Effects of hypothermia on rat brain pHi and phosphate metabolite regulation by 31P-NMR

The effects of arterial alphastat regulation on brain intracellular pH (pHi) and several phosphate metabolites were assessed in anesthetized rats during hypothermia (28.6 +/- 0.2 degrees C) and normothermia (36.2 +/- 0.2 degrees C) by using 31P high-field (8.5 T) nuclear magnetic resonance (NMR). There were significant differences in pHi and metabolite ratios at the two temperatures under conditions of equal minute ventilation. During hypothermia, the brain pHi was 0.09 U higher, the phosphocreatine-to-inorganic phosphate (PCR/Pi) ratio 49% larger, and Pi-to-ATP 20% lower than at normothermia. These changes were fully reversible on warming the animal. The change in brain pHi/temperature was -0.011U/degrees C (95% confidence interval -0.007 to -0.016). The brain's ability to regulate its pHi and phosphate metabolism during hypercapnic acid-base stress was studied by using 10% CO2 ventilation. Hypothermic rats showed a larger fall in brain pHi (0.145 +/- 0.01 U, 7.15-7.01) with 10% CO2 than normothermic rats (0.10 +/- 0.02 U, 7.06-6.96). Similarly ventilated rats had a larger fall in arterial pH with 10% CO2 at hypothermia (0.36 +/- 0.04 U) than normothermia (0.24 +/- 0.01 U), so the delta brain pH/delta arterial pH was the same at both temperatures. The brain PCr-to-Pi ratio decreased approximately 20% during 10% CO2 breathing in both hypothermic and normothermic animals. Brain pHi and metabolite ratios returned to base line 30-50 min after CO2 washout in both groups. In summary, lowering body temperature while maintaining constant ventilation leads to changes in brain pHi and metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Effects of hypercapnia on brain pHi and phosphate metabolite regulation by 31P-NMR

The ability of brain cells to regulate intracellular pH (pHi) and several phosphate metabolites was evaluated during 1 h of hypercapnia (inspiratory CO2 fraction of 0.10 and 0.05) in anesthetized rats by 31P high-field (145.6 MHz) nuclear magnetic resonance spectroscopy. Body temperature was maintained at 37 +/- 0.5 degrees C. Fully relaxed spectra were obtained for controls and 30-50 min after CO2 loading and CO2 withdrawal. Spectra were taken serially every 2.5 min after gas mixtures were changed. Brain pHi decreased 0.10 +/- 0.02 units [7.06 +/- 0.01 (SE)] to 6.96 +/- 0.01 (P less than 0.001) after 30-50 min of 10% CO2 breathing, and arterial pH decreased 0.24 +/- 0.01 units. Brain pHi decreased by 0.045 +/- 0.01 units (7.05 +/- 0.01 to 7.01 +/- 0.01, P less than 0.05) during 5% CO2 breathing. Brain pHi returned to control values after 30-50 min of CO2 washout in both groups. In three of six animals breathing 10% CO2, there was an undershoot in brain pHi by 0.07-0.09 units between 2.5 and 20 min of hypercapnia. Three animals exhibited an overshoot in pHi by 0.06-0.11 units between 7.5 and 17.5 min during CO2 washout. Phosphocreatine-to-Pi and Pi-to-beta-ATP ratios changed during hypercapnia and returned to base line after withdrawal of CO2. The findings of a smaller brain pHi change than arterial pH change and undershoots and overshoots in pHi support the view that pHi regulation involves active processes such as transmembrane ion transport.     

Spinotrapezius muscle microcirculatory function: effects of surgical exteriorization

Intravital microscopy facilitates insights into muscle microcirculatory structural and functional control, provided that surgical exteriorization does not impact vascular function. We utilized a novel combination of phosphorescence quenching, microvascular oxygen pressure (microvascular PO(2)), and microsphere (blood flow) techniques to evaluate static and dynamic behavior within the exposed intact (I) and exteriorized (EX) rat spinotrapezius muscle. I and EX muscles were studied under control, metabolic blockade with 2,4-dinitrophenol (DNP), and electrically stimulated conditions with 1-Hz contractions, and across switches from 21 to 100% and 10% inspired O(2). Surgical preparation did not alter spinotrapezius muscle blood flow in either I or EX muscle. DNP elevated muscle blood flow approximately 120% (P < 0.05) in both I and EX muscles (P > 0.05 between I and EX). Contractions reduced microvascular PO(2) from 30.4 +/- 4.3 to 21.8 +/- 4.8 mmHg in I muscle and from 33.2 +/- 3.0 to 25.9 +/- 2.8 mmHg in EX muscles with no difference between I and EX. In each O(2) condition, there was no difference (each P > 0.05) in microvascular PO(2) between I and EX muscles (21% O(2): I = 37 +/- 1; EX = 36 +/- 1; 100%: I = 62 +/- 5; EX = 51 +/- 9; 10%: I = 20 +/- 1; EX = 17 +/- 2 mmHg). Similarly, the dynamic behavior of microvascular PO(2) to altered inspired O(2) was unaffected by the EX procedure [half-time (t(1/2)) to 100% O(2): I = 23 +/- 5; EX = 23 +/- 4; t(1/2) to 10%: I = 14 +/- 2; EX = 16 +/- 2 s, both P > 0.05]. These results demonstrate that the spinotrapezius muscle can be EX without significant alteration of microvascular integrity and responsiveness under the conditions assessed.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Preservation of ischemia and isoflurane-induced preconditioning after brain death in rabbit hearts

We sought to determine whether brain death-induced catecholamine release preconditions the heart, and if not, whether it precludes further protection by repetitive ischemia or isoflurane. Anesthetized rabbits underwent 30 min of coronary occlusion and 4 h of reperfusion. The effect on infarct size of either no intervention (controls), ischemic preconditioning (IPC), or isoflurane inhalation (Iso) was evaluated with or without previous brain death (BD) induced by subdural balloon inflation. Plasma catecholamine levels were measured at several time points. Although it dramatically increase plasma catecholamine levels, BD failed to reduce infarct size that averaged 0.49 +/- 0.34 without BD versus 0.45 +/- 0.27 g with BD. IPC and Iso, alone as well as after BD, significantly reduced infarct size that averaged 0.11 +/- 0.04, 0.21 +/- 0.15, 0.10 +/- 0.09, and 0.22 +/- 0.10 g in IPC, Iso, BD + IPC, and BD + Iso groups, respectively (means +/- SD, P < 0.05 vs. controls). BD-induced catecholamines "storm" does not precondition the rabbit heart that however retains the ability to be protected by repetition of brief ischemia or isoflurane inhalation.     

Reflex control of the cutaneous circulation during passive body core heating in humans.

The impact of body core heating on the interaction between the cutaneous and central circulation during blood pressure challenges was examined in eight adults. Subjects were exposed to -10 to -90 mmHg lower body negative pressure (LBNP) in thermoneutral conditions and -10 to -60 mmHg LBNP during heat stress. We measured forearm vascular conductance (FVC; ml. min(-1). 100 ml(-1). mmHg(-1)) by plethysmography; cutaneous vascular conductance (CVC) by laser-Doppler techniques; and central venous pressure, arterial blood pressure, and cardiac output by impedance cardiography. Heat stress increased FVC from 5.7 +/- 0.9 to 18.8 +/- 1.3 conductance units (CU) and CVC from 0.21 +/- 0.07 to 1.02 +/- 0.20 CU. The FVC-CVP relationship was linear over the entire range of LBNP and was shifted upward during heat stress with a slope increase from 0. 46 +/- 0.10 to 1.57 +/- 0.3 CU/mmHg CVP (P < 0.05). Resting CVP was lower during heat stress (6.3 +/- 0.6 vs. 7.7 +/- 0.6 mmHg; P < 0. 05) but fell to similar levels during LBNP as in normothermic conditions. Data analysis indicates an increased capacity, but not sensitivity, of peripheral baroreflex responses during heat stress. Laser-Doppler techniques detected thermoregulatory responses in the skin, but no significant change in CVC occurred during mild-to-moderate LBNP. Interestingly, very high levels of LBNP produced cutaneous vasodilation in some subjects.     

Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain.

31P-NMR spectroscopic studies were performed in vivo on brains of rats administered cocaine. Cocaine.HCl (1-5 mg/kg) administered systemically to lightly anesthetized rats resulted in significant and progressive deficits in whole brain intracellular free Mg ([Mg2+]i). Intracellular pH (pHi) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i was noted. Both [Mg2+]i and pHi returned to normal in most rats. Brains of rats that exhibited stroke-like events, however, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of Pi up until death. These observations are consistent with the tenet that injection of cocaine can result in severe cerebral vasospasm, ischemia and rupture of cerebral blood vessels as a consequence of depletion of brain [Mg2+]i.     

Ischemia-reperfusion injury in isolated rat hindquarters

The purpose of this study was to determine the suitability of the maximally vasodilated (papaverine) isolated rat hindquarters preparation to study the effects of ischemia and reperfusion on the microvasculature of skeletal muscle. The osmotic reflection coefficient for plasma proteins (sigma) and total vascular resistance (RT, mmHg.ml-1.min.100 g-1) were determined before ischemic periods of 30, 60, 120, 180, and 240 min in intact (with skin) and 30, 60, and 120 min in skinned hindquarters and again after 60 min of reperfusion. In both intact and skinned hindquarters, reductions in sigma and increases in RT were observed during reperfusion and were correlated with the ischemic period duration. After 120 min of ischemia in intact and skinned hindquarters, sigma was reduced from preischemia values of 0.92 +/- 0.02 and 0.89 +/- 0.02 to 0.61 +/- 0.03 and 0.57 +/- 0.03, respectively, whereas RT was increased from preischemia levels of 8.9 +/- 0.3 and 8.1 +/- 0.1 to 28.4 +/- 2.9 and 74.2 +/- 16.8, respectively. The increases in RT were associated with proportional increases in skeletal muscle vascular resistance. Thus, in isolated rat hindquarters, increasing the duration of ischemia results in progressive increases in the permeability to plasma proteins (decreased sigma) and RT, which are associated primarily with skeletal muscle.     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

Glucoregulatory and hormonal responses to repeated bouts of intense exercise in normal male subjects.

Glucose turnover and its regulation were studied during and after two identical bouts of intense exhaustive exercise separated by 1 h to define differences in response. Six lean young postabsorptive male subjects exercised at approximately 100% maximal O2 uptake (3.7 +/- 0.3 l/min) for 13.0 +/- 0.7 min for the first (EX1) and 13.2 +/- 0.8 min for the second (EX2) bout. Plasma glucose increased during EX1 and peaked at 7.0 +/- 0.6 mmol/l in early recovery but to 5.8 +/- 0.5 mmol/l (P less than 0.05) after EX2, and both the hyperglycemic and the hyperinsulinemic responses were less after EX2 (P less than 0.015, analysis of variance). The hyperglycemia was due to lesser increments in glucose utilization (Rd) (3-fold resting) than glucose production (Ra) (7-fold) toward exhaustion and for 7 min of recovery. The rise in Rd was more rapid (P less than 0.05) and metabolic clearance rate was greater during (P = 0.015) and from 9 to 60 min after EX2, and Ra also remained higher during recovery (P less than 0.05). Marked and similar increments in plasma norepinephrine (18-fold) and epinephrine (14-fold) occurred with both bouts. Plasma glucagon increments were small and not different. Therefore, 1) more circulating glucose was used with EX2, 2) greater metabolic clearance rate during and after EX2 suggests local muscle adaptations due to EX1, and 3) significant correlations (P less than 0.002) between plasma norepinephrine and Ra (r = 0.82) and Ra - Rd (r = 0.52) and between epinephrine and Ra (r = 0.71) and Ra - Rd (r = 0.48) suggest a major regulatory role for the catecholamine responses.     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

In vivo 31P-NMR studies on energy metabolism in and catecholamine effect on rat liver during hypovolemic shock

In vivo 31P-NMR spectroscopy (31P-MRS) was used to study the metabolism of phosphate compounds in rat liver under various conditions. The changes in hepatic concentrations of ATP and inorganic phosphate (Pi) or intracellular pH (pHi) were monitored during hypovolemic shock with or without the infusion of catecholamines. Rapid decreases in the ATP level and pHi with a concomitant increase of Pi were observed upon induction of the hypovolemic shock. Dopamine infusion markedly improved the liver ATP concentration and intracellular acidosis, but epinephrine or norepinephrine were without effects. The present results suggest that dopamine increases abdominal blood flow and improves the energy metabolism in the liver during hypovolemic shock.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Oxidative metabolism and anaerobic glycolysis during repeated exercise

The purpose of the present study was to examine aerobic and muscle anaerobic energy production during supramaximal repeated exercise. Eight subjects performed three 2-min bouts of cycling (EX1-EX3) at an intensity corresponding to about 125 % of VO2 max separated by 15 min of rest. Ventilatory variables were measured breath by breath during the exercise and a muscle biopsy was taken before and after each exercise bout. Blood samples were collected before and after each cycling period and during the recovery periods. Total work in the first 2 min bout of cycling, EX1, [46.3 +/- 2.1 KJ] was greater than in the second, EX2, (p < 0.01) and in the third, EX3, (p < 0.05). The ATP utilization [4.0 +/- 1.4 mmol x (kg dry weight)(-1), EX1] during the three exercise bouts was the same. The decrement in muscle phosphocreatine (PCr) [46.8 +/- 8.5 mmol x (kg dry weight)(-1), EX1] was also similar for the three exercise bouts. Muscle lactate accumulation was greater (p < 0.05) during EX1 compared to EX2 and EX3. The total oxygen consumption was the same for the three exercise bouts, but when it is corrected for the total work performed, oxygen uptake during EX2 (153 +/- 9 ml x KJ(-1)) and EX3 (150 +/- 9 ml x KJ(-1)) was higher (p < 0.01 and p < 0.05, respectively) than during EX1 (139 +/- 8 ml x KJ(-1)). The present data suggest that oxidative metabolism does not compensate for the reduction of anaerobic glycolysis during repeated fatiguing exercise.     

Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), beta-nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue PCO2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by 1H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.     

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regular exercise enhances blood pressure lowering effect of acetylcholine by increased contribution of nitric oxide

This study is aimed to test the hypothesis, that short-term daily bouts of exercise alter the endothelial regulation of peripheral vascular resistance by nitric oxide. Rats ran on a treadmill once a day, 5 days a week, for an average of three weeks with gradually increasing intensity (EX), while a control group remained sedentary (SED). Dose dependent reductions in mean arterial blood pressure (resting MABP; SED: 120.0 +/- 3.4 and EX: 127.8 +/- 4.0 mm Hg) of pentobarbital anesthetized rats to intravenous endothelium independent dilator sodium nitropmsside (SNP; 0.6-3.0 microg/kg) were not different in EX and SED animals. In contrast, dose dependent reductions in MABP to endothelium dependent dilator acetylcholine (ACh) were significantly enhanced in EX compared to those in SED rats (at 0.5 and 1.0 microg/kg ACh: 60.3 +/- 2.4 and 66.5 +/- 1.8 vs 52.8 +/- 2.0 and 59.8 +/- 1.7 mmHg, respectively, p<0.01). There was no significant difference in the heart rate (HR) response to ACh and SNP in the two groups of rats. Intravenous administration of 20 mg/kg Nomega-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor) elicited a similar increase (approximately 30%) in the MABP in the two groups and eliminated the difference between ACh-induced blood pressure lowering responses in EX and SED rats (at 0.5 and 1.0 microg/kg ACh: 44.6 +/- 4.7 and 56.3 +/- 4.4 vs 50.9 +/- 4.5 and 59.4 +/- 3.6 mm Hg, respectively). Thus, we suggest that the enhanced acetylcholine-induced decrease in systemic blood pressure following regular daily exercise is primarily due to the augmented synthesis of nitric oxide in the endothelium of peripheral vasculature. This change in the function of endothelium could be important in the adaptation of circulation to exercise training.     

31P-NMR spectroscopy and the metabolic properties of different muscle fibers

To study the in vivo recruitment of different fiber types and their metabolic properties, 31P-nuclear magnetic resonance spectroscopy (31P-NMRS) of the human calf muscle was performed in seven normal sedentary subjects. In the exhaustive exercise protocol used, the work load was increased every minute during 5 min. This resulted in a prominent split of the Pi resonance in all subjects, indicating pH compartmentation in the muscles studied. From the chemical shift of the Pi peaks relative to phosphocreatine (PCr) at the end of the exercise, intracellular pH (pHi) averaged 6.92 +/- 0.05 (SD) in compartment 1 and 6.23 +/- 0.15 in compartment 2. The recovery of both Pi resonances after exercise could be followed easily in five of these subjects. The recovery rate of the Pi peak is a good estimate of the oxidative metabolism at the end of the exercise. A monoexponential regression analysis showed that the mean initial recovery rate S0 was 2.49 +/- 0.17%/s in compartment 1 and only 0.87 +/- 0.12%/s in compartment 2, indicating aerobic function three times higher in compartment 1 at the end of exercise. The mean relative ATP fraction dropped significantly (P less than 0.001), from 20.0 +/- 1.0% of the total 31P signal integral before exercise to 14.0 +/- 1.6% at the end of exercise. The simultaneous visualization of two compartments, in good order, one with high pHi and fast recovery and another with low pHi and slow recovery, is rationalized by the different metabolic behavior of type I and II fibers in human calf muscle in response to exhaustive exercise. This study demonstrates that 31P-NMRS is an excellent noninvasive procedure to quantify aerobic metabolism in both fiber types simultaneously.