Noninvasive quantification of muscarinic receptors in vivo with positron emission tomography in the dog heart.

The in vivo quantification of myocardial muscarinic receptors has been obtained in six closed-chest dogs by using positron emission tomography. The dogs were injected with a trace amount of 11C-labeled methylquinuclidinyl benzilate (MQNB), a nonmetabolized antagonist of the muscarinic receptor. This was followed 30 minutes later by an injection of an excess of unlabeled MQNB (displacement experiment). Two additional injections of unlabeled MQNB with [11C]MQNB (coinjection experiment) and without [11C]MQNB (second displacement experiment) were administered after 70 and 120 minutes, respectively. This protocol allowed a separate evaluation of the quantity of available receptors (B'max) as well as the association and dissociation rate constants (k+1 and k-1) in each dog. The parameters were calculated by using a nonlinear mathematical model in regions of interest over the left ventricle and the interventricular septum. The average value of B'max was 42 +/- 11 pmol/ml tissue, the rate constants k+1, k-1, and Kd were 0.6 +/- 0.1 ml.pmol-1.min-1, 0.27 +/- 0.03 ml.pmol-1.min-1, and 0.49 +/- 0.14 pmol.ml-1, respectively, taking into account the MQNB reaction volume estimated to 0.15 ml/ml tissue. Although [11C]MQNB binding would appear irreversible, our findings indicate that the association of the antagonist is very rapid and that the dissociation is far from negligible. The dissociated ligand, however, has a high probability of rebinding to a free receptor site instead of escaping into the microcirculation. We deduce that the positron emission tomographic images obtained after injecting a trace amount of [11C]MQNB are more representative of blood flow than of receptor density or affinity. We also suggest a simplified protocol consisting of a tracer injection of [11C]MQNB and a second injection of an excess of cold MQNB, which is sufficient to measure B'max and Kd in humans.

Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor.

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Sequence similarity of calreticulin with a Ca2(+)-binding protein that co-purifies with an Ins(1,4,5)P3-sensitive Ca2+ store in HL-60 cells.

HL-60 cells possess a 60 kDa Ca2(+)-binding protein that is contained in a discrete subcellular compartment, referred to as calciosomes. Subcellular fractionation studies have suggested that, in HL-60 cells, this intracellular compartment is an Ins(1,4,5)P3-sensitive Ca2+ store. In order to investigate the structural relationship of the 60 kDa Ca2(+)-binding protein of HL-60 cells to other Ca2(+)-binding proteins, we have purified the protein by ammonium sulphate extraction, acid precipitation, and DEAE-cellulose and phenyl-Sepharose column chromatography. The N-terminal sequence of the protein shows 93% identity with rabbit muscle calreticulin, a recently cloned sarcoplasmic reticulum Ca2(+)-binding protein. No amino acid sequence similarity with calsequestrin was found, although the purified protein cross-reacted with anti-calsequestrin antibodies. The calreticulin-related protein of HL-60 cells might play a role as an intravesicular Ca2(+)-binding protein of an Ins(1,4,5)P3-sensitive Ca2+ store.

Plasma, tissue, and urine carnitine concentrations in healthy adult cats and kittens.

Mean carnitine concentrations [( carnitine]) were higher (P less than 0.05) in adult cats than in kittens for skeletal muscle (total and free carnitine), myocardium (free carnitine), and urine (total and free carnitine). The free/total carnitine ratio was lower (P less than 0.05) in kittens than in adults for liver, myocardium, and urine. Carnitine concentrations were similar between genders in kittens, but in adult cats, [carnitine] in plasma (total, free, and esterified carnitine) and liver (total and free carnitine) were higher (P less than 0.05) in female than in male cats. Total and free plasma [carnitine] were correlated to total and free liver [carnitine], respectively. Skeletal muscle [carnitine] was not correlated to plasma [carnitine]. Correlations in [carnitine] between plasma and myocardium, kidney, or urine were inconsistent.

Swimming causes myosin adaptations in the rat cardiac isograft.

To investigate the contributions of humoral and hemodynamic factors to cardiac adaptations associated with chronic exercise, female Fischer 344 rats were subjected to chronic swimming, infrarenal cardiac transplantation, or both. Swimming resulted in hypertrophy (11-12%) of the in situ hearts in both the unoperated and operated animals compared with the matched sedentary controls. The cardiac isograft exhibited atrophy (32-35%), which was not attenuated by swimming. The cardiac isograft was also associated with a decrease in the percent of V1 myosin isoenzyme, which was attenuated by swimming (45 +/- 5% versus 66 +/- 6%). Swimming also increased the percent of this isomyosin in the in situ hearts of operated rats. These data suggest that hemodynamic load and/or neural innervation are necessary for hypertrophy associated with chronic conditioning by swimming, whereas myosin isoenzyme control is significantly mediated by humoral factors.

Mechanism of cytokine inhibition of beta-adrenergic agonist stimulation of cyclic AMP in rat cardiac myocytes. Impairment of signal transduction.

Studies conducted in our laboratory have demonstrated that activated immune cells produce a soluble inhibitor(s) of cardiac myocyte contractile and cyclic AMP (cAMP) responses to beta-adrenergic stimulation. To examine the mechanism of this effect, metabolic assays were conducted on cultured rat cardiac myocytes incubated in the presence and absence of supernatants harvested from rat activated splenocyte cultures. Intracellular cAMP accumulation in response to isoproterenol was inhibited by up to 74% in a dose-dependent fashion by conditioned media containing soluble cytokines from activated immune cells. By use of myocyte cultures in which contaminating nonmyocyte proliferation was inhibited by nonlethal irradiation, this phenomenon was shown to be independent of mitogenic effects. Isobutylmethylxanthine, a phosphodiesterase inhibitor, did not ablate cytokine-induced inhibition of cAMP accumulation. Parameters of beta-adrenergic receptor binding and affinity were also unaffected. cAMP suppression was maintained after cholera toxin stimulation of cAMP production via stimulatory G protein ADP-ribosylation. cAMP inhibition was not apparent when cells were stimulated with forskolin, a direct adenylate cyclase activator. Importantly, pertussis toxin treatment significantly ablated cytokine-induced cAMP inhibition. Thus, interference with agonist-occupied beta-adrenergic receptor coupling to adenylate cyclase to produce cAMP and subsequent contractile responses is induced by a factor(s) elaborated by activated immune cells. This interference occurs at the level of signal transduction across the membrane, can be overridden by pertussis toxin, and may involve changes in the coupling of the stimulatory/inhibitory G proteins to adenylate cyclase. These results demonstrate a novel mechanism of cytokine-induced myocyte dysfunction and may have important pathophysiological ramifications in immune-mediated myocardial diseases.

Functional and autoradiographic evidence for endothelin 1 receptors on human and rat cardiac myocytes. Comparison with single smooth muscle cells.

This study aimed to determine whether receptors for endothelin were present on the cardiac myocyte as well as on vascular smooth muscle cells. Low- and high-resolution autoradiography was performed using 125I-endothelin 1 on intact rat myocardium and samples of human ventricle obtained from explanted hearts at the time of transplant. In addition to specific binding to the smooth muscle of the blood vessel lumen, there was considerable binding associated with cardiac myocytes. To discover whether there was any functional correlate for this binding, muscle cells were isolated enzymatically from human and rat ventricle and from rat femoral artery, and their contractile characteristics were studied. Single cardiac cells were superfused with physiological saline at 32 degrees C, and their length change was displayed continuously on a chart recorder. Endothelin 1 had a pronounced effect on shortening in both rat and human myocytes. The contraction amplitude was approximately doubled in both cases, from 4.1 +/- 0.8% cell length to 8.1 +/- 1.3% for rat (mean +/- SEM, n = 9, p less than 0.001), and from 2.1 +/- 0.5% to 4.0 +/- 0.5% in human (n = 10, p less than 0.001). In rat, the magnitude of the effect was comparable to that of the alpha-adrenoceptor agonist phenylephrine. The maximum contraction amplitude of the human cells, produced by raising extracellular calcium to greater than 10 mM, was 11.4 +/- 1.1% cell length (n = 9), significantly greater than that produced by endothelin (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary homocysteine on copper status in rats.

The effect on copper status of diets containing homocysteine, an intermediate in the transsulfuration pathway of methionine metabolism, was investigated in rats. Two groups of six male weanling Sprague-Dawley rats were provided with deionized water and pair-fed diets that were adequate (14.0 mg/kg) or deficient (1.3 mg/kg) in Cu to groups fed diets similarly adequate or deficient in Cu but containing DL-homocysteine (10 g/kg). Hemoglobin concentration, tissue Cu levels and the activities of the Cu-dependent enzymes--ceruloplasmin, superoxide dismutase and cytochrome c oxidase--were markedly lowered by Cu-deficient diets and by homocysteine. These dietary treatments also lowered the activity of glutathione peroxidase and produced concomitant increases in the activity of manganese-dependent superoxide dismutase and iron levels in the liver. However, dietary homocysteine decreased hepatic Mn and low Cu diets decreased cardiac iron content. Moreover, both dietary treatments significantly lowered kidney Fe levels. Homocysteine increased heart, liver and kidney weights (g/100 g body tissue) and greatly elevated the level of thiobarbituric acid reactive substances (TBARS) in heart tissue. These results indicate that dietary homocysteine can markedly lower Cu status in rats and result in tissue redistribution of Fe and increased cardiac levels of TBARS, a measure of lipid peroxidation.

Desensitization of myocardial beta-adrenergic receptors in cirrhotic rats.

Cardiac responses to catecholamines are known to be attenuated in chronic liver disease. To elucidate the role of beta-adrenergic receptor alteration in this phenomenon, we measured heart rate responsiveness to isoprenaline and myocardial beta-adrenergic receptor-binding characteristics in three groups of rats: those that were sham operated, those that had portal vein stenosis and those that were cirrhotic because of bile duct ligation. Responsiveness to isoprenaline was evaluated in conscious rats by the dose of isoprenaline needed to increase basal heart rate by 50 beats/min and by the maximal heart rate response. beta-Receptor characteristics in heart membranes were derived from 125I-iodocyanopindolol binding data. Compared with sham-operated controls, cirrhotic rats needed a significantly higher dose of isoprenaline to raise basal heart rate by 50 beats/min (102.3 +/- 19.1 vs. 28.3 +/- 11.3 ng/kg) and lower maximal heart rate response (104 +/- 29 vs. 158 +/- 61 beats/min). In addition, myocardial beta-receptor density was significantly lower in cirrhotic rats (26.5 +/- 4.6 vs. 37.5 +/- 10.3 fmol/mg protein) and the dissociation constant was higher (31.6 +/- 17.0 vs. 14.0 +/- 2.5 pmol/L). Analysis of beta 1/beta 2 subpopulations revealed that the decreased total beta-receptor density was entirely due to selective beta 1-receptor down-regulation. beta-Receptor affinity for agonist was not altered in cirrhotic rats. Rats with portal vein stenosis showed no significant differences in either isoprenaline responsiveness or beta-receptor characteristics when compared with controls. These results indicate that beta-adrenergic receptor down-regulation may be responsible for the myocardial hyporesponsiveness to catecholamines observed in cirrhosis.

Cardiac angiotensinogen and its local activation in the isolated perfused beating heart.

Increasing evidence suggests that the renin-angiotensin system modulates cardiovascular homeostasis both via its circulating, plasma-borne components and through locally present, tissue-resident systems with site-specific activity. The existence of such a system in the heart has been proposed, based on biochemical studies as well as on the demonstration of renin and angiotensinogen messenger RNA in cardiac tissue. We conducted the present study to determine whether biologically active angiotensin peptides may be cleaved within the heart from locally present angiotensinogen. Isolated, perfused rat hearts were exposed to infusions of purified hog renin; the coronary sinus effluent was collected and subsequently assayed for angiotensin I (Ang I) and angiotensin II (Ang II) by high-pressure liquid chromatography and specific radioimmunoassay. Both Ang I and II were undetectable under control conditions but appeared promptly after the addition of renin. Dose-dependent peak values for Ang I release ranged from 2.42 +/- 0.65 fmol/min to 1.38 +/- 0.18 pmol/min during renin infusions at concentrations between 10 microunits/ml and 5 milliunits/ml. Ang II levels measured in the perfusate reflected a mean fractional intracardiac conversion of Ang I to Ang II of 7.18 +/- 1.09%. Generation of Ang I and Ang II was inhibited in the presence of specific inhibitors of renin and converting enzyme, respectively. To investigate the source of angiotensinogen, we measured spontaneous angiotensinogen release from isolated perfused hearts. In the absence of renin in the perfusate, angiotensinogen was initially released in high, but rapidly declining, concentrations and subsequently at a low, but stable, rate. Prior perfusion with angiotensinogen-rich plasma resulted in enhanced early angiotensinogen release but did not alter the second, delayed phase, suggesting that, in addition to plasma-derived substrate, locally produced angiotensinogen may also participate in the intracardiac formation of angiotensin. Supporting this interpretation, hearts from animals pretreated with dexamethasone showed increased angiotensinogen messenger RNA concentrations as well as increased rates of angiotensinogen release not only during the early but also during the late phase. Our study newly demonstrates that Ang I and II may be formed within the isolated heart from locally present substrate, which appears to be derived in part from the circulating pool and in part from endogenous synthesis. These findings add support to the concept of a functionally active and locally integrated cardiac renin-angiotensin system and emphasize its potential physiological and pathological relevance.

Calcium overload in human giant cell myocarditis.

Myocardial calcium overload was observed in a patient with giant cell myocarditis. The myocardial calcium content estimated by atomic absorption spectrophotometry amounted to 120 mEq/kg dry weight, and the von Kossa stain disclosed multiple foci with patchy calcifications of myocardial fibres. Cytochemical examination of the ultrastructural calcium localisation using the phosphate-pyroantimonate method showed considerable variation in the subcellular calcium distribution. In normal myocytes calcium precipitates were confined to the inner leaflet of the sarcolemma, T-tubules, intercalated disks, and sporadically to mitochondria. In contrast, extensive calcification of mitochondria and loss of sarcolemmal calcium was evident in necrotic myocytes. A number of grossly normal myocytes also showed an increase of calcium precipitates in slightly swollen mitochondria. These findings suggest that myocardial calcium overload in this case started in viable myocytes and was not merely a secondary phenomenon occurring after cell death.

Histamine H1-receptor in heart: unique electrophoretic mobility and autoradiographic localization.

Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.

Essential fatty acid nutrition of the American alligator (Alligator mississippiensis).

The essential fatty acid (EFA) nutrition of young American alligators (Alligator mississippiensis) was examined by feeding a variety of fats/oils with potential EFA activity. Over a 12-wk period, alligators fed diets containing 2.5 or 5.0% chicken liver oil grew longer and heavier and converted feed to body mass more efficiently than alligators fed other fat/oil combinations that lacked or contained only trace amounts of arachidonic acid [20:4(n-6)]. Alligators fed an EFA-deficient diet (containing only coconut fat as the dietary fat) were the slowest-growing animals and converted feed to body mass least efficiently. However, over a 41-wk feeding period, alligators fed this diet showed no obvious external signs of deficiency other than being reduced in size and unthrifty. Fatty acid composition of heart, liver, muscle, skin and adipose tissue lipids was influenced markedly by dietary fat composition. Tissues varied significantly in response to dietary fat composition. Heart lipids contained the lowest levels of short- and medium-chain fatty acids and the highest levels of arachidonic acid. Arachidonic acid levels were less influenced by diet than were levels of other 20- and 22-carbon polyunsaturated fatty acids. Radiotracer studies indicated that linoleic acid was converted to arachidonic acid in the liver. Nevertheless, tissue arachidonic acid levels also appeared to be maintained by concentration from dietary sources and selective conservation. It appears that a dietary source of arachidonic acid may be required for a maximum rate of growth.

Hydroxyethyl starch macromolecules reduce myocardial reperfusion injury.

We assessed the value of a fraction of hydroxyethyl starch (HES Pz) in reducing the myocardial reperfusion injury in a canine open-chest model in which 1 hour of left anterior descending coronary artery occlusion was followed by 24 hours of reperfusion. Three treatment infusions (5% of blood volume) were compared: Ringer's lactate, serum albumin, and HES Pz (70% of the macromolecules between 100,000 and 1,000,000 d). When compared with Ringer's lactate and albumin, HES Pz significantly reduced the ratio of 24-hour infarct size to pretreatment area at risk (3% vs 19% and 16%, respectively) and myocardial water content (0.5% vs 3% and 1%). Potassium content differences between injured and normal myocardium were significantly less in the infarct regions of animals receiving HES Pz. In the canine model, HES Pz reduced 1-hour myocardial ischemia reperfusion injury significantly.

Subunits of cyclic adenosine 3',5'-monophosphate-dependent protein kinase show differential and distinct expression patterns during germ cell differentiation: alternative polyadenylation in germ cells gives rise to unique smaller-sized mRNA species.

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

Force and acid-base state of turtle cardiac tissue exposed to combined anoxia and acidosis.

We measured contractile force of ventricular strips form the turtle Chrysemys picta bellii exposed to 1 h of combined anoxia and acidosis (pH 7.0) at 20 degrees C. Strips either beat spontaneously (self-paced) or in response to electrical stimulation (paced at 12, 24, or 36 beats/min). Tissue [lactate] and intracellular pH (pHi) were measured in control strips and at the end of anoxia-acidosis. In self-paced strips, at normal extracellular Ca2+ concentration ([Ca2+]o) (1 mM), both rate and force fell significantly after 1 h of anoxia-acidosis to 54 and 17.1%, respectively, of control values. Increased [Ca2+]o to 10 mM at 30 min had a small but significant positive effect on both rate and force. Contractile force of paced strips also fell progressively during anoxia-acidosis, but the decrease varied directly with pacing frequency. Under all cases of anoxia-acidosis, pHi fell significantly from the control value of 7.53; in paced strips, acidosis was most severe at 36 beats/min (pHi 6.75), and in self-paced strips, pHi (approximately 6.85) was independent of [Ca2+]o. Based on this and previous work, we conclude that combined anoxia-acidosis, similar to that observed in vivo after prolonged anoxic submergence, profoundly depresses cardiac function. Both hypercalcemia and bradycardia improve performance in this extreme state, but these effects are not as great as when anoxia and acidosis occur alone.

Continuous fluorimetric assessment of the changes in cytoplasmic calcium concentration during exposure of rat isolated myocardium to conditions of simulated ischaemia.

1. The cytoplasmic calcium concentration ([Ca]c) of rat isolated atrial myocardium was assessed with the dye indo-1. Dye-loaded atria were superfused with physiological salt solution and excited with radiation at 360 nm, while epifluorescence emissions were collected simultaneously at 400 nm and 500 nm. The ratio of these emissions was used as a measure of [Ca]c. 2. Dye-loaded atria showed a phasic rise and fall in [Ca]c with each applied electrical pacing stimulus. The amplitudes of systolic increments in tension and [Ca]c were augmented by the presence of isoprenaline. 3. Atria superfused with a solution the composition of which resembled that found extracellularly in regions of ischaemia rapidly lost systolic increments in tension and [Ca]c, while end-diastolic [Ca]c and tension gradually rose. 4. The presence of lactate (20 mM) or flufenamate (5 microM) in the superfusate during simulated ischaemia aggravated the rises in both end-diastolic tension and end-diastolic [Ca]c. Inclusion in the superfusate of sulphinpyrazone (50 microM) or glucose (20 mM) protected against some of the deleterious effects of lactate seen during simulated ischaemia.

Tm(DOTP)5-: a 23Na+ shift agent for perfused rat hearts.

Tm(DOTP)5-, the thulium(III) complex of 1,4,7,10-tetraazacyclododecane-N,N',N',N'''-tetra(methylenephos p hon ate), is introduced as a 23Na+ shift agent for use in discrimination of the NMR resonances of intra- and extracellular 23Na+ ions in perfused rat hearts. The novel shift agent is directly compared to the widely used Dy(TTHA)3- (dysprosium(III) triethylenetetraminehexaacetate).

Influence of global ischemia on intracellular sodium in the perfused rat heart.

Intracellular [Na+], [H+], and [ATP] and mechanical performance were measured in the isovolumic perfused rat heart during ischemia. The concentration of intracellular sodium, [Na+]i, was determined by atomic absorption spectroscopy under control conditions, and [Na+]i was monitored by 23Na NMR spectroscopy at 1-min intervals under control conditions and during global ischemia. [ATP], [H+], and [Pi] were measured by 31P NMR in a separate group under identical conditions. The control [Na+]i measured by atomic absorption was 30.7 +/- 3.3 mM (mean +/- SD, n = 6), and [Na+]i measured by NMR was 6.2 +/- 0.5 mM (n = 3). Brief ischemia (10 min) was associated with a 54% increase in [Na+]i which reversed completely with reperfusion. Developed pressure also returned to control values upon reperfusion. Prolonged ischemia (30 min) produced continuous further accumulation of sodium (0.53 mM/min, r2 = 0.99). [H+] also increased approximately linearly early in ischemia (0.084 microM/min, r2 = 0.97). The rate of increase in [Na+]i was more than 4000 times greater than the increase in [H+] on a molar basis. Nevertheless, [H+]/[Na+]i increased early in ischemia because the proportional change in [H+] was greater than that in [Na+]i. These results indicate that (1) intracellular sodium measured by NMR in the functioning heart is about 20% of total intracellular sodium; (2) intracellular acidosis and accumulation of sodium develop simultaneously during global ischemia; (3) increased intracellular sodium content is not in itself an indicator of irreversible injury; and (4) recovery of mechanical performance is associated with return of [Na+]i (measured by NMR) to baseline after brief ischemia. The mechanism of the increase in sodium content detected by NMR is unknown.