A comparison of some metabolic effects of N-methylaspartate stereoisomers, glutamate and depolarization: a multinuclear MRS study.

Exposure of guinea pig brain slices to low concentrations (10 microM) of NMDA caused decreases in PCr and ATP within 30 min, with a slower decrease in NAA and increase in lactate, both detectable after 1 h. Exposure to NMDA for over 1 h or at higher concentrations caused further increases in lactate and decreases in NAA, with no further change in PCr or ATP. The L-isomer, NMLA, and the racemic mixture, NMDLA, caused similar changes in lactate and NAA, but both produced greater decreases in the energy state than NMDA, similar to those caused by prolonged exposure to glutamate. MK-801 prevented the changes in the energy state caused by NMDA, but not those caused by NMLA or by glutamate. The results are compared to previous studies on depolarization and discussed in terms of the role of the NMDA sub-type of glutamate receptor in the excitotoxic hypothesis of neuronal degeneration.

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

Contributions of cell kill and posttreatment tumor growth rates to the repopulation of intracerebral 9L tumors after chemotherapy: an MRI study.

The drought of progress in clinical brain tumor therapy provides an impetus for developing new treatments as well as methods for testing therapeutics in animal models. The inability of traditional assays to simultaneously measure tumor size, location, growth kinetics, and cell kill achieved by a treatment complicates the interpretation of therapy experiments in animal models. To address these issues, tumor volume measurements obtained from serial magnetic resonance images were used to noninvasively estimate cell kill values in individual rats with intracerebral 9L tumors after treatment with 0.5, 1, or 2 x LD10 doses of 1,3-bis(2-chloroethyl)-1-nitrosourea. The calculated cell kill values were consistently lower than those reported using traditional assays. A dose-dependent increase in 9L tumor doubling time after treatment was observed that significantly contributed to the time required for surviving cells to repopulate the tumor mass. This study reveals that increases in animal survival are not exclusively attributable to the fraction of tumor cells killed but rather are a function of the cell kill and repopulation kinetics, both of which vary with treatment dose.

Mechanism of action of lonidamine in the 9L brain tumor model involves inhibition of lactate efflux and intracellular acidification.

Malignant gliomas have been associated with a high rate of glycolytic activity which is believed necessary to sustain cellular function and integrity. Since lonidamine (LND) is believed to reduce tumor glucose utilization by inhibition of the mitochondrially-bound glycolytic enzyme hexokinase (HK), 31P magnetic resonance spectroscopy (MRS) was used to noninvasively follow the effects of LND on both tumor pH and the high-energy phosphate metabolites: ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) in subcutaneous rat 9L gliosarcomas. 31P tumor spectra acquired in 5 min intervals pre- and post LND administration of 50 and 100 mg/kg, i.p. revealed an acidotic pH shift of -0.25 and -0.45 pH units, respectively within 30 min post administration. The ATP/Pi ratio of 9L tumors decreased to 40% of control and Pi levels increased to 280% of control over a 3 hr period. LND exerted no effect on tumor blood flow and mean arterial blood pressure. Brain and muscle metabolite levels and pH were also unaffected by LND. In vitro measurements of cultured 9L tumor cell intra- and extracellular lactate, pentose phosphate pathway (PPP) and hexokinase (HK) activities suggest that the mode of action of LND involves inhibition of lactate efflux and intracellular acidification. The selective reduction of tumor energy metabolites and pH by LND may be exploitable for sensitizing gliomas to radiation, chemotherapy or hyperthermia.

Induction of cytotoxic oxidative stress by D-alanine in brain tumor cells expressing Rhodotorula gracilis D-amino acid oxidase: a cancer gene therapy strategy.

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) generated in the stereoselective deamination of D-amino acids catalyzed by D-amino acid oxidase (DAAO). H2O2 readily crosses cellular membranes and damages DNA, proteins, and lipids. The scarcity of DAAO substrates in mammalian organisms and its co-localization with catalase in the peroxisomal matrix suggested that the cytotoxicity of ROS could be harnessed by administration of D-amino acids to tumor cells ectopically expressing DAAO in the cytoplasm. To evaluate this hypothesis, the cDNA encoding the highly active DAAO from the red yeast Rhodotorula gracilis was mutated to remove the carboxy-terminal peroxisomal targeting sequence. A clonal line of 9L glioma cells stably transfected with this construct (9Ldaao17) was found to synthesize active R. gracilis DAAO. Exposure of 9Ldaao17 cells to D-alanine resulted in cytotoxicity at concentrations that were nontoxic to parental 9L cells. Depletion of cellular glutathione further sensitized 9Ldaao17 cells to D-alanine (D-Ala). This result, combined with stimulation of pentose phosphate pathway activity and the production of extracellular H2O2 by 9Ldaao17 cells incubated with D-alanine implicates oxidative stress as the mediator of cytotoxicity. These results demonstrate that expression of R. gracilis DAAO in tumor cells confers chemosensitivity to D-alanine that could be exploited as a novel cancer gene therapy paradigm.

In vivo 31P MRS evaluation of ganciclovir toxicity in C6 gliomas stably expressing the herpes simplex thymidine kinase gene.

Phosphorus MRS was evaluated as a monitor of tumour therapeutic response to the herpes simplex virus thymidine kinase suicide gene therapy paradigm. In vivo 31P spectra were obtained from subcutaneous rat C6 gliomas constitutively expressing the HSVtk gene post treatment with ganciclovir (GCV, 15 mg/kg i.p., twice-daily). Significant regression (p < 0.1) of tumour volume was observed 10 days after beginning GCV administration. However, no changes in tumour pH or energy metabolites from pre-treatment values were observed. High-resolution 31P spectra of tumour extracts revealed a statistically significant reduction in the phosphocholine to phosphoethanolamine ratio six days post-GCV administration. These results indicate that the HSVtk/GCV-induced killing of tumours is not associated with corresponding changes in 31P MRS-observable energy metabolites and pH. The observed reduction in the PE/PC ratio may provide a non-invasive in vivo indicator of therapeutic efficacy.

Noninvasive assessment of the relative roles of cerebral antioxidant enzymes by quantitation of pentose phosphate pathway activity.

Cerebral pentose phosphate pathway (PPP) plays a role in the biosynthesis of macromolecules, antioxidant defense and neurotransmitter metabolism. Studies on this potentially important pathway have been hampered by the inability to easily quantitate its activity, particularly in vivo. In this study we review the use of [1,6-13C2,6,6-2H2]glucose for measuring the relative activities of the PPP and glycolysis in a single incubation in cultured neurons and in vivo, when combined with microdialysis techniques. PPP activity in primary cerebrocortical cultures and in the caudate putamem of the rat in vivo was quantitated from data obtained by GC/MS analysis of released labeled lactate following metabolic degradation of [1,6-13C2,6,6-2H2]glucose. Exposure of cultures to H2O2 resulted in stimulation of PPP activity in a concentration-dependent fashion and subsequent cell death. Chelation of iron during H2O2 exposure exerted a protective effect thus implicating the participation of the Fenton reaction in mediating damage caused by the oxidative insult. Partial inhibition of glutathione peroxidase, but not catalase, was extremely toxic to the cultures reflecting the pivotal role of GPx in H2O2 detoxification. These results demonstrate the ability to dynamically monitor PPP activity and its response to oxidative challenges and should assist in facilitating our understanding of antioxidant pathways in the CNS.

Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress.

Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-(13)C2, 6,6-(2)H2] glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.

Dynamic measurements of cerebral pentose phosphate pathway activity in vivo using [1,6-13C2,6,6-2H2]glucose and microdialysis.

Cerebral pentose phosphate pathway (PPP) activity has been linked to NADPH-dependent anabolic pathways, turnover of neurotransmitters, and protection from oxidative stress. Research on this potentially important pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to address this need focused on the use of a novel isotopically substituted glucose molecule, [1,6-13C2,6,6-2H2]glucose, in conjunction with microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6-13C2,6,6-2H2]glucose through glycolysis produces [3-13C]lactate and [3-13C,3,3-2H2]lactate, whereas metabolism through the PPP produces [3-13C,3,3-2H2]lactate and unlabeled lactate. The ratios of these lactate isotopomers can be quantified using gas chromatography/mass spectrometry (GC/MS) for calculation of PPP activity, which is reported as the percentage of glucose metabolized to lactate that passed through the PPP. Following addition of [1,6-13C2,6,6-2H2]glucose to the perfusate, labeled lactate was easily detectable in dialysate using GC/MS. Basal forebrain and intracerebral 9L glioma PPP values (mean +/- SD) were 3.5 +/- 0.4 (n = 4) and 6.2 +/- 0.9% (n = 4), respectively. Furthermore, PPP activity could be stimulated in vivo by addition of phenazine methosulfate, an artificial electron acceptor for NADPH, to the perfusion stream. These results show that the activity of the PPP can now be measured dynamically and regionally in the brains of conscious animals in vivo.

Oxidation therapy: the use of a reactive oxygen species-generating enzyme system for tumour treatment.

Oxygen radicals induce cytotoxicity via a variety of mechanisms, including DNA damage, lipid peroxidation and protein oxidation. Here, we explore the use of a polyethylene glycol (PEG)-stabilised enzyme capable of producing reactive oxygen species (ROS), glucose oxidase (GO), for the purpose of harnessing the cytotoxic potential of ROS for treating solid tumours. PEG-GO (200 U), administered by two intratumoral injections 3 h apart, produced a significant growth delay in subcutaneous rat 9L gliomas as compared with control animals receiving heat-denatured PEG-GO. Rats were protected from systemic toxicity by subsequent i.v. administration of PEG-superoxide dismutase (PEG-SOD) and PEG-catalase. In vivo tumour metabolic changes, monitored using 31P magnetic resonance spectroscopy (31P-MRS) 6 h following initial administration of PEG-GO, revealed a 96 +/- 2% reduction in the ATP/Pi ratio and a 0.72 +/- 0.10 unit decline in intracellular pH. A 3-fold sensitisation of 9L glioma cells in vitro to hydrogen peroxide could be achieved by a 24 h preincubation with buthionine sulphoximine (BSO). This study suggests that oxidation therapy, the use of an intratumoral ROS-generating enzyme system for the treatment of solid tumours, is a promising area which warrants further exploration.

Metabolic loss of deuterium from isotopically labeled glucose.

The isotopically substituted molecule (6-13C, 1, 6, 6-2H3)glucose was evaluated to determine whether metabolic 2H loss would prevent its use in quantitating pentose phosphate pathway (PPP) activity. PPP activity causes the C1 of glucose to be lost as CO2, while C6 can appear in lactate. 2H NMR analysis of the lactate produced from this glucose can distinguish (3-2H)-lactate (from C1 of glucose) from (3-13C, 3, 3-2H2)lactate (from C6 of glucose). 2H NMR spectroscopic analysis of medium containing (6-13C, 1, 6, 6-2H3)glucose after incubation with cultured rat 9L glioma cells suggested a 30.8 +/- 2.1% PPP activity as compared with 6.0 +/- 0.8% from separate, parallel incubations with (1-13C)glucose and (6-13C)glucose. Subsequent experiments with other isotopically labeled glucose molecules suggest that this discrepancy is due to selective loss of 2H from the C1 position of glucose, catalyzed by phosphomannose isomerase. Failure to consider 2H exchange from the C1 and C6 positions of glucose can lead to incorrect conclusions in metabolic studies utilizing this and other deuterated or tritiated glucose molecules.

Measurement of pentose phosphate-pathway activity in a single incubation with [1,6-13C2,6,6-2H2]glucose.

The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.

Oxidative stress in the central nervous system: monitoring the metabolic response using the pentose phosphate pathway.

We propose that monitoring the activity of the pentose phosphate pathway (PPP) may provide an opportunity to obtain unique information regarding the metabolic response to oxidative stress since glutathione peroxidase activity is coupled, via glutathione reductase, to the PPP enzyme glucose-6-phosphate dehydrogenase. PPP activity was quantitated from data obtained from gas chromatography/mass spectrometry analysis of released lactate following metabolic degradation of (1,6-13C2,6,6-2H2)glucose. The feasibility of this approach for in vitro studies is shown using cultured rat 9L gliosarcoma cells, primary mixed cerebrocortical and primary astrocytic cultures and in vivo using intracerebral microdialysis. Exposure of 9L gliosarcoma cells to increasing concentrations of phenazine methosulfate, diamide and H2O2 correlated with increasing stimulation of the PPP, revealing the coupling of the PPP to the glutathione pathway. In all cultured cell types, the activity of the PPP was stimulated in a concentration-dependent fashion by exposure to H2O2. In primary mixed and purified astrocytic cultures, PPP activity was stimulated with H2O2 from 2.0 to 22.5 and from 5.9 to 66.7%, respectively. H2O2-induced neuronal injury was evident before saturation of the PPP occurred. H2O2 toxicity was attenuated when neurons were preincubated with the iron chelator, deferoxamine, and did not occur until saturation of the PPP. In vivo measurements of PPP activity in the conscious rat forebrain revealed basal levels of 4.5%, which was stimulated to 16.9 and 35.7% when 1 mM H2O2 and 500 microM phenazine methosulfate were added to the perfusion solution, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerol 3-phosphate and lactate as indicators of the cerebral cytoplasmic redox state in severe and mild hypoxia respectively: a 13C- and 31P-n.m.r. study.

The incorporation of 13C from [1-13C]glucose and [2-13C]acetate into selected intermediary metabolites in extracts prepared from incubated cerebral-cortex slices was monitored by using 13C-n.m.r. spectroscopy under conditions of mild and severe hypoxia. Mild hypoxia had little effect on labelling of tricarboxylic-acid-cycle-related amino acids [glutamate, glutamine and gamma-aminobutyrate (GABA)], although the pool sizes of glutamate and glutamine decreased. There were large increases in the labelling of lactate and of alanine, and an increase in the pool size of lactate. In severe hypoxia, the resonances of lactate and alanine remained high, whereas those of the other intermediates decreased greatly. The pool size of GABA increased. Calculation of percentage 13C enrichments and total label incorporated showed that lactate was not further affected by severe hypoxia, but the total label in alanine and its pool size were further increased. A new resonance appeared in the phosphomonoester region of the 13C-n.m.r. spectrum only in severe hypoxia. This was unambiguously assigned to glycerol 3-phosphate from a combination of 31P- and 13C-n.m.r. spectroscopy. The percentage 13C-enrichment was calculated from the 13C-n.m.r. spectrum, and the total label incorporated was measured by g.l.c./m.s. The results are discussed in terms of the ability of lactate dehydrogenase to maintain normal levels of NADH in mild hypoxia, but not in severe hypoxia. The pyruvate which accumulates under the latter condition is channelled into alanine, and the increased NADH is reflected by the increase in glycerol 3-phosphate. We conclude that glycerol 3-phosphate and alanine may provide novel means of monitoring severe hypoxia, whereas lactate is a reliable indicator only of mild hypoxia.

A rapid spectrophotometric estimation of individual phosphates and phosphonates: its application to chromatography of sugar phosphates and their phosphonate analogues.

A compound containing the -PO3H2 group (phosphoric acid, one of its monoesters, or an alkylphosphonic acid) may be rapidly assayed by the decrease it produces in the absorbance at 450 nm of a buffered acidic solution of Fe3+ and N(-3) [corrected]. The method has been used to follow chromatograms of sugar phosphates and their phosphonomethyl analogues.

Studies on metabolic regulation using NMR spectroscopy.

The effects of hypoxia and hypoglycaemia on cerebral metabolism and calcium have been studied using multinuclear magnetic resonance spectroscopy. 13C MRS showed that severe hypoxia did not cause any further increase in metabolic flux into lactate seen in mild hypoxia, but there was a further increase in 13C labelling of alanine and glycerol 3-phosphate. These results are discussed in terms of the ability of lactate dehydrogenase to maintain normal levels of NADH in mild hypoxia, but not in severe hypoxia. We conclude that glycerol 3-phosphate and alanine may provide novel means of monitoring severe hypoxia whereas lactate is a reliable indicator only of mild hypoxia. 19F- and 31P NMR spectroscopy showed that neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2+]i. Combined sequential insults (hypoxia, followed by hypoxia plus hypoglycaemia), or vice versa, produced a 100% increase in [Ca2+]i, whereas immediate exposure to the combined insult (hypoxia plus hypoglycaemia) resulted in a large 5-fold increase in [Ca2+]i, with severe irreversible effects on the energy state. These results are discussed in terms of metabolic adaptation to the single type of insult, which renders the tissue less vulnerable to the combined insult. The effects of this combined insult are far more severe than those caused by glutamate or NMDA, which throws doubt on the current excitoxic hypothesis of cell damage.

Neuronal-glial metabolism under depolarizing conditions. A 13C-n.m.r. study.

Time courses of incorporation of 13C from 13C-labelled glucose and/or acetate into the individual carbon atoms of amino acids, citrate and lactate in depolarized cerebral tissues were monitored by using 13C-n.m.r. spectroscopy. There was no change in the maximum percentage of 13C enrichments of the amino acids on depolarization, but the maxima were reached more rapidly, indicating that rates of metabolism in both glycolysis and the tricarboxylic acid cycle were accelerated. Although labelling of lactate and of citrate approached the theoretical maximum of 50%, labelling of the amino acids was always below 20%, suggesting that there is a metabolic pool or compartment that is inaccessible to exogenous substrates. Under resting conditions labelling of citrate and of glutamine from [1-13C]glucose was not detected, whereas both were labelled from [2-13C]acetate, which is considered to reflect glial metabolism. In contrast, considerable labelling of these two metabolites from [1-13C]glucose was observed in depolarized tissues, suggesting that the increased metabolism may be due to increased consumption of glucose by glial cells. The labelling patterns on depolarization from [1-13C]glucose alone and from both precursors [( 1-13C]glucose plus [2-13C]acetate) were similar, which also indicates that the changes are due to increased consumption of glucose rather than acetate.

Effects of N-methyl-D-aspartate on [Ca2+]i and the energy state in the brain by 19F- and 31P-nuclear magnetic resonance spectroscopy.

The effects of N-methyl-D-aspartate (NMDA) on the free intracellular Ca2+ concentration [( Ca2+]i) and the energy state in superfused cerebral cortical slices have been studied using 19F- and 31P-nuclear magnetic resonance spectroscopy. [Ca2+]i was measured using the calcium indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA). NMDA (10 microM) in the absence of extracellular Mg2+ caused the expected rise in [Ca2+]i but produced an impairment of the energy state: the phosphocreatine (PCr) content was decreased by 42%, and the Pi/PCr ratio was increased by 55%. There was no detectable change in ATP or free intracellular Mg2+ concentration. Increasing the NMDA concentration in the superfusing medium to 100 or 400 microM caused no further increase in [Ca2+]i or further decrease in PCr content, but the Pi/PCr ratio continued to rise. The impairment of the energy state preceded the effect on [Ca2+]i, and these changes were irreversible on return to control conditions. Repeating the experiments in the presence of 1.2 mM extracellular Mg2+ resulted in similar changes in the energy state, with no change in [Ca2+]i. The possibilities that the effects were due to membrane depolarisation or to the presence of 5FBAPTA within the tissues were eliminated. The results suggest that low concentrations (10 microM) of NMDA produce an impaired energy state independent of the presence of extracellular Mg2+ and that the decreased energy state is not due to the changes in [Ca2+]i, which are seen only in the absence of extracellular Mg2+.

Use of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) in the measurement of free intracellular calcium in the brain by 19F-nuclear magnetic resonance spectroscopy.

We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration [( Ca2+]i) in superfused brain slices. A mean +/- SD control value of 380 +/- 71 nM (n = 18) was obtained at 37 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 1% in the mitochondria or microsomes. The NMR-visible de-esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e, the [Ca2+]i "overshot" to values lower than the original control value. The [Ca2+]i was surpisingly resistant to changes in extracellular Ca2+ concentration.