87Rb, 23Na and 31P nuclear magnetic resonance spectroscopy of the perfused rat kidney

87Rb, 23Na and 31P nuclear magnetic resonance (NMR) were used to monitor changes in renal cations and energetics during the induction of hypoxia in the isolated perfused rat kidney. The NMR-determined unidirectional Rb+ flux in normoxic kidneys was shown to be a good measure of net intracellular K+ influx in the perfused rat kidney model. The changes in 87Rb, 23Na and 31P spectra following the induction of hypoxia are consistent with hypoxic depletion of intracellular adenosine triphosphate (ATP) and a subsequent decrease in Na-K-ATPase transport activity. The exponential rate constant for 87Rb+ efflux measured during Rb+ uptake in normoxic kidneys (0.12 +/- 0.01 min-1) was not significantly different to the rate constant for 87Rb+ efflux during the induction of hypoxia (0.16 +/- 0.07 min-1). We conclude that there is no direct effect of hypoxia on renal cellular membrane integrity and that renal cell sensitivity to hypoxia is due to an inability to sustain cellular ion gradients following depletion of intracellular ATP.     

Hyposmotic shock: effects on rubidium/potassium efflux in normal and ischemic rat hearts,assessed by 87Rb and 31P NMR

The study evaluated effects of hyposmotic shock on the rate of Rb(+)/K(+) efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl alpha-D-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb(+) efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb(+)/K(+) uptake, indicating that the activity of Na(+)/K(+) ATPase was not affected. Dimethylamiloride (DMA) (10 microM) abolished activation of the Rb(+)/K(+) efflux in the second model; however, Na(+)/H(+) exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb(+)/K(+) efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 microM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb(+)/K(+) efflux occurred, at least partially, through the DMA-sensitive cation/H(+) exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia-reperfusion-induced activation of Rb(+)/K(+) efflux.     

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.     

Acetylcholine-induced metabolic changes in the perfused rabbit mandibular salivary gland studied by 31P-NMR spectroscopy

Changes in the content of high-energy phosphates, intracellular pH (pHi) and the ratio of MgATP to total ATP ([MgATP]/[ATP]t) resulting from continuous stimulation with acetylcholine (10(-9) to 10(-4) M) were measured by 31P-NMR spectroscopy in the isolated, perfused rabbit mandibular gland at 37 degrees C. With 10(-9) to 10(-7) M acetylcholine, no significant changes in these parameters were observed. On stimulation with 10(-6) M acetylcholine, the optimal concentration for sustained secretion, the content of ATP decreased by 28 +/- 10% (mean +/- S.E.; n = 8) of its control value. pHi decreased initially by approx. 0.05 pH unit, then showed an alkalinization of 0.09 +/- 0.02 pH unit (n = 8). With 10(-5) and 10(-4) M acetylcholine, changes in ATP and pHi were similar to those induced by 10(-6) M acetylcholine: the total content of high-energy phosphates remained at approx. 70% of the control value and no decrease in [MgATP]/[ATP]t was observed. As possible causes of the reduced secretory rate observed with higher concentrations of acetylcholine (10(-5) to 10(-3) M), we can exclude depletion of high-energy phosphates, inhibition of metabolism caused by intracellular acidosis, and inhibition of ATP usage caused by a decrease in MgATP availability.     

Potassium fluxes, energy metabolism, and oxygenation in intact diabetic rat hearts under normal and stress conditions

We evaluated the function of Na(+)/K(+) ATPase and sarcolemmal K(ATP) channels in diabetic rat hearts. Six weeks after streptozotocin (STZ) injection, unidirectional K(+) fluxes were assayed by using (87)rubidium ((87)Rb(+)) MRS. The hearts were loaded with Rb(+) by perfusion with Krebs-Henseleit buffer, in which 50% of K(+) was substituted with Rb(+). The rate constant of Rb(+) uptake via Na(+)/K(+) ATPase was reduced. K(ATP)-mediated Rb(+) efflux was activated metabolically with 2,4-dinitrophenol (DNP, 50 micromol.L(-1)) or pharmacologically with a K(ATP) channel opener, P-1075 (5 micromol.L(-1)). Cardiac energetics were monitored by using (31)P MRS and optical spectroscopy. DNP produced a smaller ATP decrease, yet similar Rb(+) efflux activation in STZ hearts. In K(+)-arrested hearts, P-1075 had no effect on high-energy phosphates and stimulated Rb(+) efflux by interaction with SUR2A subunit of K(ATP) channel; this stimulation was greater in STZ hearts. In normokalemic hearts, P-1075 caused cardiac arrest and ATP decline, and the stimulation of Rb(+) efflux was lower in normokalemic STZ hearts arrested by P-1075. Thus, the Rb(+)efflux stimulation in STZ hearts was altered depending on the mode of K(ATP) channel activation: pharmacologic stimulation (P-1075) was enhanced, whereas metabolic stimulation (DNP) was reduced. Both the basal concentration of phosphocreatine ([PCr]) and [PCr]/[ATP] were reduced; nevertheless, the STZ hearts were more or equally resistant to metabolic stress.     

丹参对心肌低氧/复氧损伤的保护作用的研究

目的：研究中药丹参（ＳＭ）对心肌低氧／复氧损伤的保护作用。方法：运用＾３１Ｐ－ＮＭＲ技术对离体灌流大鼠心脏的高能磷酸化合物含量及细胞内的ｐＨ值（ｐＨｉ）进行动态跟踪。结果：丹参注射液能明显减轻低氧期间心肌高能磷酸合物含量的下降,促使复氧期间ＰＣｒ、ＡＴＰ相对含量的恢复,减少低氧及复氧阶段心肌ｐＨｉ的下降。结论：丹参参改善低氧及复氧期间心肌能量代谢水平,减轻心肌低氧／复氧损伤,并能显著改善细胞内酸碱     

Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia)

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.     

Intracellular pH and oxygen chemoreception in the cat carotid body in vitro.

To test the hypothesis that O2 chemoreception in the carotid body (CB) is mediated by cellular acidosis, we simultaneously measured responses of the chemosensory and intracellular pH (pHi) to agents that are known to change pHi and studied the effects of hypoxia and ischemia on these variables in the cat CB. The CB was perfused and superfused in vitro with a modified Tyrode's solution at 36.0 +/- 0.5 degrees C with or without CO2-HCO3- (pH 7.40) and equilibrated at a given PO2. Chemosensory discharges were recorded from the whole carotid sinus nerve. To measure pHi changes, the CB was loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and the fluorescence (excitation 420-490 nm, emission greater than 515 nm) was detected by an intensified charged coupled device camera with an epifluorescence macroscope. Boluses of Tyrode's solution (0.5 ml, free of CO2-HCO3-) containing sodium acetate or NH4Cl prolonged perfusion of acid Tyrode's solution (pH 7.20-6.50), and boluses of Tyrode's solution with CO2-HCO3- were used. A decrease of fluorescence indicated pHi turning acid, and an increase of fluorescence indicated a change in alkaline pHi. Chemosensory activity varied inversely with the fluorescence change after application of these agents. Interruption of perfusate flow or application of hypoxic perfusate resulted in large increases in chemosensory discharge without any change in the fluorescence. The results indicated that chemosensory responses to brief ischemia and hypoxia were not mediated by a fall of pHi of CB cells, whereas those to CO2 and extracellular acidity were associated with decreases in pHi.     

Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR

The relationships between pHi (intracellular pH) and phosphate compounds were evaluated by nuclear magnetic resonance (NMR) in normo-, hypo-, and hypercapnia, obtained by changing fractional inspired concentration of CO2 in dogs anesthetized with 0.75% isoflurane and 66% N2O. Phosphocreatine (PCr) fell by 2.02 mM and Pi (inorganic phosphate) rose by 1.92 mM due to pHi shift from 7.10 to 6.83 during hypercapnia. The stoichiometric coefficient was 1.05 (r2 = 0.78) on log PCr/Cr against pHi, showing minimum change of ADP/ATP and equilibrium of creatine kinase in the pH range of 6.7 to 7.25. [ADP] varied from 21.6 +/- 4.1 microM in control (pHi = 7.10) to 26.8 +/- 6.3 microM in hypercapnia (pHi = 6.83) and 24.0 +/- 6.8 microM in hypocapnia (pHi = 7.17). ATP/ADP X Pi decreased from 66.4 +/- 17.1 mM-1 during normocapnia to 25.8 +/- 6.3 mM-1 in hypercapnia. The ADP values are near the in vitro Km; thus ADP is the main controller. The velocity of oxidative metabolism (V) in relation to its maximum (Vmax) as calculated by a steady-state Michaelis-Menten formulation is approximately 50% in normocapnia. In acidosis (pH 6.7) and alkalosis (pH 7.25), V/Vmax is 10% higher than the normocapnic brain. This increase of V/Vmax is required to maintain cellular homeostasis of energy metabolism in the face of either inhibition at extremes of pH or higher ATPase activity.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.     

31-P NMR characterization of the metabolic anomalies associated with the lack of glycogen phosphorylase activity in human forearm muscle.

Exercise-induced changes in phosphorus-containing metabolites and intracellular pH (pHi) have been studied in the finger flexor muscles of 3 patients with glycogen phosphorylase deficiency (McArdle's disease) in comparison to 14 healthy volunteers. At rest, no difference was observed for PCr/Pi ratio and pHi while patients exhibited a higher PCr/ATP ratio (5.91 +/- 0.98 vs 4.02 +/- 0.6). At end-of-exercise, PCr/Pi was abnormally low (0.51 +/- 0.19 vs 1.64 +/- 0.37) whereas no acidosis was observed. The slow recovery of PCr/Pi ratio indicates an impairment of oxidative capacity accompanying the defect in the glycogenolytic pathway. The failure to observe a transient Pi disappearance at the onset of recovery (an index of glycogen phosphorylase activity) can be used in conjunction with the lack of exercise acidosis as a diagnostic index of McArdle's disease.     

Temperature and pH dependence of energy balance by (31)P- and (1)H-MRS in anaerobic frog muscle

The temperature (T)-dependence of energy consumption of resting anaerobic frog gastrocnemii exposed to different, changing electrochemical gradients was assessed. To this aim, the rate of ATP resynthesis (delta approximately P/deltat) was determined by (31)P- and (1)H-MRS as the sum of the rates of PCr hydrolysis (delta[PCr]/deltat) and of anaerobic glycolysis (delta[La]/ deltat, based on a approximately P/La ratio of 1.5). The investigated T levels were 15, 20 and 25 degrees C, whereas initial extracellular pH (pHe) values were 7.9, 7.3 and 7.0, i.e. higher, equal or lower, respectively, than intracellular pH (pHi). The latter was changing with T according to the neutrality point (dpH/dT=-0.0165 pH units/ degrees C). Both rates of PCr hydrolysis and of lactate accumulation and that of their sum, expressed as delta approximately P/deltat, were highly T-dependent. By contrast, the pHe-dependence of the muscle energy balance was nil or extremely limited at 15 and 20 degrees C, respectively, but remarkable at 25 degrees C (with a depression of the ATP resynthesis rate up to 25% with a decrease of pHe from 7.9 to 7.0). The pHe-dependent reduction of metabolic rate was associated with a down-regulation of anaerobic glycolysis due to reduced activity of ion-transporters controlling acid-base balance and/or to a shift from Na(+)/H(+) to a more efficient Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Uncoupling of glycogenolysis from P-metabolite concentrations, both as function of T (>or=20 degrees C) and of pHe (相似文献   

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

The coronary vasodilator mediator released by hypoxia and isoprenaline is not affected by cyclo-oxygenase inhibition

Donor and recipient guinea-pig hearts were perfused in series, the recipient being perfused with regassed donor effluent. Coronary perfusion pressure and force and rate of contraction were measured. Exposure of donor hearts to hypoxia (1.5 min) and to isoprenaline (5 ng) caused the appearance of vasodilator material in recipient hearts, the direct beta-adrenoceptor effects of isoprenaline carried over in the effluent being antagonized in the recipient by propranolol. Cyclo-oxygenase was inhibited by infusion of meclofenamate (60 micrograms X min-1) which consistently abolished the vasodilator responses to arachidonic acid added to the donor. The vasodilator responses of the donor to hypoxia and isoprenaline were unaffected by meclofenamate. The falls in perfusion pressure of the recipient in response to material released by these procedures were also not significantly different before (hypoxia, 11.5 +/- 2.6mm Hg; isoprenaline, 10.3 +/- 1.3mm Hg) and during the infusion (hypoxia, 10.2 +/- 4.1; isoprenaline, 11.0 +/- 1.3mm Hg). The coronary vasodilator responses to hypoxia and isoprenaline and the vasodilator material released by these procedures do not therefore appear to be due to products of arachidonic acid via cyclo-oxygenase pathways. Furthermore, since there was also no potentiation of the responses, there does not appear to be a concomitant release of a prostanoid to inhibit the major vasodilator material. Adenosine, as the likely candidate for this predominant vasodilator mediator, is discussed.