Cyclooxygenase-1 and -2 knockout mice demonstrate increased cardiac ischemia/reperfusion injury but are protected by acute preconditioning.

BACKGROUND: The purpose of this study was to examine the effects of cyclooxygenase (COX) deficiency on baseline functional characteristics and on recovery of left ventricular developed pressure (LVDP) after 20 minutes of global ischemia and 40 minutes of reperfusion in untreated and preconditioned hearts. METHODS AND RESULTS: Compared with hearts from wild-type (WT) and COX-2(-/-) mice, baseline cardiac prostaglandin (PG) E(2) and 6-keto-PGF(1alpha) levels were significantly decreased in hearts from COX-1(-/-) mice. After ischemia, cardiac PGE(2) levels increased in WT, COX-1(-/-), and COX-2(-/-) mice (P<0.05). Recovery of function (LVDP) after global ischemia in hearts from COX-1(-/-) and COX-2(-/-) mice was significantly less than in WT hearts. Pretreatment of WT mice with indomethacin for 2 days before ischemia significantly decreased LVDP recovery; however, perfusion of WT hearts with indomethacin for 40 minutes before ischemia did not significantly alter LVDP recovery. Postischemic recovery of LVDP in COX-1(-/-) and COX-2(-/-) was unchanged by perfusion with 5 micromol/L PGE(2), PGD(2), PGF(2alpha), or carboprostacyclin. Hearts from COX-2(-/-) mice showed an increase in ischemic contracture compared with hearts from WT and COX-1(-/-) mice; however, hearts did not differ in intracellular pH, ATP, or inorganic phosphate during ischemia. Ischemic preconditioning significantly improved postischemic LVDP recovery in COX-1(-/-), COX-2(-/-), and WT mice. CONCLUSIONS: Genetic disruption or 2-day chemical inhibition of COX-1 and COX-2 decreases recovery of LVDP after ischemia; however, acute perfusion with indomethacin is not detrimental. These data are consistent with protection due to the altered expression of some protein that is modulated by COX or its metabolites.

Development and evaluation of a boronate inhibitor of gamma-glutamyl transpeptidase.

Gamma-glutamyl transpeptidase (gamma-GT) plays a central role in the metabolism of glutathione and is also a marker for neoplasia and cell transformation. We have investigated the compound L-2-amino-4-boronobutanoic acid (ABBA) as a structural analog of the putative ternary complex formed by the enzyme, L-serine, and borate, proposed to function as a transition state analog inhibitor. ABBA was found to be a potent inhibitor of the enzyme, with Ki = 17 nM using typical assay conditions (pH 8, gamma-glutamyl-p-nitroanilide substrate, 20 mM glycyl-glycine acceptor). ABBA is a stable amino acid analog with pK values determined from 13C and 11B NMR to be 2.3, 11.0 (amino titration), and 7.9 (boronate titration). The structural similarity to glutamate suggested that it might function as a glutamate analog for some glutamate-dependent enzymes or receptors. Transamination of pyruvate by ABBA to yield alanine in the presence of glutamic pyruvic transaminase was demonstrated by 13C NMR. The 2-keto-4-boronobutanoic acid transamination product is apparently fairly labile to hydrolysis, leading to formation of 2-ketobutanoic acid plus borate. The latter is also subsequently transaminated to yield 2-aminobutanoic acid. Both of these metabolites were observed in the 13C NMR spectrum. However, the corresponding transamination of oxaloacetate by ABBA in the presence of glutamic oxaloacetic transaminase was not observed. Effects of ABBA on the growth of cultured rat liver cell lines ARL-15C1 (nontumorigenic, low gamma-GT activity) and ARL-16T2 (tumorigenic, high gamma-GT activity) were also investigated, both in standard Williams Media as well as in a low cysteine growth medium. A high concentration (1 mM) of ABBA inhibited the growth of both cell lines in both media, with the degree of inhibition greater in the low cysteine medium. Alternatively, growth inhibition by 10 microM ABBA could be observed only in the low cysteine media. In general, there were no significant differences between the two cell lines in terms of sensitivity to ABBA.

Interligand Overhauser effects in type II dihydrofolate reductase.

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to the increased clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The type II enzymes contain four identical subunits which form a homotetramer containing a single active site pore accessible from either end. Although the crystal structure of the complex of R67 DHFR with folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], the nature of the ternary complex which must form with substrate and cofactor is unclear. We have performed transferred NOE and interligand NOE (ILOE) studies to analyze the ternary complexes formed from NADP(+) and folate in order to probe the structure of the ternary complex. Consistent with previous studies of the binary complex formed from another type II DHFR, the ribonicotinamide bond of NADP(+) was found to adopt a syn conformation, while the adenosine moiety adopts an anti conformation. Large ILOE peaks connecting NADP(+) H4 and H5 with folate H9 protons are observed, while the absence of a large ILOE connecting NADP(+) H4 and H5 with folate H7 indicates that the relative orientation of the two ligands differs significantly from the orientation in the chromosomal enzyme. To obtain more detailed insight, we prepared and studied the folate analogue 2-deamino-2-methyl-5,8-dideazafolate (DMDDF) which contains additional protons in order to provide additional NOEs. For this analogue, the exchange characteristics of the corresponding ternary complex were considerably poorer, and it was necessary to utilize higher enzyme concentrations and higher temperature in order to obtain ILOE information. The results support a structure in which the NADP(+) and folate/DMDDF molecules extend in opposite directions parallel to the long axis of the pore, with the nicotinamide and pterin ring systems approximately stacked at the center. Such a structure leads to a ternary complex which is in many respects similar to the gas-phase theoretical calculations of the dihydrofolate-NADPH transition state by Andres et al. [(1996) Bioorg. Chem. 24, 10-18]. Analogous NMR studies performed on folate, DMDDF, and R67 DHFR indicate formation of a ternary complex in which two symmetry-related binding sites are occupied by folate and DMDDF.

Leukocyte-type 12-lipoxygenase-deficient mice show impaired ischemic preconditioning-induced cardioprotection.

To investigate the role of 12-lipoxygenase in preconditioning, we examined whether hearts lacking the "leukocyte-type" 12-lipoxygenase (12-LOKO) would be protected by preconditioning. In hearts from wild-type (WT) and 12-LOKO mice, left ventricular developed pressure (LVDP) and (31)P NMR were monitored during treatment (+/-preconditioning) and during global ischemia and reperfusion. Postischemic function (rate-pressure product, percentage of initial value) measured after 20 min of ischemia and 40 min of reperfusion was significantly improved by preconditioning in WT hearts (78 +/- 12% in preconditioned vs. 44 +/- 7% in nonpreconditioned hearts) but not in 12-LOKO hearts (47 +/- 7% in preconditioned vs. 33 +/- 10% in nonpreconditioned hearts). Postischemic recovery of phosphocreatine was significantly better in WT preconditioned hearts than in 12-LOKO preconditioned hearts. Preconditioning significantly reduced the fall in intracellular pH during sustained ischemia in both WT and 12-LOKO hearts, suggesting that attenuation of the fall in pH during ischemia can be dissociated from preconditioning-induced protection. Necrosis was assessed after 25 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride. In WT hearts, preconditioning significantly reduced the area of necrosis (26 +/- 4%) compared with nonpreconditioned hearts (62 +/- 10%) but not in 12-LOKO hearts (85 +/- 3% in preconditioned vs. 63 +/- 11% in nonpreconditioned hearts). Preconditioning resulted in a significant increase in 12(S)-hydroxyeicosatetraenoic acid in WT but not in 12-LOKO hearts. These data demonstrate that 12-lipoxygenase is important in preconditioning.

Carbon-13 nuclear magnetic resonance study of metabolism of propionate by Escherichia coli.

We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1, 2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.

Decreased intracellular pH is not due to increased H+ extrusion in preconditioned rat hearts.

Ischemic preconditioning reduces intracellular acidification during a subsequent, prolonged period of ischemia. This may reflect decreased anaerobic glycolysis or increased H+ efflux. To distinguish between these hypotheses, we monitored intracellular and extracellular pH during a sustained period of ischemia to determine whether the preconditioned hearts had increased H+ efflux compared with nonpreconditioned hearts. At the end of 20 min of ischemia, intracellular pH in nonpreconditioned hearts was 5.90 +/- 0.08 and extracellular pH was 5.51 +/- 0.21, whereas in preconditioned hearts, intracellular pH was 6.50 +/- 0.06 and extracellular pH was 6.62 +/- 0.06. To investigate whether an Na+/H+ exchange inhibitor would alter the reduced acidification during ischemia, we preconditioned hearts with and without dimethylamiloride (DMA). Intracellular pH during ischemia was similar in preconditioned hearts with and without DMA treatment (pH 6.42 +/- 0.02 vs. 6.45 +/- 0.03, respectively). These data do not support the hypothesis that enhanced proton efflux is responsible for the more alkaline intracellular pH during sustained ischemia in preconditioned hearts.

Dynamic frequency shifts of complexed ligands: An NMR study of D--1-13C,1-2H-glucose complexed to the Escherichia coli periplasmic glucose/galactose receptor.

The 13C multiplet structure of D--1-13C,1-2H-glucose complexed to the Escherichia coli periplasmic glucose/galactose receptor has been studied as a function of temperature. Asymmetric multiplet patterns observed are shown to arise from dynamic frequency shifts. Multiplet asymmetry contributions resulting from shift anisotropy-dipolar cross correlations were found to be small, with optimal fits of the data corresponding to small, negative values of the correlation factor, chiCD-CSA. Additional broadening at higher temperatures most probably results from ligand exchange between free and complexed states. Effects of internal motion are also considered theoretically, and indicate that the order parameter for the bound glucose is >/=0.9.

Inhibition of glucose transport in human red blood cells by adenosine antagonists.

Previous studies have suggested that adenosine antagonists can interfere with normal glucose uptake in perfused rat heart. In the present studies, fluorine-19 nuclear magnetic resonance spectroscopy was used to study the effect of the adenosine antagonist, BW-A1433U, on the equilibrium exchange of fluorinated glucose analogs in human erythrocytes. Studies of the equilibrium exchange of both 2-fluoro-2-deoxy-D-glucose and 3-fluoro-3-deoxy-D-glucose with either one-dimensional magnetization transfer or two-dimensional exchange spectroscopy were performed, and significant inhibition was observed in all cases. From concentration-dependent studies, an inhibition constant for the equilibrium exchange measured at 37 degrees C of 24 microM was determined.

Mg2+ and other polyvalent cations catalyze nucleotide fluorolysis.

The reaction of fluoride with adenosine triphosphate has been studied as a nonenzymatic analog of the pyruvate kinase-catalyzed fluorokinase reaction. The production of fluorophosphate, as well as adenosine 5'-O-fluorophosphate (FAMP) and adenosine 5'-O-(2-fluorodiphosphate) (betaFADP) was found to be dependent on the presence of polyvalent metal ions. All ions tested showed significant activity. Two catalytic regimes for the cations could be distinguished: a less specific enhancement of product formation at lower fluoride/cation ratios, and a considerably more active and specific (for fluorophosphate production) enhancement at high fluoride/cation ratios. A comparison of the results with studies of cation-catalyzed nucleotide hydrolysis indicates that the fluorolysis mechanisms are analogous to the hydrolysis by hydroxyl ions observed at high pH. In addition to these nonenzymatic studies, experiments performed using several commercially available kinases indicated significant fluorokinase activity for two: glycerokinase and acetate kinase, although the activities were much below that of pyruvate kinase. With the exception of the concentrations used in these studies, these reactions proceed under physiological conditions, yielding products at sufficient concentrations to be readily detected by 19F NMR spectroscopy.

Fluorine-19 NMR studies of glucosyl fluoride transport in human erythrocytes.

Fluorine-19 magnetization transfer studies have been used to measure the transport rate of glucopyranosyl fluorides under equilibrium exchange conditions. Although rate constants and permeabilities could be determined for beta-D-glucopyranosyl fluoride, the exchange rate for alpha-D-glucopyranosyl fluoride was found to be too slow for determination using this method. The time-dependent decomposition of the beta-glucopyranosyl fluoride also limits the accuracy of the numerical results for this species; however, it is clear that the permeabilities of the alpha and beta forms differ significantly, i.e., P beta > P alpha. This observation is in contrast to recent observations for n-fluoro-n-deoxyglucose, for which P alpha > P beta for n = 2, 3, 4, or 6. The difference can be explained in terms of a simple alternating conformation model in which one of the conformations (with an external sugar-binding site) exhibits a preference for the beta form of the molecule, while the second conformation (with an internal sugar binding site) exhibits a preference for the alpha form. Fluorine/hydroxyl substitutions unmask these preferences by selectively reducing the binding to one of the conformations, depending on the specific site of fluorination.

Anomeric dependence of fluorodeoxyglucose transport in human erythrocytes.

The transport of several n-fluoro-n-deoxy-D-glucose derivatives across the human erythrocyte membrane has been studied under equilibrium exchange conditions using one- and two-dimensional nuclear magnetic resonance (NMR) techniques. This approach is based on the intracellular 19F shift, which was found to depend on the anomeric form and on the F/OH substitution position. Since the transport behavior of both glucose anomers can be followed simultaneously, this approach is particularly sensitive to differences in anomeric permeability. For 2-, 3-, 4-, and 6-fluorodeoxyglucose analogs, the alpha anomers permeate more rapidly, and the P alpha/P beta ratio is dependent on the position of fluorination, with values of 1.1, 1.3, 2.5, and 1.6, respectively, obtained at 37 degrees C. These results have been analyzed in terms of a simple alternating conformation model for the glucose transporter. Although mutarotase activity has been reported for red cells, mutarotation behavior for all anomers was found to be completely negligible on the transport and spin-lattice relaxation time scales. Metabolic transformation of the fluorinated glucose analogs, primarily to fluorinated gluconate and sorbitol analogs, is very slow and does not significantly interfere with the transport measurements. A mean ratio of 2.6 was found for the extracellular/intracellular fluorine spin-lattice relaxation rates.

Uridine diphospho sugars and related hexose phosphates in the liver of hexosamine-treated rats: identification using 31P-[1H] two-dimensional NMR with HOHAHA relay.

The effects of administration of galactosamine (GalN) and glucosamine (GlcN) on the levels of UDP-sugars and hexose monophosphates in rat livers were studied by a variety of 31P NMR methods. The flux of metabolites in the liver was monitored by in vivo NMR and showed elevated levels of UDP-sugars, and even greater increases in resonances at 4.6 ppm for GlcN treatment and at 2.0 ppm for GalN treatment. The individual compounds corresponding to these changes were identified in PCA liver extracts by 31P-[1H] two-dimensional relay spectroscopy with a HOHAHA-type 1H spin-lock. This method of transferring proton magnetization allows for nearly all of the proton chemical shifts to be observed for the hexose moiety of a UDP-sugar present in a complex mixture. The UDP-sugars in the extracts from treated rats were predominantly UDP-hexosamines. Relay spectra were also used to determine that GalN-1-P was the major component (16.0 mumol/g of liver) of the GalN-treated liver, while both alpha and beta anomers of GlcNAc-6-P were readily identified as the major hexose monophosphates in the GlcN experiment. Spectra from the 1H dimension of relay experiments conducted on extracts were nearly superimposable on relay spectra obtained under the same conditions for mixtures of standard compounds of known structure. UDP-GlcN and UDP-GalN were not commercially available, but their presence was established in the extracts after GalN treatment by obtaining relay spectra for a mixture of the compounds produced in situ enzymatically, without purification.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic resonance imaging studies of the brains of anesthetized rats treated with manganese chloride.

An understanding of the distribution of manganese ions in the brain is of interest in connection with the development of an understanding of the neurotoxicity of this element. Information about the time dependent biodistribution of manganese ions in the brains of intact rats subsequent to single IP injections of MnCl2 has been obtained from magnetic resonance imaging (MRI) studies. The enhanced MRI contrast is based on the reduction in the spin lattice relaxation time (T1) of water protons which exchange into the coordination sphere of the manganese ions. These studies indicate rapid and significant accumulations of water accessible manganese in the ventricles, the pineal gland, and the pituitary gland. The rapid appearance of high levels of manganese in the ventricular cerebrospinal fluid indicates that manganese readily crosses the filtration barrier of the choroid plexus and is thereafter apparently absorbed by the ependymal surfaces of the ventricles and transported to the subarachnoid space.

Determination of membrane potential and cell volume by 19F NMR using trifluoroacetate and trifluoroacetamide probes.

The distribution of ionic species between intra- and extracellular compartments forms one basis for the determination of cell membrane potential. It is shown that fluorine-19 NMR studies of erythrocytes in the presence of trifluoroacetate, a stable, relatively nontoxic anion with pK = -0.3, provide a sensitive probe of membrane potential. Since such measurements are based on ion concentrations, the parallel use of the neutral analogue trifluoroacetamide to provide information on intra/extracellular volume ratios was also explored. In both cases, separate 19F resonances corresponding to intra- and extracellular ions were observed, with the intracellular resonance shifted downfield by approximately 0.2 ppm and the intracellular peak typically somewhat broader than the extracellular resonance. Studies with the band 3 anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) indicate that both transmembrane diffusion and flux involving the band 3 anion exchanger contribute to the observed transport of the trifluoroacetate anion. Intra/extracellular volume ratios determined on the basis of trifluoroacetamide intensity ratios were in good agreement with determinations based on measured hematocrits. On the basis of the high sensitivity of 19F NMR and the capability of monitoring volume changes simultaneously, the time resolution for these measurements can approach the lifetime of intracellular trifluoroacetate ions and hence be limited by the trifluoroacetate flux rate.

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat.

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.

NMR observability of ATP: preferential depletion of cytosolic ATP during ischemia in perfused rat liver.

The extent to which cellular metabolites are NMR observable is of fundamental importance in the interpretation of in vivo NMR studies. Analysis of ischemic rat liver shows that ATP resonances measured by 31P NMR decrease considerably faster than total tissue ATP measured in extracts. This discrepancy demonstrates that, in liver, ATP is not 100% observable. Furthermore, the data are consistent with the supposition that in situ mitochondrial ATP resonances are not normally observable by in vivo NMR techniques. The specificity of the NMR measurement for cytosolic ATP indicates that 31P NMR can be a valuable tool for the specific measurement of ATP in this compartment.

Metabolism of excess methionine in the liver of intact rat: an in vivo 2H NMR study.

L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo 2H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-[methyl-2H3]methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small 2H resonances assigned to N1-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.

In vivo 31P NMR studies of the hepatic response to L-ethionine in anesthetized rats.

Phosphorus-31 surface coil spectroscopy has been used to study the effects of L-ethionine administration on the hepatic metabolism of the anesthetized Sprague-Dawley rat. ATP levels were found to decrease by approximately 30% 3 to 4 hr after administration of 1 mg/g body wt of L-ethionine to the anesthetized rat by gastric gavage. ATP levels returned to control values approximately 8 hr postadministration. The relatively small decrease in ATP level was confirmed by extraction and conventional enzyme assay and is a consequence of the mode of administration of the ethionine. Hepatic inorganic phosphate levels rose concomitantly with the ATP fall. There were no significant changes in either cellular pH or Mg2+ levels as monitored by the 31P shifts of sensitive metabolites. In vivo 31P NMR spectroscopy provides a promising approach to study the effects of hepatotoxicants on cellular ATP, pH, and Mg levels.

Cytosolic free calcium levels in sickle red blood cells.

In this study, we used a recently developed nuclear magnetic resonance (NMR) technique to measure ionized calcium in sickle erythrocytes. The NMR technique, which involves 19F NMR studies of a fluorinated calcium chelator quinMF, [2-(2-amino-4-methyl-5-fluorophenoxy)methyl-8-aminoquinoline-N,N,N',N'- tetraacetic acid] provides a novel approach to the study of ionized calcium in erythrocytes since the presence of hemoglobin precludes the use of fluorescent calcium indicators. The mean value for ionized calcium in oxygenated sickle erythrocytes was 18 +/- 2 nmol/L (SE). Experiments with normal RBCs gave a mean value of 21 +/- 2 nmol/L (SE). After 1 hour of deoxygenation, mean values for ionized calcium in sickle erythrocytes did not increase as compared with values obtained under oxygen. To investigate whether deoxygenation stimulated endocytosis, sickle erythrocytes were deoxygenated for 1 hour in the presence of impermeant FBAPTA (1,2 bis-(2-amino-5-fluorophenoxy) ethane N,N,N',N'-tetraacetic acid). Cells were then separated from the extracellular medium and assayed for the presence of FBAPTA; they had incorporated significant quantities of the extracellular FBAPTA. This incorporation was not observed with normal erythrocytes. These data are consistent with at least a portion of the elevation in total cell calcium in sickle erythrocytes arising as a consequence of an endocytotic process in which extracellular calcium ions are incorporated into vesicles. Additional experiments show that these intracellular vesicles accumulate Ca2+ on further deoxygenation, consistent with a transient increase in ionized cell calcium. These studies represent the first use of NMR spectroscopy to evaluate endocytotic processes.