NMR-Based Identification of Intra- and Extracellular Compartments of the Brain Pi Peak

Abstract: The P_i peak in a ³¹P NMR spectrum of the brain can be deconvoluted into six separate Lorentzian peaks with the same linewidth as that of the phosphocreatine peak in the spectrum. In an earlier communication we showed that the six P_i peaks in normal brain represent two extracellular and four intracellular compartments. In that report we have identified the first of the extracellular peaks by marking plasma with infused P_i, thereby substantially increasing the amplitude of the single peak at pH 7.35. 2-Deoxyglucose-6-phosphate (2-DG-6-P) was placed in the brain interstitial space by microdialysis. The resulting 2-DG-6-P peak was deconvoluted into three separate peaks. The chemical shift of the principle 2-DG-6-P peak gave a calculated pH of 7.24 ± 0.02 for interstitial fluid pH, a value that agreed well with the pH of the second extracellular P_i peak at pH 7.25 ± 0.01. We identified the intracellular compartments by selectively stressing cellular energy metabolism in three of the four intracellular spaces. A seizure-producing chemical, flurothyl, was used to activate the neuron, thereby causing a demand for energy that could not be completely met by oxidative phosphorylation alone. The resulting loss of high-energy phosphate reserves caused a significant increase in intracellular P_i only in those cells associated with the P_i peak at pH 6.95 ± 0.01. This suggests that this compartment represents the neuron. Ammonia is detoxified in the astrocyte (glutamine synthetase) by incorporating it into glutamine, a process that requires large amounts of glucose and ATP. The intraarterial infusion of ammonium acetate into the brain stressed astrocyte energy metabolism resulting in an increase in the P_i of the cells at pH of 7.05 ± 0.01 and 7.15 ± 0.02. This finding, coupled with our observation that these same cells take up infused P_i probably via the astrocyte end-foot processes, lead us to conclude that these two compartments represent two different types of astrocytes, probably protoplasmic and fibrous, respectively. As a result of this study, we now believe the brain contains four extracellular and four intracellular compartments.     

In Vivo Microdialysis of 2-Deoxyglucose 6-Phosphate into Brain

Abstract : A unique method for simultaneously measuring interstitial (pH_e) as well as intracellular (pH_i) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of ³¹P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the ³¹P spectrum. pH_e and pH_i values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pH_e was 7.24 ± 0.01, whereas the average pH_i was 7.05 ± 0.01 (n = 7). The average pH_e value and the average CSF bicarbonate value (23.5 ± 0.1 mEq/L) were used to calculate an interstitial Pco₂ of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO₂ (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (Pco₂ < 30 mm Hg), by increasing the ventilation rate and volume. pH_e responded inversely to arterial Pco₂ and was well described (r² = 0.91) by the Henderson-Hassel-balch equation, assuming a pK_a for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO₂ of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pH_i responded inversely and linearly to arterial Pco₂. The intracellular effect was muted as compared with pH_e (slope = -0.0025, r² = 0.60). pH_e and pH_i values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pH_e decreases more rapidly than pH_i during the first 5 min of ischemia. After 12 min of ischemia, pH_e and pH_i values were not significantly different (6.44 ± 0.02 and 6.44 ± 0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/³¹P-NMR method for measurement of pH_e and pH_i are discussed.     

A nonradioisotope, enzymatic microplate assay for in vivo evaluation of 2-deoxyglucose uptake in muscle tissue

To determine 2-deoxy-D-glucose (2DG) and 2-deoxy-D-glucose 6-phosphate (DG6P) in mouse tissue after injection of 2DG, we have developed a novel assay. This assay is a simple procedure involving incubation of samples with four independent, single reaction mixtures followed by measurement of fluorescence. From differences between the values obtained with the four reactions, each of glucose, glucose 6-phosphate, 2DG and DG6P were able to be quantified in a sensitive manner. Using this assay system, glucose and 2DG in blood and DG6P-accumulation in muscle were easily determined. Therefore, this assay may be useful for measuring in vivo glucose uptake without the use of radioisotopes.     

An enzymatic fluorimetric assay to quantitate 2-deoxyglucose and 2-deoxyglucose-6-phosphate for in vitro and in vivo use

Previously, we developed a microplate assay to quantitate 2-deoxyglucose (2DG) and 2-deoxyglucose-6-phosphate in samples for in vitro and in vivo use. In this assay system, four different reaction mixtures were used, and the difference in the reactivity of the two types of glucose-6-phosphate dehydrogenase (G6PDH) variants was used. Because G6PDH from tolura yeast was no longer available, we modified our assay system for the use of G6PDH from Leuconostoc. Using this improved assay system, concentrations of glucose, 2DG, glucose-6-phosphate, and 2-deoxyglucose-6-phosphate were easily measured. This assay may be useful for measuring uptake of 2DG without the use of radioisotopes.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Effects of 2-deoxyglucose on Saccharomyces cerevisiae as observed by in vivo 31P-NMR

Saccharomyces cerevisiae cells were treated with 2-deoxyglucose (1 mM) and the effects induced in the levels of phosphorus compounds and in the internal pH were monitored using 31P-NMR. Upon incubation with 2-deoxyglucose a strong decrease in the polyphosphate level was observed and the cytoplasmic pH decreased by about 0.4 units. This shows that 2-deoxyglucose strongly interferes with the cell conditions and consequently, the results of experiments in which 2-deoxyglucose was used to obtain deenergized cells should be carefully reanalysed.     

Metabolic Fluxes Between [14C]2-Deoxy-D-Glucose and [14C]2-Deoxy-D-Glucose-6-Phosphate in Brain In Vivo

The rates of the phosphorylation and dephosphorylation of 2-deoxyglucose were measured in rat brain in vivo using tracer kinetic techniques. The rate constant for each reaction was estimated from two separate experiments with different protocols for tracer administration. Tracer amounts of [1-14C]2-deoxyglucose (1 microCi) were injected through the internal carotid artery (intraarterial experiment), or through the atrium (intravenous experiment). Brains were sampled by freeze-blowing at various times after the injection. In the intraarterial experiment, the rate constant for the forward reaction from 2-deoxyglucose to 2-deoxyglucose phosphate was calculated by dividing the initial rate of 2-deoxyglucose phosphate production by the 2-deoxyglucose content in brain. The rate constant for the reverse reaction from 2-deoxyglucose phosphate to 2-deoxyglucose was calculated from the decay constant of 2-deoxyglucose phosphate. The rate constants estimated were 10.1 +/- 1.4%/min (SD) and 3.00 +/- 0.01%/min (SD), respectively, for the forward and reverse reactions. In the intravenous experiment, rate constants for both reactions were estimated by compartmental analysis. By fitting data to program SAAM-27, the rate constants for the forward and reverse reactions were estimated as 11.4 +/- 0.4%/min (SD) and 5.1 +/- 0.4%/min (SD), respectively. The rate constants determined were compared to those for the reactions between glucose and glucose-6-phosphate, estimated previously from labeled glucoses. It is concluded that the rate of glucose utilization measured by the 2-deoxyglucose method reflects the rate of the hexokinase reaction and not the rate of glucose utilization or brain energy utilization.     

Apparent Absence of a Translocase in the Cerebral Glucose-6-Phosphatase System

In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent-disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose-6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

Intracellular pH and Catecholamine Synthesis in Cultured Bovine Adrenal Medullary Cells: Effect of Extracellular Na+ Removal

Incubation of cultured bovine adrenal medullary cells in Na+-free sucrose medium or in Na+-free Cs+ medium enhanced the synthesis of 14C-catecholamines from [14C]tyrosine about two- to threefold or sixfold, respectively. The increment of 14C-catecholamine synthesis produced by Na+-free medium was partially dependent on the presence of Ca2+ in the medium. Dibutyryl cyclic AMP also stimulated the synthesis of 14C-catecholamines in adrenal medullary cells, and the effects of Na+ removal and dibutyryl cyclic AMP (5 mM) on the synthesis were almost additive. The intracellular pH measured by using a weak acid 5,5-dimethyloxazolidine-2,4-dione was 7.14 in control cells and when Na+ was replaced by sucrose or Cs+, it shifted down to 6.56 or 5.66, respectively. The fall in intracellular pH and the stimulation of 14C-catecholamine synthesis were similarly dependent on the concentration of Na+ in the medium. The optimal pH of soluble tyrosine hydroxylase was 5.5-6.0 both in control cells and in cells incubated in Na+-free medium. These results suggest that removal of extracellular Na+ increases the synthesis of catecholamines, at least in part, by shifting the intracellular pH toward the optimal pH of tyrosine hydroxylase.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Characterization and Location of Met5-Enkephalin-Arg6-Phe7 Stored in Various Rat Brain Regions

A specific antiserum against met⁵-enkepha-lin-arg⁶-phe⁷ was raised and used to study the distribution and characterization of met⁵-enkephalin-arg⁶-phe⁷-like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH-terminus of the peptide, as it fails to cross-react with met⁵-enkeph-alin, met³-enkephalin-arg⁶, met⁵-enkephalin-arg⁶-arg⁷, met⁶-enkephalin-lys⁶, and leu-enkephalin. However, it cross-reacts with phe-met-arg-phe by about 10% and with phe-met-arg-phe-NH₂ to an insignificant degree. The highest content of met⁵-enkephalin-arg⁶-phe⁷ was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met⁵-enkephalin-arg⁶-phe⁷ in striatum can be released in a Ca²⁺-dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu-roregulatory role for met⁵-enkephalin-arg⁶-phe⁷. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.     

Glutamate Increases Glycogen Content and Reduces Glucose Utilization in Primary Astrocyte Culture

The glycogen content of primary cultured astrocytes was approximately doubled by incubation with 1 mM L-glutamate or L-aspartate. Other amino acids and excitatory neurotransmitters were without effect. The increase in glycogen level was not blocked by the glutamate receptor antagonist kynurenic acid but was completely blocked by the glutamate uptake inhibitor threo-3-hydroxy-D,L-aspartate and by removal of Na+ from the medium. Incubation with radiolabeled glucose and glutamate revealed that the increased glycogen content was derived almost entirely from glucose. Glutamate at 1 mM was also found to cause a 53 +/- 12% decrease in glucose utilization and a 112 +/- 69% increase in glucose-6-phosphate levels. These results suggest that the glycogen content of astrocytes is linked to the rate of glucose utilization and that glucose utilization can, in turn, be affected by the availability of alternative metabolic substrates. These relationships suggest a mechanism by which brain glycogen accumulation occurs during decreased neuronal activity.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Early Events in Free Radical-Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na⁺ ([Na⁺]_i) and intracellular Ca²⁺ ([Ca²⁺]_i) concentrations, and basal glutamate release of synaptosomes. Both H₂O₂ and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na⁺]_i over an incubation period of 15 min. A steady rise of the [Ca²⁺]_i due to peroxides was also observed that was external Ca²⁺ dependent and detected only at an inwardly directed Ca²⁺ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H₂O₂. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.     

Imaging of the Degeneration of Neurons and Their Processes in Rat or Cat Brain by 45CaCl2 Autoradiography or 55CoCl2 Positron Emission Tomography

The possibility of using radiolabeled divalent cations to visualize nerve cell degeneration in the brain was investigated after intoxication with neurotoxins. At different survival times after the intracerebral injection of kainic acid or 6-hydroxydopamine, autoradiographs were made from brain sections of rats that had received 45CaCl2 intravenously 24 h before death. Brain sections, adjacent to those used for autoradiography, of the 6-hydroxydopamine-treated rats were used for histofluorescence of catecholamines to check the neurochemical effect of the treatment. These experiments show that radioactive Ca accumulates in brain tissue during a particular phase of degeneration. Not only could degenerating cell bodies be traced by 45Ca autoradiography, but also degenerating nerve terminals in the striato-nigral and nigro-striatal projection systems. In positron emission tomography (PET) studies, 55CoCl2 was used as a marker for Ca2+. Unilateral lesions of the cat forebrain, produced by kainic acid, could be imaged in vivo by PET with 55CoCl2. PET with this radiolabel may provide diagnostic potentials for human neurodegenerative disorders.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.