ATPase is sensitive to aluminum. AlCl₃ (1–100 μM) inhibits the K⁺-dependent-p-nitrophenylphosphatase (K⁺-NPPase) activity of the Na⁺, K⁺-ATPase in a dose-dependent manner. AlCl₃ (100 μM) decreases theV _max by 14% but does not alter theK _M, suggestive of noncompetitive inhibition. The enzyme from aged brain displays a greaterV _max, but shows the same susceptibility to AlCl₃ as the enzyme from younger brain. In summary, disruption of the Na⁺, K⁺-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Biogenesis of surface domains in fly photoreceptor cells: Fine-structural analysis of the plasma membrane and immunolocalization of Na+,K+ ATPase and α-spectrin during cell differentiation

Electron microscopic examination of pupal and adult blowfly (Calliphora erythrocephala) retina provides novel details on the biogenesis of the photoreceptor surface, particularly regarding the development of the microvillar rhabdomere and associated structures, such as the submicrovillar endoplasmic reticulum. Localization of the Na⁺,K⁺-ATPase on the surface of developing photoreceptors has also been examined by immunofluorescence confocal microscopy and immunogold electron microscopy. Na⁺,K⁺-ATPase has a nonpolarized distribution in midpupal photoreceptors that are determined by fate but that are not yet completely differentiated. Large amounts of Na⁺,K⁺-ATPase are synthesized and delivered to the cell surface throughout the second half of pupal life. At certain time points in late pupal development, specific membrane domains become cleared of Na⁺,K⁺-ATPase in the photoreceptors R1–R6. However, the distribution of Na⁺,K⁺-ATPase remains nonpolarized in R7/R8, even after eclosion. Because the membrane-associated cytoskeleton plays a direct role in the establishment and maintenance of membrane domains in a variety of systems, it is of interest to study the distribution of α-spectrin and its possible association with Na⁺,K⁺-ATPase. The localization of α-spectrin resembles the distribution pattern of Na⁺,K⁺-ATPase in midpupal and adult photoreceptors. However, changes in Na⁺,K⁺-ATPase localization in late pupal photoreceptors precede the redistribution of α-spectrin by several days. Biochemical studies of cellular membranes demonstrate further that Na⁺,K⁺-ATPase can be solubilized by Triton X-100, although α-spectrin remains in a macromolecular complex. These results indicate that the development and the maintenance of the polarized Na⁺,K⁺-ATPase distribution in blowfly photoreceptors are not tightly coupled to α-spectrin. © 1997 Wiley-Liss Inc.     

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Initial clearance of extracellular K⁺ ([K⁺]_o) following neuronal excitation occurs by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺,K⁺-ATPases. Subsequently, astrocytic K⁺ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K⁺]_o increase and allowing neuronal Na⁺,K⁺-ATPase-mediated re-uptake. Na⁺,K⁺-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K⁺]_o increases to open L-channels for Ca²⁺, activating [K⁺]_o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K⁺]_o is decreased to levels mainly affecting astrocytic membrane potential or Na⁺,K⁺-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K⁺]_o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.