High-resolution longitudinal assessment of flow and permeability in mouse glioma vasculature: Sequential small molecule and SPIO dynamic contrast agent MRI.

The poor prognosis associated with malignant glioma is largely attributable to its invasiveness and robust angiogenesis. Angiogenesis involves host-tumor interaction and requires in vivo evaluation. Despite their versatility, few studies have used mouse glioma models with perfusion MRI approaches, and generally lack longitudinal study design. Using a micro-MRI system (8.5 Tesla), a novel dual bolus-tracking perfusion MRI strategy was implemented. Using the small molecule contrast agent Magnevist, dynamic contrast enhanced MRI was implemented in the intracranial 4C8 mouse glioma model to determine K(trans) and v(e), indices of tumor vascular permeability and cellularity, respectively. Dynamic susceptibility contrast MRI was subsequently implemented to assess both cerebral blood flow and volume, using the macromolecular superparamagnetic iron oxide, Feridex, which circumvented tumor bolus susceptibility curve distortions from first-pass extravasation. The high-resolution parametric maps obtained over 4 weeks, indicated a progression of tumor vascularization, permeability, and decreased cellularity with tumor growth. In conclusion, a comprehensive array of key parameters were reliably quantified in a longitudinal mouse glioma study. The syngeneic 4C8 intracerebral mouse tumor model has excellent characteristics for studies of glioma angiogenesis. This approach provides a useful platform for noninvasive and highly diagnostic longitudinal investigations of anti-angiogenesis strategies in a relevant orthotopic animal model.

1H-NMR spectroscopy can accurately quantitate the lipolysis and oxidation of cardiac triacylglycerols.

Triacylglycerol metabolism in isolated, perfused hearts from rats fed a diet containing 20% rapeseed oil (RSO) was studied using 1H-NMR spectroscopy. RSO-induced elevation in cardiac triacylglycerols is associated with an increase in the peak area of fatty acid 1H-NMR resonances. The ratio of methyl, gamma-methylene or methylene protons adjacent to a carbon-carbon double bond to the number of methylene protons in these hearts measured by 1H-NMR spectroscopy gives values similar to those derived from previously reported chemical analyses. In addition, the triacylglycerol content of these hearts determined by chemical analysis directly correlates with their content of 1H-NMR visible fatty acid resonances. This quantitative relationship allows the real-time measurement of the rates of cardiac triacylglycerol lipolysis using 1H-NMR spectroscopy. Rates of triacylglycerol lipolysis measured using 1H-NMR spectroscopy are similar to those previously measured by chemical methods. Triacylglycerol lipolysis measured using 1H-NMR spectroscopy occurs at a significantly faster rate in hearts perfused in the presence or absence of glucose when compared to hearts perfused with glucose and acetate or medium-chain fatty acids. Finally, the rate of triacylglycerol lipolysis in glucose perfused hearts is linearly related to work output. These results demonstrate that 1H-NMR spectroscopy can accurately quantitate triacylglycerol content and metabolism in the rapeseed oil-fed rat model. 1H-NMR spectroscopic or imaging techniques may be useful in the real-time evaluation of cardiac triacylglycerol content and metabolism.

Ischemic dysfunction and impaired recovery in hypertensive hypertrophied hearts is associated with exaggerated intracellular sodium accumulation.

The primary objectives of this study were to determine if hypertrophied spontaneously hypertensive rat (SHR) hearts exhibited a greater increase in intracellular sodium (Na+i) compared with Wistar-Kyoto (WKY) control rats during low flow ischemia, and to determine whether Na+i accumulation in these hearts was associated with greater ischemic dysfunction and damage. In addition, intracellular pH and high energy phosphates were monitored to assess the relationships between changes in these variables and changes in Na+i. Interleaved 31P and 23Na spectra were acquired in perfused hearts from 8- to 10-month-old rats during low flow ischemia and reperfusion, while left ventricular pressures were monitored continuously. The majority of SHR (n = 13) exhibited an increase in Na+ similar to that for WKY and did not demonstrate exaggerated ischemic dysfunction or damage. However, a subgroup of SHR (n = 7) exhibited exaggerated Na+i accumulation during ischemia, compared with WKY, that was associated with contractile failure and a greater increase in left ventricular end diastolic pressure during ischemia, and slower recovery of developed pressure during reperfusion. Greater Na+i accumulation in this SHR subgroup preceded significantly greater depletion of high energy phosphates compared with WKY. In conclusion, increased Na+i accumulation was observed in all hypertrophied hearts with greater ischemic dysfunction compared with WKY. These results suggest that impaired Na+i handling may indeed contribute to the greater susceptibility of hypertrophied hearts to ischemic dysfunction and damage.

NMR study of rat diaphragm exposed to metabolic and compensated metabolic acidosis.

When exposed to hypercapnia, several muscles deteriorate with respect to their mechanical performance. Exposure to metabolic acidosis and, perhaps surprisingly, to compensated metabolic acidosis has the same effect on the diaphragm. The mechanisms involved in these effects remain unclear. If the diaphragmatic intracellular pH (pHi) is assumed to decrease with hypercapnia, to remain unchanged during metabolic acidosis, and to increase during compensated metabolic acidosis, it would appear that different mechanisms must be responsible for the depreciation in the diaphragm's mechanical performance. The present experiments using 31P nuclear magnetic resonance (31P-NMR) spectroscopy were undertaken to determine the effect of metabolic acidosis and compensated metabolic acidosis on pHi and on high-energy phosphate metabolites in the resting rat diaphragm. A whole diaphragm was slightly stretched while being stitched onto a fiberglass mesh. The area approximated that at functional residual capacity. It was superfused in the NMR sample tube with a phosphate-free Krebs-Ringer bicarbonate solution [( HCO3-] = 6 meqO equilibrated with either 95% O2-5% CO2 or 98.75% O2-1.25% CO2). Spectra were acquired during 15-min intervals for control (30 min of normal Krebs-Ringer bicarbonate superfusate, equilibrated with 95% O2-5% CO2), for 120 min of exposure to either form of acidosis and for 60 min of recovery with normal superfusate. The pHi decreased rapidly during metabolic acidosis but did not change significantly during compensated metabolic acidosis. In both forms of acidosis, phosphocreatine declined gradually but not significantly, whereas ATP and inorganic phosphate did not change at all. The results suggest that HCO3- passes freely through the diaphragmatic sarcolemma, very much like the cardiac sarcolemma.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chromium picolinate on growth, body composition, and tissue accretion in pigs.

An experiment was conducted to evaluate the effect of dietary chromium picolinate (CrP) on growth and body composition of pigs. Twenty-four barrows (three from each of eight litters) were randomly allotted within litter to one of three treatments: 1) basal (B) diet from 19.1 to 106.4 kg BW (Control); 2) B from 19.1 to 57.2 kg BW and then B + 200 ppb of chromium as CrP from 57.2 to 106.4 kg BW (CrP-F); and 3) B + 200 ppb of chromium as CrP from 19.1 to 106.4 kg BW (CrP- GF). Average daily gain and ADFI were reduced (P < .08) and first rib fat thickness was increased (P < .08) in pigs fed CrP-GF compared with pigs fed the Control diet. Specific gravity of the carcass was not affected (P > .10) by treatment. Tenth rib fat was reduced (P < .01) in pigs fed CrP-F compared with pigs fed CrP-GF, and percentage of muscle was increased in pigs fed CrP-F (P < .09) compared with pigs fed either the Control or CrP-GF diets. Leaf fat (P < .05) and lung weights (P < .08) were reduced in pigs fed CrP-F compared with pigs fed CrP-GF. As determined by physical-chemical separation, pigs fed CrP-GF had an increased (P < .07) percentage of intermuscular fat compared with pigs fed the Control or CrP-F diets. Pigs fed CrP-F had a lesser (P < .07) percentage of total fat and a greater (P < .07) percentage of muscle than pigs fed the Control or CrP-GF diets. As determined by mechanical-chemical separation, pigs fed CrP-F had a greater (P < .10) percentage of moisture than pigs fed the Control diet and a lesser (P < .10) percentage of fat and a greater (P < .06) percentage of ash than pigs fed the Control or CrP-GF diets. Pigs fed CrP-GF had an increased (P < .04) daily fat accretion compared with pigs fed CrP-F. Sensory and shear force values were not affected by CrP, with the exception that meat from pigs fed CrP-GF had a greater (P < .10) shear force value than meat from pigs fed CrP-F. These results suggest that dietary supplementation of CrP in the finishing phase of pig production may increase muscle and decrease fat deposition; however, not all measures of muscling or fatness were improved by CrP.

Quadratic relationship between early-post-mortem glycolytic rate and beef tenderness.

Post-mortem (PM) glycolytic rate in beef M. longissimus thoracis et lumborum (LTL) was controlled by applying low-voltage electrical stimulation (ES) for 1 min at different stages along the slaughter line. The ES treatments were as follows: (1) No electrical stimulation (NES); (2) 75 V to one side of the carcass immediately after splitting; (3) 20-40 V during exsanguination; (4) 75 V either during or following exsanguination. The study utilized 40 bulls and 40 steers. Loin steaks were aged in vacuum pouches 2, 4, 8 and 16 days PM. Quadratic equations utilizing pH at 3 h (pH(3)) gave the best estimate of Warner-Bratzler (WB) shear force for 2, 4, 8 and 16 day steaks. The rate of glycolysis is the primary determinant of LTL tenderness in this study. Temperature may only be important through its influence on early PM glycolytic rate. Optimum tenderness was produced by stimulating carcasses or sides to produce a pH(3) of 6·0. ES application to the carcass either just before or after splitting (treatment 2) produced more desirable and consistent pH(3) responses than either NES or ES during exsanguination. Aging has a differential effect whereby the WB shear values from tougher (leaner bulls) 2 day steaks improve to a greater degree, so they are not different from more tender (fatter steers) steaks by 16 day PM.

23Na-NMR measurements of intracellular sodium in intact perfused ferret hearts during ischemia and reperfusion.

23Na nuclear magnetic resonance (NMR) spectroscopy was utilized to measure intracellular Na+ in perfused ferret hearts exposed to the shift reagent dysprosium triethylenetramine-hexa-acetic acid [Dy(TTHA)3-]. The intracellular Na+ signal was small under normal perfusion conditions; resolution was enhanced by using a Jump-Return NMR pulse protocol. During 20 min of total global ischemia at 30 degrees C, intracellular Na+ concentration ([Na+]i) increased steadily to a peak value fivefold greater than control. [Na+]i declined monotonically back to control levels within 9 min of reperfusion. In contrast, the mean contractile pressure only recovered to 54% of control levels. Thus major alterations in Na+ homeostasis occur during severe ischemia. [Na+] recovers rapidly during reperfusion and is therefore dissociated from the lingering postischemic depression of contractile function known as "stunning."

The functional recovery of post-ischemic myocardium requires glycolysis during early reperfusion.

Glycolysis normally provides only a small fraction of myocardial ATP production, but ATP from glycolysis may be preferentially used to support membrane activities such as ion pumping. Since ion homeostasis is disturbed during ischemia, glycolysis may be particularly important in the recovery of postischemic myocardium. This hypothesis was investigated in isovolumic, isolated rabbit hearts, perfused with 16 mM glucose, 5 mM pyruvate or 5 mM acetate. Global left ventricular function (rate-pressure product, RPP) and unidirectional ATP synthesis rate (P(i)-->ATP flux, 31P NMR) were measured before and after 20 min global ischemia. Control hearts with intact glycolysis were compared with hearts which had glycolysis inhibited by iodoacetate (150 microM), 2-deoxyglucose (10 mM) or prior glycogen depletion. In normal hearts, inhibition of glycolysis had no effect on function when pyruvate or acetate was present as as a carbon substrate. In post-ischemic hearts, reperfusion with glucose (n = 7) resulted in moderate recovery of function to about 65% of pre-ischemic levels after 1 h reperfusion. Administration of iodoacetate at the onset of reperfusion to hearts receiving pyruvate or acetate resulted in much worse functional recovery and a marked rise in left ventricular end-diastolic pressure (LVEDP). With pyruvate (n = 7), RPP recovered to 27% of pre-ischemic levels, while mean LVEDP increased to 34 mmHg (vs 16 mmHg with glucose); with acetate (n = 6), RPP returned to 31% of pre-ischemic levels, while mean LVEDP rose to 32 mmHg. The ratio of P(i)-->ATP flux to atoms of oxygen consumed (P:O ratio) was 2.14 +/- 0.36 in hearts reperfused with iodoacetate and pyruvate, consistent with partial mitochondrial uncoupling. However, if inhibition of glycolysis with iodoacetate was delayed until after 30 min reperfusion, recovery of hearts reperfused with pyruvate was similar to hearts perfused with glucose, and there was no evidence of mitochondrial uncoupling (P:O ratio = 2.95 +/- 0.33). Inhibition of glycolysis during reperfusion with 2-deoxyglucose yielded results similar to reperfusion with iodoacetate. The worst recovery was observed in hearts with combined glycolytic inhibition by pre-ischemic glycogen depletion and iodoacetate during reperfusion (RPP = 13% of pre-ischemic levels). These findings indicate that glycolysis plays a crucial role during early reperfusion in the functional and metabolic recovery of post-ischemic myocardium.

Ca2+ transients in perfused hearts revealed by gated 19F NMR spectroscopy.

Gated acquisition of 19F nuclear magnetic resonance spectra from perfused ferret hearts loaded with the fluorinated Ca2+ indicator 5,5'-F2-BAPTA allows direct quantitation of the cyclical changes in the intracellular free Ca2+ concentration ([Ca2+]i) that underlie contraction in intact hearts. [Ca2+]i increased from approximately 200 nM in diastole to approximately 1 microM or higher in early systole. Although the 19F spectra that report [Ca2+]i changed dramatically and reproducibly during the cardiac cycle, no changes were detectable in gated phosphorus spectra. We exploited the ability to control the coronary arterial flow of our hearts to investigate the mechanism of the fall in contractility that results from a decrease in perfusion even when the flow suffices to sustain normal high energy phosphate concentrations. Under these conditions, the amplitude of Ca2+ transients falls markedly along with the decline in pressure. This down-regulation of Ca2+ transients constitutes a novel protective mechanism that minimizes energy demand during low-flow ischemia.

Simultaneous multicompartment intracellular Ca2+ measurements in the perfused heart using 19F NMR spectroscopy.

Although Ca2+ transport regulation at subcellular organelles is of great interest, only limited methodology has been available for measuring organellar [Ca2+] levels. The present study employs the 19F NMR resonance frequency of 4F-BAPTA to measure free [Ca2+]. In 4F-BAPTA loaded perfused rabbit hearts, two 19F NMR resonances were clearly observed. The frequency of one was consistent with cytosolic [Ca2+] levels. Responses to agents that after sarcoplasmic reticulum function identified the other resonance as originating from that organelle. The experiments demonstrate the utility of NMR shift indicator methodology in obtaining simultaneous multi-compartment intracellular [Ca2+] measurements and in enabling organellar [Ca2+] measurements to be made from within intact living tissue.

Nuclear magnetic resonance studies of cationic and energetic alterations with oxidant stress in the perfused heart. Modulation with pyruvate and lactate.

The postischemic generation of oxygen-derived free radicals may contribute to myocardial reperfusion injury by affecting sarcolemmal ion transport. Recent evidence indicates that exposure to reactive oxygen intermediates induces rapid increases in myocardial cytosolic free Ca2+ (Ca2+i). The mechanism is undetermined but may involve disturbances in Na+ homeostasis. We tested this hypothesis by interleaving 23Na and 31P nuclear magnetic resonance (NMR) measurements of Na+i and high-energy phosphates in glucose-perfused rat hearts exposed to hydroxyl radicals generated from H2O2 and Fe3+. In separate experiments, K+i and Ca2+i were measured with 39K and 19F NMR, respectively. The hearts rapidly exhibited contracture. Threefold Na+i increases and substantial K+i depletion were observed. Glycolytic inhibition was indicated by rapid sugar phosphate accumulation and cellular energy depletion. Notably, however, severe functional and energetic deterioration and substantial elevation of Ca2+i occurred before substantial Na+i accumulation or K+i depletion was observed. Further experiments investigated the ability of pyruvate to scavenge H2O2 and to protect the myocardium from oxidant stress. Pyruvate (1 or 2.5 mmol/L) dramatically attenuated functional and energetic alterations and alterations in Na+i and K+i, whereas acetate (2.5 mmol/L) offered no protection. Unlike pyruvate, lactate (5 mmol/L) has little or no capacity to scavenge H2O2 but has similar protective effects. In conclusion, pyruvate effectively protects against H2O2/Fe3+, largely by direct H2O2 scavenging. Protection with lactate may involve intracellular pyruvate augmentation. Without exogenous pyruvate or lactate, myocardial Na+ homeostasis can be substantially altered by oxidant stress, possibly via cellular energy depletion. Excess Na+i accumulation may, in turn, hasten metabolic and functional deterioration, but a causal link with the initial alterations in function or Ca2+i was not supported.

High-resolution 23Na-NMR studies of human erythrocytes: use of aqueous shift reagents.

Aqueous shift reagents were used to clearly distinguish intra-and extracellular 23Na-nuclear magnetic resonance (NMR) signals in samples consisting of whole blood or suspensions of washed human erythrocytes (both fresh and outdated). The lanthanide chelates Dy(PPP)2(7-) and Tm( TTHA )3- were used to shift the extracellular signals upfield, and Dy( TTHA )3- and Tm(PPP)2(7-) were similarly used to shift extracellular resonances downfield. The absolute intensities of the signals were used along with the measured hematocrit to simultaneously determine the intra- and extracellular Na+ concentrations. The results were generally within 5% of the values determined by more time-consuming centrifugation-flame emission photometry measurements on the same samples. Thus the 23Na-NMR signals from both intra- and extracellular cations suffer no NMR invisibility within experimental error. The lower level of intracellular Na+ in fresh erythrocytes (less than 12 mM) is easily distinguished from the higher level (approximately 30 mM) in erythrocytes that have been stored (in the cold) outside the body for some weeks.

Comparative costs of a cooperative care program versus hospital inpatient care for gynecology patients.

The cost of gynecologic care delivered in a cooperative care (co-op) unit was compared with the cost for similar patients treated in a traditional hospital inpatient unit. No significant differences were found. This finding was in direct contrast to our earlier study of obstetric patients where overall savings were achieved. The authors did, however, find cost savings (approximately $450) for those co-op patients cared for by physicians who were frequent users of the unit. Most of the savings for this group was achieved through a reduction in the cost of routine services, which includes nursing care. If the current nursing shortage leads to an increase in co-op units, nursing administrators need to be aware that potential cost savings may depend on physician familiarity with the co-op concept. A major role of nursing therefore, is to provide information on the benefits of cooperative care both to physicians and to potential patients.

A defect in the oxidative metabolism of human polymorphonuclear leukocytes that remain in circulation early in hemodialysis.

Human granulocytes harvested from uremic volunteers 15 min after the initiation of dialysis (at the nadir of neutropenia) were compared to predialysis controls. These intradialysis cells had a significant defect in peak luminol-enhanced chemiluminescence in response to opsonized zymosan, f-Met-Leu-Phe, and phorbol myristate acetate relative to predialysis control cells from the same patients. This defect could not be explained by a decrease in PMN myeloperoxidase concentration. H2O2 secretion by intradialysis cells (2 patients) was also depressed relative to predialysis controls. The ability to perform an independent function, orientation (polarization), was normal in both pre- and intradialysis cells relative to control. Whereas 125I-labeled formyl peptide binding studies demonstrated identical values for affinity and receptor number for predialysis and normal control cells, intradialysis cells displayed a 27% decrease in receptor number. This decrease in available receptor number. This decrease in available receptors may be related to the decreased chemiluminescence observed in response to f-Met-Leu-Phe. Furthermore, the results are consistent with the hypothesis that a defective PMN population remains in the circulation during the neutropenia of hemodialysis.

High-resolution NMR studies of transmembrane cation transport: use of an aqueous shift reagent for 23Na.

23Na NMR studies of large unilamellar vesicles of egg lecithin in salt solutions are reported. A shift reagent, the dysprosium nitrilotriacetate ion Dy[N(CH2CO2)3]3-2 has been used to distinguish between 23Na+ inside and outside the vesicles. When both are present and the shift reagent is present on only one side, two clearly distinct resonances are observed. Creation of a Na+ concentration gradient and subsequent catalysis of passive transport induced by the introduction of gramicidin can be monitored easily by using the relative intensities of the two resonances. We report the observation of transport both out of and into vesicles.

23Na and 31P nuclear magnetic resonance studies of ischemia-induced ventricular fibrillation. Alterations of intracellular Na+ and cellular energy.

To clarify the role of Na+i, pHi, and high-energy phosphate (HEP) levels in the initiation and maintenance of ischemia-induced ventricular fibrillation (VF), interleaved 23Na and 31P nuclear magnetic resonance spectra were collected on perfused rat hearts during low-flow ischemia (51 minutes, 1.2 mL/g wet wt). When untreated, 50% of the hearts from normal (sham) rats and 89% of the hypertrophied hearts from aorticbanded (band) rats (P < .01 versus sham) exhibited VF. Phosphocreatine content was significantly higher in sham than band hearts during control perfusion (53.3 +/- 1.6 versus 39.8 +/- 2.0 mumol/g dry wt). Before VF at 20 minutes of ischemia, Na+i accumulation was greater in hearts that eventually developed VF than in hearts that did not develop VF for both band and sham groups (144% versus 128% of control in sham; P < .005) and was the strongest metabolic predictor of VF; ATP depletion was also greater for VF hearts in the sham group. Infusion of the Na(+)-H+ exchange inhibitor 5-(N,N-hexamethylene)-amiloride prevented VF in sham and band hearts; reduced Na+i accumulation but similar HEP depletion were observed compared with VF hearts before the onset of VF. Rapid changes in Na+i, pHi, and HEP began with VF, resulting in intracellular Na+i overload (approximately 300% of control) and increased HEP depletion. A delayed postischemic functional recovery occurred in VF hearts, which correlated temporally with the recovery of Na+i. In conclusion, alterations in Na+i were associated with spontaneous VF transitions, consistent with involvement of excess Na+i accumulation in VF initiation and maintenance and with previously reported alterations in Ca2+i with VF.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of K(atp)channel activation on myocardial cationic and energetic status during ischemia and reperfusion: role in cardioprotection.

The role of cation and cellular energy homeostasis in ATP-sensitive K(+)(K(ATP)) channel-induced cardioprotection is poorly understood. To evaluate this, rapidly interleaved(23)Na and(31)P NMR spectra were acquired from isolated rat hearts exposed to direct K(ATP)channel activation from nicorandil or pinacidil. Nicorandil attenuated ATP depletion and intracellular Na(+)(Na(+)(i)) accumulation, delayed the progression of acidosis during zero-flow ischemia and prevented ischemic contracture. The K(ATP)channel inhibitor 5-hydroxydecanoate abolished these effects. Pinacidil did not alter Na(+)(i)accumulation, ATP depletion or pH during ischemia under the conditions employed. Both agonists greatly improved the post-ischemic functional recovery. Both agonists also dramatically improved the rate and extent of the reperfusion recoveries of Na(+)(i), PCr and ATP. The Na(+)(i)and PCr reperfusion recovery rates were tightly correlated, suggesting a causal relationship. Separate atomic absorption tissue Ca(2+)measurements revealed a marked reperfusion Ca(2+)uptake, which was reduced two-fold by pinacidil. In conclusion, these results clearly indicate that while K(ATP)channel-induced metabolic alterations can vary, the functional cardioprotection resulting from this form of pharmacological preconditioning does not require attenuation of acidosis, cellular energy depletion, or Na(+)(i)accumulation during ischemia. Rather than preservation of cationic/energetic status during ischemia, the cardioprotective processes may involve a preserved capability for its rapid restoration during reperfusion. The enhanced reperfusion Na(+)(i)recovery may be enabled by the improved reperfusion cellular energy state. This accelerated Na(+)(i)recovery could play an important cardioprotective role via a potential causal relationship with the reduction of reperfusion tissue Ca(2+)uptake and resultant reperfusion injury.

Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion.

Calcium has been implicated as a mediator of cell injury in ischemia and reperfusion, but direct measurements of Ca2+ are required to refine this idea. We used nuclear magnetic resonance spectroscopy and the Ca2+ indicator 5F-BAPTA to measure [Ca2+]i in perfused ferret hearts. Several lines of evidence are presented to show that loading with the acetoxymethyl ester of 5F-BAPTA is not significantly complicated by accumulation of partially de-esterified metabolites, compartmentalization into mitochondria, or disproportionate uptake into endothelial cells. During 20 minutes of total global ischemia at 30 degrees C, time-averaged [Ca2+]i increased significantly, reaching peak values roughly three times control at 15-20 minutes. Reperfusion resulted in a persistent elevation of [Ca2+]i during the first 5 minutes, but not afterward. Although the nonlinear response of 5F-BAPTA to [Ca2+] leads to underestimation of the true time-averaged [Ca2+]i, the measured alterations of intracellular Ca2+ homeostasis during ischemia are large compared with the likely errors in quantification. Phosphorus nuclear magnetic resonance spectroscopy of 5F-BAPTA-loaded hearts reveals changes during ischemia similar to those recorded previously in hearts not containing a Ca2+ indicator. Developed pressure recovers to only 50% of control values during reflow, indicating that the presence of 5F-BAPTA in the cytosol does not protect against stunning, at least when the extracellular calcium concentration has been raised to 8 mM. We conclude that 5F-BAPTA provides useful measurements that reveal that time-averaged [Ca2+]i rises during ischemia and returns to control levels soon after reperfusion.

Acylcarnitine accumulation does not correlate with reperfusion recovery in palmitate-perfused rat hearts.

Carnitine palmitoyltransferase-I (CPT-I) inhibitors improve postischemic myocardial function either by decreasing muscle long-chain acylcarnitines (LCAC) during ischemia or by increasing oxidation of alternate substrates such as glucose during reperfusion. These possibilities were evaluated using oxfenicine, a CPT-I inhibitor, and alternate substrates that bypass carnitine-dependent metabolism. Isolated rat hearts subjected to 20 min of ischemia followed by 40 min of reperfusion with 1.8 mM palmitate as exogenous substrate recovered little function during reperfusion. Hearts made ischemic and reperfused with palmitate and 2.4 mM hexanoate as exogenous substrates had significantly improved reperfusion function compared to palmitate-perfused hearts. Addition of 2 mM oxfenicine to palmitate-hexanoate-perfused hearts gave an additional small improvement in reperfusion function. At the end of ischemia, the LCAC content of hearts perfused with palmitate or hexanoate and palmitate was identical. Palmitate-, hexanoate, and oxfenicine-perfused hearts had significantly decreased LCAC content at the end of ischemia compared with hexanoate-palmitate-perfused hearts. Therefore, depressed reperfusion function in long-chain fatty acid-perfused hearts can be ameliorated by alternate substrates, including medium-chain fatty acids. LCAC accumulation during ischemia apparently plays only a minor role in the postischemic dysfunction of long-chain fatty acid-perfused hearts.