1H-MRS detected lipolysis in diabetic rat hearts requires neutral lipase.

PURPOSE: Triacylglycerol (TAG) lipolysis increases in diabetic hearts. However, it is not known which pathway for lipolysis catalyzes this process. Thus, using 1H-magnetic resonance spectroscopy (MRS), we determined whether TAG lipolysis in diabetic rat hearts requires acid lipase or neutral lipase activity. METHODS: Rats were given IP injections of 110 mg streptozotocin (STZ)/kg. Forty-eight to 72 h after this treatment, all rats exhibited ketotic diabetes. The hearts of these ketotic rats were isolated, perfused isovolumically, and analyzed using 1H-MRS. RESULTS: The content of methylene protons (CH2)n--and otherfatty acid protons, measured using 1H-MRS, increased in hearts isolatedfrom STZ-treated compared to untreated rats. This increase in heart--(CH2)n--was directly related to the chemical content of heart TAGs. If isolated diabetic hearts were perfused with either glucose or glucose plus the acid lipase inhibitor methylamine, then heart content of TAG, measured as (CH2)n, decreased at rates of approximately 130 nmol TAG/gdw/min throughout a 55-min perfusion. If diabetic hearts were pretreated with the neutral lipase inhibitor diethyl-p-nitro-phenylphosphate (DNPP) and perfused with glucose, then heart TAG content, measured as (CH2)n, did not change during perfusion. CONCLUSIONS: 1H-MRS can detect the TAG and the net lipolysis of TAG in diabetic rat hearts. Net TAG lipolysis in diabetic rat hearts requires neutral lipase.

23Na NMR demonstrates prolonged increase of intracellular sodium following transient regional ischemia in the in situ pig heart.

This study tests the hypothesis that Na+i increases during regional ischemia in the in situ pig heart. An extracorporeal shunt was created between the carotid artery and the left anterior descending artery of 14 open chest pigs. 23Na and 31P NMR spectroscopy measured myocardial Na+i and high energy phosphates (HEPs). The protocol consisted of three 40 min periods: pre-ischemia (shunt pressure, 76 +/- 23 mmHg (S.D.)), ischemia (shunt pressure, 25 +/- 7 mmHg), and post-ischemia (shunt pressure, 53 +/- 11 mmHg). The pre-ischemia Na+i concentration was 6.7 +/- 4.2 mM. Phosphocreatine (PCr) was 15.3 +/- 0.5 mM, ATP 9.4 +/- 0.4 mM, inorganic phosphate (Pi) 1.5 +/- 0.2 mM, and pHi 7.16 +/- 0.09. At the end of ischemia Na+i had increased to 10.5 +/- 2.8 mM (p < 0.0002); PCr decreased to 5.9 +/- 2.1 mM (p < 0.0002); ATP was 6.5 +/- 0.5 mM (p < 0.003); Pi had increased to 6.3 +/- 1.0 mM (p < 0.0002), and pHi was 6.41 +/- 0.06 (p < 0.0002). During the first 10 min of the reperfusion, Na+i increased further to 12.4 +/- 2.8 mM (p < 0.025), whereas HEPs all returned to pre-ischemic values. Na+i increases during regional ischemia in the in situ pig heart, suggesting reduced Na+/K+ ATPase activity. While ATP probably does not limit Na+/K+ ATPase activity, increases in Pi and decreases in pHi may reduce Na+/K+ ATPase activity. Additional Na+i increases during reperfusion suggest either augmented Na+ influx or decreased Na+ efflux.

1H NMR measurement of triacylglycerol accumulation in the post-ischemic canine heart after transient increase of plasma lipids.

This study tests the hypothesis that increased levels of plasma lipids can accelerate accumulation of myocardial triacylglycerols in post-ischemic but viable myocardium. Two groups of dogs underwent 90 min of left anterior descending coronary artery (LAD) occlusion followed by 240 min of reperfusion. The first group of saline-treated dogs (n = 7) had physiological levels of plasma lipids during reperfusion: a second group treated with Liposyn and heparin (n = 5) experienced increased plasma lipids during reperfusion. The transmural content of triacylglycerols was determined during ischemia and reperfusion using 1H NMR one-dimensional chemical shift imaging (1D CSI), and at the end of reperfusion using Oil Red-O staining and chemical assay. TTC staining was used to identify the extent of irreversibly injured myocardium. Subepicardial and plasma triacylglycerol content, measured both by 1D CSI and chemically, did not change during reperfusion in saline-treated dogs. Infusing dogs with Liposyn and heparin for 90 min during reperfusion transiently elevated their plasma triacylglycerols, which returned to normal levels following Liposyn wash-out. During Liposyn wash-out, myocardial triacylglycerols measured by 1D CSI preferentially increased in the subepicardium of area-at-risk myocardium (P < 0.05). Triacylglycerol content, measured chemically, also increased in area-at-risk compared to non-ischemic subepicardium (P < 0.001). Significant endocardial damage occurred in both groups, but elevated levels of plasma lipids did not increase the size of the area-at-risk. Therefore, elevated plasma lipids caused a preferential accumulation of triacylglycerols in area-at-risk myocardium during reperfusion without exacerbating irreversible ischemic injury. These results are consistent with either inhibited fatty acid oxidation or mis-matched fatty acid extraction and oxidation in area-at-risk myocardium.

Model systems for modulating the free energy of ATP hydrolysis in normoxically perfused rat hearts.

This study has two objectives; first, to develop perfusion conditions that decrease the free energy of ATP hydrolysis, Delta GATP, in isolated hearts; and, second, to modulate the Delta GATP in these perfused hear models. To accomplish the first goal, a series of inhibitors was employed to restrict acetyl-CoA oxidation. The second goal was accomplished by increasing work demand. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either; (i) no inhibitors (G group hearts); (ii) 0.3 mm bromo-octanoate (BrO), an inhibitor of beta-oxidation (GB group); (iii) 0.4 mm amino-oxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (GA group); (iv) BrO and AOA (GBA group hearts); or (v) BrO, AOA, and 4 mm butyrate, an alternate substrate (GBA-Bu). Pacing hearts at 300 beats per min (beats/min), at 450 beats/min, and at 450 beats/min in the presence of 80 microgram/l dobutamine allowed three increasing levels of work demand to be attained. The Delta GATP values of the five groups of hearts were calculated for each workstate using the concentrations of high energy phosphate metabolites measured by 31P NMR spectroscopy. At the highest levels of workload demand, the G, GB, and GBA-Bu group hearts had Delta GATP values >/=-53 kJ/mol ATP. At the highest levels of workload demand, the GA and GBA hearts had Delta GATP values 20 min. The G, GB, and GBA-Bu hearts attained RPPs of >/=54x10(3) mmHg/min at the highest levels of workload demand. The GA and GBA hearts attained RPPs of

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.

1H NMR spectroscopic imaging of myocardial triglycerides in excised dog hearts subjected to 24 hours of coronary occlusion.

BACKGROUND: Myocardial ischemic insult causes depression of fatty-acid beta-oxidation and increased fatty-acid esterification with triglyceride (TG) accumulation. This accumulation has been demonstrated to occur in the territory with diminished blood flow surrounding an infarct, ie, the region at risk. To evaluate whether the extent of TG accumulation in the canine heart after 24 hours of ischemia could be detected, we applied myocardial 1H nuclear magnetic resonance (NMR) spectroscopic imaging (SI). METHODS AND RESULTS: Seven adult mongrel dogs underwent 24 hours of left anterior descending coronary artery occlusion. Postmortem, the hearts were excised and the size and location of the infarct were determined. With a Philips 1.5-T clinical NMR imaging/spectroscopic system, two-dimensional (2D) 1H NMR SI was performed. TG 1H NMR chemical shift images were reconstructed from the frequency domain spectra by numerical integration. A statistically significant (P < .05) increase in TG signal intensity was demonstrated in the region at risk compared with the nonischemic control region. There was an intermediate quantity of TG in the infarct region. Biochemical determination of tissue TG content (milligrams per gram wet weight) in the control, at-risk, and infarct regions confirmed the 1H NMR measurements. Histological evaluation with oil red O staining also demonstrated graded TG accumulation in myocytes. The highest TG levels were found in the at-risk region and the lowest levels in the control region. CONCLUSIONS: By use of 2D 1H NMR SI, the present study confirms and extends previous work that demonstrates preferential accumulation of TG in the reversibly injured myocardium after 24 hours of coronary occlusion. This study provides an important step toward the clinical application of TG imaging. When TG imaging is ultimately possible, resultant data would have diagnostic, prognostic, and therapeutic implications.

Water-suppressed one-dimensional 1H NMR chemical shift imaging of the heart before and after regional ischemia.

This study tests the hypothesis that brief periods of ischemia result in an increase in myocardial lipids during early reperfusion. We conducted 1H NMR spectroscopy to serially measure myocardial lipids before and after regional ischemia. Localized 1H NMR spectra (spatial resolution of 1.25 mm) were obtained using a one-dimensional chemical shift imaging technique. Two regions, the subendocardium and the subepicardium, were estimated by summing spectral areas from three slices (3.75 mm). Two groups of dogs that underwent a 45 min ischemia and 4 h reperfusion were studied: a group in which the myocardium beneath the surface coil underwent ischemia and reperfusion; and a group in which the ischemic event was distant from the tissue under the surface coil. Microsphere measurements showed significant blood flow reductions in the subepicardium and subendocardium in the ischemic zones during coronary occlusion. Flow returned to baseline values during reperfusion. In the ischemic zone group, the subendocardium, the triglyceride resonance areas decreased by 24% (p < 0.05) during reperfusion. However, subepicardial triglyceride areas were unchanged. Subendocardial creatine areas were also unchanged. The non-ischemic zone group subendocardial triglycerides decreased by 33% (p < 0.05) following ischemia and reperfusion in the remote region. In contrast to the ischemic group, the subepicardial triglyceride resonance areas decreased by 42% (p < 0.05). Subendocardial creatine areas were unchanged. These data show that triglycerides of the ischemic-reperfused subendocardium do not increase during 4 h of reperfusion. Furthermore, they show that the triglycerides resonance areas of the non-ischemic region decrease following remote ischemia and reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of crossclamping the descending aorta on the high-energy phosphates of myocardium and skeletal muscle. A phosphorus 31-nuclear magnetic resonance study.

The study was designed to test the hypothesis that a moderate decrease in upper body oxygen consumption observed during crossclamping of the thoracic aorta represents tissue hypoxia (possibly as a result of microcirculatory disorders) and results in adenosine triphosphate homeostasis disturbances. We averaged phosphorus 31-nuclear magnetic resonance spectroscopy measurements for 10 minutes with the use of a surface coil on the left ventricle and on the deltoid muscle during a 1-hour period before aortic crossclamping, during aortic crossclamping, and after aortic unclamping. Skeletal muscle creatine phosphate levels decreased 3.1% (p < 0.01), whereas the ratio of creatine phosphate to adenosine triphosphate decreased 2.2% (p < 0.05); glycolytic intermediates increased 70% (p < 0.01) and intracellular inorganic phosphate decreased 9% (p < 0.01). Myocardial creatine phosphate decreased 15% (p < 0.01), whereas the ratio of creatine phosphate to adenosine triphosphate decreased 5.3% (p < 0.01); glycolytic intermediates did not change, but intracellular inorganic phosphate almost doubled (p < 0.05). These data suggest that observations of reduced upper body oxygen consumption after aortic crossclamping are consistent with the effects of skeletal muscle hypoxia. Changes in myocardial metabolites may result from transient ischemia caused by the increased wall stress.

Extracellular volume and transsarcolemmal proton movement during ischemia and reperfusion: a 31P NMR spectroscopic study of the isovolumic rat heart.

We have measured, directly and simultaneously, changes in extracellular volume and intra- and extracellular pH during ischemia in the isolated rat heart using 31P NMR spectroscopy. Hearts were perfused with buffer containing 15 mM sodium phenylphosphonate at pH 7.4. Wash in and wash out experiments showed that phenylphosphonate entered only the extracellular (interstitial, vascular and chamber) space of the heart and had no adverse effects on myocardial energetics, contractile function or coronary flow rate. Hearts were subjected to 28 min of total, global ischemia, during which the phenylphosphonate resonance area in the 31P NMR spectra decreased by 83%, indicating that extracellular fluid had moved rapidly from the heart to the bath surrounding the heart, partly as a result of vascular collapse. A separate, morphological study confirmed that 95% of the vasculature had collapsed by 28 min ischemia. Intra- and extracellular pH were determined from the chemical shifts of the P(i) and the phenylphosphonate resonances, respectively. In the pre-ischemic rat heart, intracellular pH was 7.15 +/- 0.03 and extracellular pH was 7.39 +/- 0.03. By 4 min of ischemia, intra- and extracellular pH were the same and decreased concomitantly throughout the remainder of ischemia to final values of 6.09 +/- 0.19 and 6.16 +/- 0.23, respectively. On reperfusion, the extracellular volume and pH returned to pre-ischemic levels within 1 min, but restoration of intracellular pH took > 2.5 min. Thus, a large volume of extracellular fluid moves out of the rat heart to the surrounding bath and the intra- and extracellular pH become the same during total, global ischemia.

Aqueous shift reagents for high-resolution cation NMR. VI. Titration curves for in vivo 23Na and 1H2O MRS obtained from rat blood.

Frequency shift/concentration calibration curves applicable to the use of shift reagents (SRs) for in vivo 23Na MRS studies can be obtained from experiments with whole blood. Here, they are reported for titrations of rat blood with the SRs DyTTHA3- and TmDOTP5-. There are a number of considerations that must be made in order to derive accurate calibration curves from the experimental data. These include the effects of bulk magnetic susceptibility (BMS, since the SRs are paramagnetic), the effects of water flux (since addition of the SR stock solution to blood renders the plasma hyperosmotic), and the consequences of restricted distribution of the SR anion in the erythrocyte suspension. We give in some detail the BMS shift theory that obtains in this case and show also how it applies to excised perfused organ as well as in vivo studies. Also, we report significant effects of adjuvant Ca2+ additions in the TmDOTP5- titrations. These are very important to the successful use of this SR in vivo. Finally, our considerations of BMS lead naturally to an understanding of its manifestations in the shifts of the 1H2O resonance frequencies of cell suspensions and tissues induced by SRs. Since these are being increasingly reported, and often misinterpreted, we devote an experiment and some discussion to this subject. We show that this phenomenon cannot be used to quantitatively discriminate intra- and extracellular 1H2O signals.

Water-suppressed one-dimensional 1H NMR spectroscopic imaging of myocardial metabolites in vivo.

Excitation by conventional B1 pulses in surface coil chemical-shift imaging experiments yields resonance intensities that vary greatly with position. B1-compensated semi-selective pulses overcome much of this problem. These pulses are employed for excitation and refocusing in 1H NMR spectroscopic localization in the intact heart.

Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis.

Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.

Magnetic susceptibility shift selected imaging: MESSI.

Paramagnetic compounds are often used to enhance contrast in MRI by virtue of their increase in the kinetics of the relaxation of water 1H magnetization. Here, we demonstrate a method for contrast enhancement which is based on the resonance frequency shifts caused by the bulk magnetic susceptibility (BMS) effects of such compounds. This involves the frequency selective excitation in the absence of field gradients, during the imaging sequence, of a portion of the water 1H resonance which is rendered inhomogeneous by BMS shifts only. The image which results is of that portion of the sample which gives rise to the portion of the spectrum excited. A phantom sample which simulates some aspects of tissue, particularly blood vessels with different orientations in the magnetic field, was prepared. The contrast enhancement exhibited here avoids some of the distortions attendant to the use of paramagnetic reagents. This new approach can, in principle, utilize the natural BMS differences found in all tissue.

31P and 23Na NMR spectroscopy of normal and ischemic rat skeletal muscle. Use of a shift reagent in vivo.

23Na NMR spectroscopy was used 1, to define the distribution of the shift reagent for cations, triethylenetetraminehexaacetatedysprosium(III), DyTTHA3-, in the living rat; 2, to define the characteristics of the Na resonances reporting intra- and extracellular Na+ in skeletal muscle in vivo; and 3, to calculate the Na+ concentrations in the intra- and extracellular spaces of the gastrocnemius muscle during well-perfused and ischemic conditions. The concentration of DyTTHA3- infused intravenously into the jugular vein of the living rat reached a maximum value of 8-9 mM in the extracellular space of the muscle after ca 40 min of infusion. This allowed excellent discrimination of extra- and intracellular Na signals (Nao and Nai, respectively) and did not spoil the resolution of concurrent 31P NMR spectra. Infusion of shift reagent changed neither hemodynamic performance of the rat nor the high-energy phosphate content of skeletal muscle. Shift reagent enters ca 15% (v/w) of the rat body weight; this corresponds to almost all of the "fast" or rapidly permeable extracellular space. It is excreted from the body with a pseudo-first order rate constant of 0.0158 min-1. In resting muscle, we estimate that [Na+]i is 3-5 mM and, in muscle perfused with the sodium salt of the shift reagent, that [Na+]o in the fast exchangeable extracellular space is 166 mM. During 11 h of ischemia at 37 degrees C, the area of the Nai+ signal area monotonically increased sixfold. Based on estimates for maximum changes in fluid shifts reported by the decrease in the area of the Nao signal area, we calculate that the lower limit for [Na+]i after 11 h of ischemia is 27 mM. The NMR-visibility factors for the extracellular and intracellular Na+ signals are essentially the same. This study demonstrates that the shift reagent DyTTHA3- is acutely non-toxic and that the 23Na NMR spectra obtained can be used to quantitate [Na+]o and [Na+]i in tissues in vivo. Using this technique, we found that the transmembrane sodium gradient fell from ca 35 in well-perfused skeletal muscle to less than 6 during prolonged ischemia.

Bulk magnetic susceptibility shifts in NMR studies of compartmentalized samples: use of paramagnetic reagents.

The bulk magnetic susceptibility (BMS) shift of a nuclear resonance frequency caused by a paramagnetic compound is of importance in vivo NMR, both magnetic resonance spectroscopy and magnetic resonance imaging. However, since it is a rather complicated phenomenon, it has been the source of many misinterpretations in the literature. We have reworked and organized the theory of the BMS shift. This includes accounting for the important effects of local susceptibility. We have conducted experiments on phantom samples in order to illustrate the principles involved. Our phantoms consist of capillaries and coaxial cylinders. They simulate the situations of blood vessels oriented parallel and perpendicular to the magnetic field and the interstitial spaces surrounding them. In most of our experiments, the paramagnetic compound was one of several different hyperfine shift reagents for cation resonances. These were chosen to cover a range of potencies, in both magnitude and sign, of the shifts they produce. However, we also used a reagent which was incapable of inducing a hyperfine shift and thus could cause only a BMS shift. Although we report only 23Na spectra in this paper, the latter samples simulate the cases where one observes the water 1H resonance in experiments employing hyperfine shift reagents for cations. There have been a number of such investigations recently reported in the literature. The principles considered in this paper allow us to offer new interpretations for the results of several experiments published in the last few years.

Organic osmolytes in inner medulla of Brattleboro rat: effects of ADH and dehydration.

Inner medullary methylamine [glycerophosphorylcholine (GPC) and glycine betaine (betaine)] and polyol [sorbitol and myo-inositol (inositol)] osmolytes were measured in water-restricted and antidiuretic hormone (ADH)-infused Brattleboro (DI) rats. Compared with DI rats allowed water ad libitum, rats dehydrated for 3 days had higher urinary osmolality (Uosmol) (812 vs. 239 mosmol/kgH2O) and plasma osmolality (Posmol) (333 vs. 296 mosmol/kgH2O). Dehydration reduced betaine content (36 vs. 66 nmol/mg protein) but had no significant effect on GPC, sorbitol, or inositol. In separate protocols, DI rats, allowed water ad libitum, were infused for either 3 or 12 days with either ADH in saline (+ADH) or saline alone (-ADH). Compared with -ADH controls, 3- or 12-day ADH-infused rats were antidiuretic (Uosmol, 1,000-1,300 mosmol/kgH2O) but not dehydrated (Posmol, 297-300 mosmol/kgH2O). Three days of ADH infusion caused an increase in GPC (340%), betaine (80%), and sorbitol (248%) but not in inositol. After 12 days of ADH, further increases were observed in GPC (730%) and sorbitol (870%); inositol was also elevated (170%), whereas betaine was unchanged. Consequently, the total osmolyte content was significantly higher in +ADH than in -ADH [449 vs. 256 (3 days) and 778 vs. 199 (12 day) nmol/mg protein], whereas total osmolyte levels in dehydrated and control rats were similar (222 vs. 219 nmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

ATP synthesis and degradation rates in the perfused rat heart. 31P-nuclear magnetic resonance double saturation transfer measurements.

A limitation of magnetization transfer techniques for studying enzyme kinetics in vivo has been the difficulty of treating systems with more than two exchanging species. This problem was addressed in the original papers describing saturation transfer. Since then, a number of approaches have been devised to study these complex situations. Here, we present a method based on the transient saturation transfer experiment in which spin-lattice relaxation time constants and reaction rates are obtained from the same magnetization transfer data. This technique is particularly suitable for biological samples. We apply the method to evaluate flux balance in the three-site linear exchange network composed of ATP, creatine phosphate, and inorganic phosphate in the isolated, perfused rat heart and show that the method yields reasonable values for the reaction velocities of ATP synthesis and degradation.

Accumulation of major organic osmolytes in rat renal inner medulla in dehydration.

Osmotically active organic solutes, osmolytes, exist at high concentrations in the renal inner medulla; however, their modulation during antidiuresis remains largely undefined. Renal osmolyte levels were measured by nuclear magnetic resonance spectroscopy and biochemical assays in perchloric acid extracts from normal and dehydrated (3 days) rats. Dehydration increased urine osmolality from 1,503 to 3,748 mosmol/kg and inner medullary urea content from 2,036 to 4,405 nmol/mg protein. In addition, inner medullary trimethylamines [glycerophosphorylcholine (GPC) and betaine] and polyhydric alcohols (inositol and sorbitol) significantly increased by 95 and 78%, respectively. Ninhydrin-positive substances (amino acids), although abundant, were unchanged. Renal cortex also contained GPC, betaine, and inositol but only inositol increased with dehydration. Analysis of correlations among inner medullary osmolytes showed that only GPC was consistently elevated by dehydration and was not directly correlated with the other osmolytes. In contrast, betaine and inositol contents were linearly related to each other and both tended to rise only when sorbitol content was unchanged. In conclusion, the major osmolytes in the rat renal inner medulla can increase during antidiuresis but they are regulated in a complex manner.

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase.

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo.

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)