Fall In Intracellular pH Research Articles

Mathematical modeling of the role of carbonic anhydrase II and IV on the influx of CO2 in a Xenopus oocyte

Exposing oocytes to a solution containing CO2/HCO3 causes the familiar fall in intracellular pH (pHi) and a rise in surface pH (pHS) followed by a decay. Musa‐Aziz et al (ASN, 2005) examined the effects of carbonic anhydrases (CAs) on pH transients caused by CO2 influx. They found that injecting CAII into oocytes or expressing CAIV on the oocyte surface accelerates the initial rate of pHi acidification (dpHi/dt) and amplifies the height of the pHS spike (ΔpHS). They proposed that both enzymes enhance CO2 influx by maximizing CO2 gradients across the plasma membrane. We use a mathematical model of a Xenopus oocyte—which accounts for CO2, carbonic acid (H2CO3), HCO3, protons, and a multitude of non‐HCO3 buffer pairs—to investigate the above findings. We simulate the experimental protocol in which the CO2/HCO3 solution is delivered from the bulk to the oocyte surface by raising exponentially the concentrations of CO2, H2CO3 and HCO3 in the bulk and assuming that initially no CO2, H2CO3 and HCO3 are present in the extracellular unconvected fluid. We use the experimental data for 1.5% CO2/10mM HCO3 (pHo=7.5) to find parameter values that simultaneously match ΔpHS, the time that it takes for pHS to reach its peak (tP), dpHi/dt, and the time delay in the pHi decay. We validate the model against data collected when exposing oocytes to 5% CO2/33mM HCO3 and to 10% CO2/66mM HCO3. The model confirms that CAII and CAIV enhance CO2 influx.

Blockade of Na+/H+ exchange enhances phrenic burst frequency under control conditions but not in response to hypercapnia in arterially‐perfused adult rat

Increased levels of CO2 (hypercapnic acidosis, HA) produce a marked increase in ventilation. Numerous studies have suggested that this hypercapnia‐induced respiratory excitation results from the fall in intracellular pH (pHi), and that Na+/H+ exchange (NHE) plays an important role in regulating pHi recovery during HA. In CO2 chemosensitive respiratory neurons, however, pHi recovery is not observed during HA. The current study was therefore undertaken to determine the role of NHE in respiratory activity under control conditions and in response to HA. To address this issue, we examined phrenic nerve discharge in response to increasing CO2 from 5% to 10% in an arterially‐perfused adult rat preparation (n=5) under control conditions and during NHE blockade using dimethyl‐amiloride hydrochloride (DMA, 283 µM). Under control conditions, HA increased burst frequency by ~34% (P<0.01) due to a marked reduction in TE (~25%, P<0.01) and a small decrease in TI (~15%, P=0.1). Perfusion with DMA elicited a robust increase in burst frequency of ~65% (P<0.001) due solely to a reduction in TE (P<0.001). Under these conditions, burst frequency was not further altered by HA (P=0.2) although TI was still decreased by ~15% (P<0.05). These results indicate that interference with pHi regulation is capable of enhancing phrenic burst frequency under control conditions but is ineffective in altering burst frequency during HA. Supported by NS045321

A Ca(2+)-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions.

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesized that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) phosphorylation by signalling cascades downstream of rises in intracellular [Ca(2+)] and decreased energy charge via AMP-activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis correlated more closely with changes in eEF2 than 4EBP1 phosphorylation. Using a combination of Ca(2+) release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca(2+) and energy turnover-related mechanisms. Furthermore, eEF2 kinase (eEF2K) inhibition completely blunted increases in eEF2 phosphorylation and partially blunted (i.e. 30-40%) the suppression of protein synthesis during contractions. The 3- to 5-fold increase in skeletal muscle eEF2 phosphorylation during contractions in situ was rapid and sustained and restricted to working muscle. The increase in eEF2 phosphorylation and eEF2K activation were downstream of Ca(2+)-calmodulin (CaM) but not other putative activating factors such as a fall in intracellular pH or phosphorylation by protein kinases. Furthermore, blunted protein synthesis and 4EBP1 dephosphorylation were unrelated to AMPK activity during contractions, which was exemplified by normal blunting of protein synthesis during contractions in muscles overexpressing kinase-dead AMPK. In summary, in fast-twitch skeletal muscle, the inhibition of eEF2 activity by phosphorylation downstream of Ca(2+)-CaM-eEF2K signalling partially contributes to the suppression of protein synthesis during exercise/contractions.

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to Solutions Containing NH3/NH4 +

Others have shown that exposing oocytes to high levels of NH(3)/NH(4)(+) (10-20 mM) causes a paradoxical fall in intracellular pH (pH(i)), whereas low levels (e.g., 0.5 mM) cause little pH(i) change. Here we monitored pH(i) and extracellular surface pH (pH(S)) while exposing oocytes to 5 or 0.5 mM NH(3)/NH(4)(+). We confirm that 5 mM NH(3)/NH(4)(+) causes a paradoxical pH(i) fall (-DeltapH(i) approximately equal 0.2), but also observe an abrupt pH(S) fall (-DeltapH(S) approximately equal 0.2)-indicative of NH(3) influx-followed by a slow decay. Reducing [NH(3)/NH(4)(+)] to 0.5 mM minimizes pH(i) changes but maintains pH(S) changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates -DeltapH(S) at both NH(3)/NH(4)(+) levels. During removal of 0.5 or 5 mM NH(3)/NH(4)(+), failure of pH(S) to markedly overshoot bulk extracellular pH implies little NH(3) efflux and, thus, little free cytosolic NH(3)/NH(4)(+). A new analysis of the effects of NH(3) vs. NH(4)(+) fluxes on pH(S) and pH(i) indicates that (a) NH(3) rather than NH(4)(+) fluxes dominate pH(i) and pH(S) changes and (b) oocytes dispose of most incoming NH(3). NMR studies of oocytes exposed to (15)N-labeled NH(3)/NH(4)(+) show no significant formation of glutamine but substantial NH(3)/NH(4)(+) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH(i) and pH(S) demonstrate that NH(3) flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane-AmtB-enhances the flux of a gas across a biological membrane.

The effect of O2 tension on pH homeostasis in equine articular chondrocytes

To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions. Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescein diacetate. The pH homeostasis was unaffected when comparing tissue maintained at 20% O(2) (the level in water-saturated air at 37 degrees C) with that at 5% O(2) (which approximates the normal level in healthy cartilage); however, an O(2) tension of <5% caused a fall in intracellular pH (pH(i)) and slowed pH(i) recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na(+)/K(+) pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H(2)O(2), Co(2+), or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. The ability of articular chondrocytes to carry out pH homeostasis is compromised when O(2) tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O(2), such as in chronic inflammation.

Rapid intracellular acidification and cell death by H 2O 2 and alloxan in pancreatic β cells

Pancreatic β-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic β-cell line NIT-1 cells exposed to H 2O 2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H 2O 2 and alloxan caused a rapid fall in intracellular pH and suppressed Na +/H + exchanger activity in the NH 4Cl prepulse method. H 2O 2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 ± 5.8% vs 21.1 ± 10.6%, P < 0.05; apoptosis 8.0 ± 1.9% vs 44.5 ± 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H 2O 2-treated β cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.

Strain-specific effects of acidic pH on contractile state of aortas from Wistar and Wistar Kyoto rats

The effects of acidosis were investigated on the resting and precontracted aortas from Wistar and Wistar Kyoto (WKY) rats. Decrease in pH from 7.4 to 6.5, having no effect on the resting tension of Wistar aorta, induced a marked contraction of WKY aorta. Acidic pH markedly relaxed the contraction to 300 nM phenylephrine in Wistar aorta, whereas in WKY aorta, it produced a biphasic response, an initial relaxation followed by potentiation of the contraction. In aortas loaded with fura 2-AM, phenylephrine caused an increase in intracellular Ca 2+ ([Ca 2+] i) and a contraction in both Wistar and WKY rats. pH 6.5 produced a decrease in [Ca 2+] i to a near-basal level and almost abolished the phenylephrine-induced contraction in Wistar rat aorta. However, in WKY aorta, a biphasic response, an initial decline and later a recovery of [Ca 2+] i level, was observed. Interestingly, at similar sustained [Ca 2+] i, the contractile response to phenylephrine in WKY aorta was potentiated under acidic pH conditions. Acidic pH-induced inhibition of the contraction to phenylephrine was unaffected by iberiotoxin, 4-aminopyridine, and glibenclamide (Ca 2+-activated, delayed rectifier and ATP-sensitive K + channel inhibitors, respectively), in aortas from both Wistar and WKY. Decrease in extracellular pH was associated with a rapid fall in intracellular pH (pH i) and the intracellular acidification profile was not different in both strains. All these results show that acidic pH induces strain-specific inhibitory and excitatory effects on the contractile state of aortas from Wistar and WKY rats, respectively. The sustained and transient relaxant responses to acidic pH in Wistar and WKY aortas, respectively, are due to decrease in [Ca 2+] i levels, but this decrease in [Ca 2+] i is independent of the activation of K + channels.

Leukocyte-type 12-lipoxygenase-deficient mice show impaired ischemic preconditioning-induced cardioprotection.

To investigate the role of 12-lipoxygenase in preconditioning, we examined whether hearts lacking the "leukocyte-type" 12-lipoxygenase (12-LOKO) would be protected by preconditioning. In hearts from wild-type (WT) and 12-LOKO mice, left ventricular developed pressure (LVDP) and (31)P NMR were monitored during treatment (+/-preconditioning) and during global ischemia and reperfusion. Postischemic function (rate-pressure product, percentage of initial value) measured after 20 min of ischemia and 40 min of reperfusion was significantly improved by preconditioning in WT hearts (78 +/- 12% in preconditioned vs. 44 +/- 7% in nonpreconditioned hearts) but not in 12-LOKO hearts (47 +/- 7% in preconditioned vs. 33 +/- 10% in nonpreconditioned hearts). Postischemic recovery of phosphocreatine was significantly better in WT preconditioned hearts than in 12-LOKO preconditioned hearts. Preconditioning significantly reduced the fall in intracellular pH during sustained ischemia in both WT and 12-LOKO hearts, suggesting that attenuation of the fall in pH during ischemia can be dissociated from preconditioning-induced protection. Necrosis was assessed after 25 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride. In WT hearts, preconditioning significantly reduced the area of necrosis (26 +/- 4%) compared with nonpreconditioned hearts (62 +/- 10%) but not in 12-LOKO hearts (85 +/- 3% in preconditioned vs. 63 +/- 11% in nonpreconditioned hearts). Preconditioning resulted in a significant increase in 12(S)-hydroxyeicosatetraenoic acid in WT but not in 12-LOKO hearts. These data demonstrate that 12-lipoxygenase is important in preconditioning.

Effect of intracellular pH on spontaneous Ca2+ sparks in rat ventricular myocytes.

1. A fall of intracellular pH (pHi) typically depresses cardiac contractility. Among the many mechanisms underlying this depression, an inhibitory effect of acidosis upon the sarcoplasmic reticulum (SR) Ca2+ release channel has been predicted, but not so far demonstrated in the intact cardiac myocyte. In the present work, pHi was manipulated experimentally while confocal imaging was used to record spontaneous 'Ca2+ sparks' (local SR Ca2+ release events) in rat isolated myocytes loaded with the fluorescent Ca2+ indicator fluo-3. In other experiments, whole cell (global) pHi or [Ca2+]i was measured by microfluorimetry (using, respectively, intracellular carboxy SNARF-1 and indo-1). 2. Reducing pHi (i) increased whole cell intracellular [Ca2+] transients induced either electrically or by addition of caffeine, whereas (ii) it decreased spontaneous Ca2+ spark frequency. Conversely, raising pHi increased spontaneous Ca2+ spark frequency. 3. Blocking sarcolemmal Ca2+ influx with 10 mM Ni2+, or reducing external pH by 1.0 unit, had no effect on the pHi-dependent changes in spontaneous Ca2+ spark frequency. 4. Decreasing pHi over the range 7.78-7.20, decreased Ca2+ spark frequency exponentially as a function of pHi, with frequency declining by approximately 33 % for a 0.2 unit fall in pHi. In contrast, over the same pHi range, Ca2+ spark amplitude was unaffected. Intracellular acidosis produced a slight slowing of Ca2+ spark relaxation. 5. The results indicate that, in the intact myocyte, a reduced pHi decreases the probability of opening of the SR Ca2+ release channel. This phenomenon may contribute to the negative inotropic effects of acidosis.

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC).

Our group recently cloned the electrogenic Na+-HCO-3 cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with approximately 22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5' fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM HCO-3 hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl- from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 microM) greatly reduces the hyperpolarization caused by adding CO2/HCO-3. In oocytes expressing hkNBC, the effects of adding CO2/HCO-3 and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-HCO-3 cotransporter and that hkNBC is also electrogenic.

Cardioprotective actions of KC 12291 II. Delaying Na+ overload in ischemia improves cardiac function and energy status in reperfusion

The novel blocker of voltage-gated Na+ channels KC 12291 (1-(5-phenyl-1,2,4-thiadiazol-3-yl-oxypropyl)-3-[N-methyl-N- [2-(3,4-dimethoxyphenyl)ethyl] amino] propane hydrochloride) delays myocardial Na+ overload in ischemia. To test whether KC 12291 displays cardioprotective properties in the intact heart, cardiac function, energy status and intracellular pH (31P NMR) as well as ion homeostasis (23Na NMR) were investigated during low-flow ischemia (100 microl/min for 36 min) followed by reperfusion. In the well-oxygenated, isolated perfused guinea pig heart, KC 12291 (1 microM) had no effect on left ventricular developed pressure (LVDP; 54+/-19 mmHg). KC 12291 delayed the onset and decreased the extent of ischemic contracture and markedly improved the recovery of LVDP in reperfusion [39+/-14 mmHg (n=4) vs 2+/-2 mmHg in controls (n=5)]. KC 12291 did not influence the rapid drop in phosphocreatine (PCr) following onset of ischemia but attenuated the decline in ATP. It also diminished the ischemia-induced fall in intracellular pH [6.39+/-0.2 (n=6) vs 6.18+/-0.20 in controls (n=6)]. In reperfusion, KC 12291 remarkably enhanced the recovery of PCr (84.8+/-9.6% vs 51.1+/-8.8% of baseline) and ATP (38.2+/-12.9% vs 23.7+/-9.3% of baseline). It also accelerated the recovery of intracellular pH. KC 12291 not only reduced the extent of ischemia-induced Na+ overload, but also enhanced Na+ recovery. It is concluded that KC 12291 delays contracture and reduces ATP depletion and acidosis in ischemia, and markedly improves the functional, energetic and ionic recovery in reperfusion. Blocking voltage-gated Na+ channels in ischemia to delay Na+ overload may thus constitute a promising therapeutic approach for cardioprotection.

Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons.

Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by approximately 20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta cells during rapid but not slow firing rates. This effect of internal beta on spike repolarization could be blocked by cadmium. High beta also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.

Changes in ventricular repolarization during acidosis and low-flow ischemia.

Myocardial ischemia, primarily a metabolic insult, is also defined by altered cardiac mechanical and electrical activity. We have investigated the metabolic contributions to the electrophysiological changes during low-flow ischemia (7.5% of the control flow) using 31P NMR spectroscopy to monitor metabolic parameters, suction electrodes to study epicardial monophasic action potentials, and 86Rb as a tracer for K+-equivalent efflux during low-flow ischemia in the Langendorff-perfused ferret heart. Shortening of the action potential duration at 90% repolarization (APD90) was most marked between 1 and 5 min after induction of ischemia, at which time it shortened from 261 +/- 4 to 213 +/- 8 ms. The period of marked APD90 shortening was accompanied by a fivefold increase in the rate of 86Rb efflux, both of which were inhibited by the ATP-sensitive K+ (KATP)-channel blockers glibenclamide and 5-hydroxydecanoate (5-HD), as well as by a significant fall in intracellular pH (pHi) from 7.14 +/- 0.02 to 6.83 +/- 0.03 but no change in intracellular ATP concentration ([ATP]i). We therefore investigated whether a fall in pHi could be the metabolic change responsible for modulating cardiac KATP channel activity in the intact heart during ischemia. Both metabolic (30 mM lactate added to extracellular solution) and respiratory (PCO2 increased to 15%) acidosis caused an initial lengthening of APD90 to 112 +/- 1.5 and 113 +/- 0.9%, respectively, followed by shortening during continued acidosis to 106 +/- 1.2 and 106 +/- 1.4%, respectively. The shortening of APD90 during continued acidosis was inhibited by glibenclamide, consistent with acidosis causing activation of KATP channels at normal [ATP]i. The similar responses to metabolic (induced by adding either l- or d-lactate) and respiratory acidosis suggest that lactate has no independent metabolic effect on action potential repolarization.

Effects of CO2-induced acidification on the fatigue resistance of single mouse muscle fibers at 28 degrees C.

The role of reduced muscle pH in the development of skeletal muscle fatigue is unclear. This study investigated the effects of lowering skeletal muscle intracellular pH by exposure to 30% CO2 on the number of isometric tetani needed to induce significant fatigue. Isolated single mouse muscle fibers were stimulated repetitively at intervals of 4-2.5 s by using 80-Hz, 400-ms tetani at 28 degrees C in Tyrode solution bubbled with either 5 or 30% CO2. Stimulation continued until tetanic force had fallen to 40% of the initial value. Exposure to 30% CO2 caused a significant fall in intracellular pH of approximately 0.3 pH unit but did not cause any significant changes in initial peak tetanic force. During the course of repetitive stimulation, intracellular pH fell by approximately 0.3 pH unit in both normal and acidified fibers. The number of tetani needed to reduce force to 40% of the initial value was not significantly different in 5 and 30% CO2 Tyrode. The sole effect of acidosis was to reduce the rate of relaxation of force, especially in fatigued fibers. It is concluded that, at 28 degrees C, acidosis per se does not accelerate the development of fatigue during repeated tetanic stimulation of isolated mouse skeletal muscle fibers.

Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism.

1. The fall of intracellular pH (pH1) following the reduction of extracellular pH (pH0) was investigated in guinea-pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy-SNARF-1 (to monitor pH1). Cell superfusates were buffered either with a 5% CO2-HCO3- system or were nominally CO2-HCO3-free. 2. Reduction of pH0 from 7.4 to 6.4 reversibly reduced pH1 by about 0.4 pH units, independent of the buffer system used. 3. In HCO3(-)-free conditions, acid loading in low pH0 was not dependent on Na(+)-H+ exchange or on the presence of Na+. It was unaffected by high-K+ solution, by voltage-clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2+) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H(+)-or Ca(2+)-conductance channels or influx via a K(+)-H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, alpha-cyano-4-hydroxy cinnamate. 4. In HCO3(-)-free conditions, acid loading in low pH0 was reversibly inhibited (by up to 85%) by Cl(-)0 removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl(-)-HCO3-exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel Cl(-)-dependent, acid influx pathway. 5. After switching to CO2-HCO3(-)-buffered conditions, acid loading was doubled. It was still not inhibited by Na(+)-free or high-K+ solutions but was once again inhibited (by 78%) in Cl(-)-free solution. The HCO3(-)-stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl(-)-HCO3-exchange, while we suggest the other to be a novel Cl(-)-OH-exchanger (although we do not rule out the alternative configuration of H(+)-Cl-co-influx). The proposed dual acid-loading mechanism accounts for most of the sensitivity of pH1 to a fall of pH0.

The molecular basis of adaptation to ischemia in the heart: the role of stress proteins and anti-oxidants in the ischemic and reperfused heart.

When blood flow to the myocardium is interrupted, for example during a coronary occlusion or coronary artery spasm, the resulting ischemia is characterised by certain metabolic and ultrastructural perturbations including a fall in intracellular pH, decreased ATP levels, decreased glucose levels, increased lactate levels, membrane damage, electromechanical uncoupling and uncoupling of mitochondrial oxidative phosphorylation [1]. This hypoxic phase, with its associated oxygen depletion, therefore leads to significant metabolic stress. When blood flow and, therefore, oxygen are returned to the hypoxic tissue during reperfusion, oxidative stress occurs due to oxygen-derived free radical formation which causes further damage to membranes (via lipid peroxidation), nucleic acids and proteins. In addition, reperfusion is associated with intracellular and mitochondrial calcium overload which causes further damage and uncoupling of respiration and contraction. If ischemia is prolonged, inevitably the changes become irreversible and cell death and tissue necrosis occur.

Mechanism of butyrate-induced vasorelaxation of rat mesenteric resistance artery

1. The vasorelaxant effect of the sodium salt of the short chain fatty acid, butyrate, on preconstricted rat small mesenteric arteries (mean inner diameter approximately 300 microns) was characterized. Isometric force development was measured with a myograph, and intracellular pH (pHi) was simultaneously monitored, in arteries loaded with the fluorescent dye BCECF in its acetomethoxy form. Sodium butyrate (substituted isosmotically for NaCl) was applied to arteries after noradrenaline (NA) or high K+ contractures were established. 2. Arteries preconstricted with a concentration of NA inducing an approximately half maximal contraction were relaxed by 91.5 +/- 6.3% by 50 mmol l-1 butyrate. This concentration of butyrate did not, however, cause a significant relaxation of contractures to a maximal (5 mumol l-1) NA concentration, and also failed to relax significantly contractures stimulated by high (45 and 90 mmol l-1) K+ solutions. Contractures elicited with a combination of NA (at a submaximal concentration) and 45 mmol l-1 K+ were, however, markedly relaxed by butyrate. 3. Investigation of the concentration-dependency of the butyrate-induced relaxation of the half maximal NA response revealed an EC50 for butyrate of approximately 22 mmol l-1. 4. Sodium butyrate (50 mmol l-1) caused pHi to decrease from 7.25 +/- 0.02 to 6.89 +/- 0.08 (n = 4, P < 0.001). However, the vasorelaxant effect of butyrate on the submaximal NA contracture was not significantly modified when this fall in intracellular pH was prevented by the simultaneous application of NH4Cl. 5. Butyrate-induced relaxation was also unaffected by endothelial denudation and inhibition of NO synthase with N omega-nitro-L-arginine methyl ester (100 mumol l-1). 6. The relaxation of the NA contracture by 50 mmol l-1 sodium butyrate was abolished in arteries pretreated with the cyclic AMP antagonist Rp-cAMPS (25 mumol l-1). 7. We conclude that the butyrate-induced relaxation of the NA contracture is independent of intracellular acidification. The ability of Rp-cAMPS to abolish the butyrate relaxation indicates that stimulation of the cyclic AMP second messenger system may play an important role in mediating this effect.

Monitoring of mitochondrial NADH levels by surface fluorimetry as an indication of ischaemia during hepatic and renal transplantation.

One of the major causes of dysfunction in transplanted organs is ischaemia-reperfusion (IR) injury. Impairment of mitochondrial function is likely to be central to many of the known consequences of ischaemia; these include loss of cellular homeostasis involving a fall in intracellular pH (Fuller et al., 1988), mitochondrial calcium loading and cellular swelling (Caiman et al., 1973), accumulation of reduced pyridine nucleotides, inhibition of mitochondrial electron transfer, and a fall in ATP levels (Hardy et al., 1991). In irreversibly damaged cells, respiratory control is lost and is accompanied by oxidation of cytochromes a and a3 and NADH (Taegtmeyer et al., 1985). The latter was attributed originally to substrate deficiency (Chance and Williams, 1955) but more recent studies indicate that an enzymological defect develops resulting in an inability to metabolise NADH-linked substrates (Taegtmeyer et al., 1985 and Hardy et al., 1991). In vitro studies of the respiratory chain (RC) complexes have been made in several tissues including cardiac and renal tissue, subjected to ischaemia-reperfusion injury and it was found that complexes I and IV are major defective sites (Hardy et al., 1991 and Veitch et al., 1992). Return of function may, therefore, relate to preservation of inner mitochondrial membrane integrity, and the structure and activities of the RC complexes. The integrity of oxidative metabolic pathways and capacity to resynthesise ATP rather than the immediate post-ischaemic ATP levels appears to determine the return of function (Taegtmeyer et al., 1985).

907-102 Failure of Endothelin-1 Receptor Blockade During Ischemia and Reperfusion to Prevent Ischemic Damage in Isolated Rabbit Hearts

To evaluate the contribution of endogenous Endothelin-l (ET-1) production to ischemia/reperfusion injury, the effects of REA001, a potent ET-1 receptor antagonist, were delineated in paced isolated rabbit hearts subjected to 60 min low-flow ischemia (6% of baseline) followed by reperfusion. Underlying energetic changes were evaluated by use of 31 P NMR to measure ATP, CrP. P i , and pH. Results of 9 isovolumic controls were compared with those of 17 hearts treated 10 -5 M REA001, added to the perfusate 6 minutes before ischemia and continued until the end of ischemia (n = 9) or until that of reperfusion (n = 8). REA001 did not significantly influence the time of onset of ischemic contracture (32 ± 4 vs 21 ± 4 min. p = 0.08) or its amplitude (30 ± 5 vs 35 ± 6 mmHg, p = ns). Similarly, after 60 min of ischemia, REA001 did no t prevent the rise in P i (to 58 ± 6 vs 72 ± 7 ∼mol/g dw, p = ns), and the fall in intracellular pH (to 6.3 ± 0.1 vs 6.2 ± 0.1, p = ns), and did not decrease the severity of CrP (11 ± 1 vS 8 ± 2 μmol/g dw, p = ns) and ATP (9 ± 1 vs 8 ± 2 μmol/g dw, p = ns) depletion. With reperfusion, recovery of developed pressure was similar among hearts from the control group (55 ± 29%) and those treated with REA001 either during ischemia (74 ± 16%) or during both ischemia and reperfusion (64 ± 19%, p = ns ANOVA). Similarly, REA001 did not modify metabolic recovery, as the concentrations of CrP. ATP and P i remained similar in the 3 groups of hearts throughout reperfusion. Our data indicate that ET-1 receptor blockade during ischemia and reperfusion does not influence ischemia-reperfusion injury in isolated rabbit hearts. These results suggest that endogenous production of ET-l is not an important determinant of ischemic/reperfusion injury.

Effects of pH and inorganic phosphate on rigor tension in chemically skinned rat ventricular trabeculae.

1. Ventricular trabeculae from rat heart were chemically skinned with Triton X-100, which disrupts all cellular membranes including the sarcoplasmic reticulum. In the effective absence of Ca2+ (10(-9) M), trabeculae developed a maintained rigor contracture when ATP was withdrawn from the bathing solution. 2. The final level of tension obtained following withdrawal of ATP was dependent upon the pH of the bathing solution during development of rigor. Rigor tension at pH 5.5 was 10.1 +/- 0.9% (n = 8, mean +/- S.E.M.) of that at pH 7.0. Bathing the preparation in alkaline solution increased rigor force. At pH 8.0, rigor force increased to 218 +/- 6.7% (n = 4) of control responses developed at pH 7.0. The rate of development of rigor tension increased as the pH of the bathing solution was increased. Once established, rigor tension was unaffected by subsequent changes in pH. These effects of pH were fully reversible within the range 5.5-8.0. 3. The final level of rigor tension was slightly reduced when inorganic phosphate (P(i)) was included in the bathing solution prior to withdrawal of ATP. P(i) concentrations of 10, 20 and 30 mM reduced rigor tension to 87 +/- 2, 83 +/- 3 and 82 +/- 4% respectively. There was no significant effect of P(i) on the rate of development of rigor. The effect of P(i) at pH 6.0 was not significantly different from that observed at the control pH of 7.0. 4. These results suggest that the fall of intracellular pH and, to a lesser extent, the rise in [P(i)] that occurs during ischaemia will partially inhibit the development of a rigor contracture.