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PubMed1991

Calcium buffer solutions and how to make them: a do it yourself guide.

In measurements of the intracellular free calcium concentration ([Ca2+]) using either microelectrodes or fluorescent probes, calibration is normally carried out in EGTA calcium buffer solutions. In the first part of the article the general properties of calcium buffer solutions are discussed, the equations used to calculate the apparent calcium binding constant (Kapp) are derived, and the difficulties in the calculation are discussed. The effects of the purity of EGTA as well as the influence of calcium contamination on the buffer solutions are explained. Because of the difficulties in calculating Kapp, and the importance of EGTA purity and calcium contamination, it is suggested that it is easier to measure all three under the appropriate experimental conditions using the method of Bers (1982). In the second part a do-it-yourself guide to the preparation of EGTA calcium buffer solutions is given. An experimental example is provided using the Bers method to measure purity, contamination, and Kapp. It is concluded that unless all three factors are known it is not possible to prepare accurate EGTA calcium buffer solutions.

Buffers↗
PubMed1986

Calcium and proton buffering and diffusion in isolated cytoplasm from Myxicola axons.

Ion-selective electrodes recorded the pH (7.49 +/- 0.05, n = 8) and pCa (6.72 +/- 0.03, n = 40) in samples (approximately 1 microliter) of isolated Myxicola axoplasm mounted within 760-micron diameter plastic tubes. We determined the interactions between Ca2+ and H+ on axoplasmic buffers by microinjecting CaCl2 or HCl into the axoplasmic samples at a distance 75-125 micron from the tips of the electrodes (distance = r). When axoplasmic pH was lowered 0.97 +/- 0.095 from its resting value (measured at r = 125 micron) by injecting 4 nmol HCl, pCa dropped 0.30 +/- 0.05 (n = 6). When expressed in units of concentration, these data show that a HCl injection of approximately 4 mmol/l axoplasm increased H+ and Ca2+ activity by approximately 0.3 microM. Lowering axoplasmic pCa 2.20 +/- 0.43 (r = 75 micron) (n = 3) by injecting 40 pmol CaCl2 had only a small effect on pH. In other experiments, two Ca2+ electrodes measured the Ca2+ activity 125 and 375 micron from the site of CaCl2 injection. Evidence of Ca2+ buffering was obtained when the Ca2+ activity at these two locations was below that expected for simple Ca2+ diffusion away from the injection site. Centrifuged axoplasm (100,000 g) taken from the bottom of the centrifuge tube had a somewhat greater Ca2+ buffering capacity than that taken from the top of the tube. Electron microscopic studies of the centrifuged axoplasm showed a greater concentration of mitochondria and other axoplasmic vesicles in the bottom of the centrifuge tube. Ruthenium red (20-40 micrograms/ml) greatly reduced Ca2+ buffering. The mitochondrial inhibitors CN (2 mM) and oligomycin (a mixture of oligomycin A, B, and C, 5 micrograms/ml) also reduced Ca2+ buffering but were not as effective as ruthenium red. Axoplasm in which ATP and mitochondrial substrates were removed by dialysis was unable to lower free Ca2+ when the concentration of this ion was elevated to approximately 10 microM. In the presence of oligomycin to block mitochondrial ATPase, and with Mg2+ -ATP as the only source of energy, axoplasm lowered Ca2+ activity slowly; with succinate as the only metabolic substrate, axoplasm rapidly lowered the Ca2+ activity from approximately 10 microM to below 1 microM.

Animals↗
PubMed1993

Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.

Alkanesulfonates↗
PubMed1994

Regulation of cell volume and [Ca2+]i in attached human fibroblasts responding to anisosmotic buffers.

Alteration of the cell volume of attached fibroblasts with anisosmotic buffers was used to examine the relationship between cell membrane perturbation and intracellular Ca2+ concentration ([Ca2+]i) and to study pathways that may be involved in transducing this response. Human periodontal ligament gingival fibroblasts grown on cover slips were loaded with fura 2-acetoxymethyl ester. The relative cell volume change of single fibroblasts was estimated by measurement of fluorescence intensity at the isosbestic wavelength (356 nm), and [Ca2+]i was calculated from ratiometric fura 2 emission with excitation at 345 and 380 nm. Isotonic buffer (300 mosmol/kgH2O) was substituted with either hypertonic (600 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) buffer after baseline recordings. Attached cells exhibited a rapid decrease in cell volume and [Ca2+]i after hypertonic buffer treatment, which was associated with an increase in filamentous actin staining. In contrast, cells treated with hypotonic buffers demonstrated an increase in cell volume (mean approximately 10%), a significant decrease in filamentous actin staining, and a rapid transient elevation in [Ca2+]i (mean approximately 280 nM). This [Ca2+]i rise was significantly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, gadolinium ions (P < 0.05), and inhibitors of actin assembly. These results indicate that [Ca2+]i fluxes in response to hypotonic cell swelling in attached fibroblasts are mediated by stretch-activated ion channels and are dependent on actin filaments.

Actins↗
PubMed1994

Elevated extracellular Mg2+ increases Mg2+ buffering through a Ca-dependent mechanism in cardiomyocytes.

To investigate the physiological basis for the pharmacological action of extracellular magnesium (Mg2+O), cultured ventricular myocytes were exposed to either 0.8 mM (physiological) or 5.0 mM Mg2+ (therapeutic concentration) in the presence and absence of metabolic inhibitors. Metabolic inhibition, induced with 1 mM iodoacetate and 1 mM NaCN, was used to liberate Mg2+ from MgATP into the myoplasm, permitting examination of the role of elevated Mg2+O on myoplasmic Mg2+ buffering. The increase in Mg2+ activity observed in the presence of 5 mM Mg2+O was diminished compared with that observed in cells exposed to 0.8 mM Mg2+O. Furthermore, the increase in myoplasmic Mg2+ activity observed in the presence of 5 mM Mg2+O was identical to that reported previously in the absence of extracellular Ca2+. Fura 2 measurements of Ca2+ activity in these experimental conditions suggested that the Ca2+ permeability of cells conditions suggested that the Ca2+ permeability of cells exposed to 5 mM Mg2+ was less than that observed for cells exposed to 0.8 mM Mg2+. Using the Mg2+ buffer coefficient to quantitate cellular Mg2+ buffering, we observed a 63% increase in Mg2+ buffering in cells exposed to 5 mM Mg2+ compared with cells exposed to 0.8 mM Mg2+. This study demonstrates that elevated Mg2+O alters cardiomyocyte myoplasmic Mg2+ activity as the result of increased Mg2+ buffering through a Ca(2+)-sensitive mechanism.

Adenine Nucleotides↗
PubMed2003

Intracellular proton mobility and buffering power in cardiac ventricular myocytes from rat, rabbit, and guinea pig.

Intracellular pH (pHi) is an important modulator of cardiac function. The spatial regulation of pH within the cytoplasm depends, in part, on intracellular H+ (Hi+) mobility. The apparent diffusion coefficient for Hi+, DHapp, was estimated in single ventricular myocytes isolated from the rat, guinea pig, and rabbit. DHapp was derived by best-fitting predictions of a two-dimensional model of H+ diffusion to the local rise of intracellular [H+], recorded confocally (ratiometric seminaphthorhodafluor fluorescence) downstream from an acid-filled, whole cell patch pipette. Under CO2/HCO3--free conditions, DHapp was similar in all three species (mean values: 8-12.5 x 10-7 cm2/s) and was over 200-fold lower than that for H+ in water. In guinea pig myocytes, DHapp was increased 2.5-fold in the presence of CO2/HCO3- buffer, in agreement with previous observations in rabbit myocytes. Hi+ mobility is therefore low in cardiac cells, a feature that may predispose them to the generation of pHi gradients in response to sarcolemmal acid/base transport or local cytoplasmic acid production. Low Hi+ mobility most likely results from H+ shuttling among cytoplasmic mobile and fixed buffers. This hypothesis was explored by comparing the pHi dependence of intrinsic, intracellular buffering capacity, measured for all three species, and subdividing buffering into mobile and fixed fractions. The proportion of buffer that is mobile will be the main determinant of DHapp. At a given pHi, this proportion appeared to be similar in all three species, consistent with a common value for DHapp. Over the pHi range of 6.0-8.0, the proportion is expected to change, predicting that DHapp may display some pHi sensitivity.

Acid-Base Equilibrium↗
PubMed2003

Comparison of buffer and red blood cell perfusion of guinea pig heart oxygenation.

Myocardial mean myoglobin oxygen saturation was determined spectroscopically from isolated guinea pig hearts perfused with red blood cells during increasing hypoxia. These experiments were undertaken to compare intracellular myoglobin oxygen saturation in isolated hearts perfused with a modest concentration of red blood cells (5% hematocrit) with intracellular myoglobin saturation previously reported from traditional buffer-perfused hearts. Studies were performed at 37 degrees C with hearts paced at 240 beats/min and a constant perfusion pressure of 80 cmH2O. It was found that during perfusion with a hematocrit of 5%, baseline mean myoglobin saturation was 93% compared with 72% during buffer perfusion. Mean myoglobin saturation, ventricular function, and oxygen consumption remained fairly constant for arterial perfusate oxygen tensions above 100 mmHg and then decreased precipitously below 100 mmHg. In contrast, mean myoglobin saturation, ventricular function, and oxygen consumption began to decrease even at high oxygen tension with buffer perfusion. The present results demonstrate that perfusion with 5% red blood cells in the perfusate increases the baseline mean myoglobin saturation and better preserves cardiac function at low oxygen tension relative to buffer perfusion. These results suggest that caution should be used in extrapolating intracellular oxygen dynamics from buffer-perfused to blood-perfused hearts.

Animals↗
PubMed2004

Reduced oxygen supply explains the negative force-frequency relation and the positive inotropic effect of adenosine in buffer-perfused hearts.

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 +/- 1.2 nM, maximal effect (Emax) = 104 +/- 2 and 84 +/- 2 mmHg at 0.1 microM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate (r = 0.67, P < 0.01; EC50 = 3.8 +/- 1.4 nM, baseline 228 +/- 21 beats/min to a minimum of 183 +/- 22 beats/min at 0.1 microM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.

Adenosine↗
PubMed2006

Comparative shell buffering properties correlate with anoxia tolerance in freshwater turtles.

Freshwater turtles as a group are more resistant to anoxia than other vertebrates, but some species, such as painted turtles, for reasons not fully understood, can remain anoxic at winter temperatures far longer than others. Because buffering of lactic acid by the shell of the painted turtle is crucial to its long-term anoxic survival, we have tested the hypothesis that previously described differences in anoxia tolerance of five species of North American freshwater turtles may be explained at least in part by differences in their shell composition and buffering capacity. All species tested have large mineralized shells. Shell comparisons included 1) total shell CO2 concentration, 2) volume of titrated acid required to hold incubating shell powder at pH 7.0 for 3 h (an indication of buffer release from shell), and 3) lactate concentration of shell samples incubated to equilibrium in a standard lactate solution. For each measurement, the more anoxia-tolerant species (painted turtle, Chrysemys picta; snapping turtle, Chelydra serpentina) had higher values than the less anoxia-tolerant species (musk turtle, Sternotherus odoratus; map turtle, Graptemys geographica; red-eared slider, Trachemys scripta). We suggest that greater concentrations of accessible CO2 (as carbonate or bicarbonate) in the more tolerant species enable these species, when acidotic, to release more buffer into the extracellular fluid and to take up more lactic acid into their shells. We conclude that the interspecific differences in shell composition and buffering can contribute to, but cannot explain fully, the variations observed in anoxia tolerance among freshwater turtles.

Animals↗
PubMed1983

Theoretical analysis of "metabolic indices" of blood buffer system.

The well-known base excess (BE) and buffer base (BB) curves of Siggaard-Andersen's nomogram are based on their empirical finding that the blood buffer lines in the pH-log PCO2 coordinates with the same BE (or BB) always pass through a single point irrespective of the hemoglobin concentration. We have analytically derived the BE and BB curves on the assumption of a two-compartmental model of blood buffer system comprised of plasma and red blood cells. These hyperbolic functions of BE and BB curves fitted the Siggaard-Andersen's observation very well. The simple formulas derived from the buffer equation with the parameters obtained on the least-square basis were shown to estimate "metabolic indices" of blood as accurately as the computer reading of the nomographic curves. These results seem to indicate that the buffer equation based on the two-compartmental model describes the acid-base status of blood satisfactorily, and that it can entirely replace the Siggaard-Andersen's nomogram.

Acid-Base Equilibrium↗
PubMed1985

Buffering capacity of deproteinized human vastus lateralis muscle.

The in vitro deproteinized vastus lateralis muscle buffer capacity, carnosine, and histidine levels were examined in 20 men from 4 distinct populations (5 sprinters, 800-m runners; 5 rowers; 5 marathoners; 5 untrained). Needle biopsies were obtained at rest from the vastus lateralis muscle. The buffer capacity was determined in deproteinized homogenates by repeatedly titrating supernatant extracts over the pH range of 7.0-6.0 with 0.01 N HCl. Carnosine and histidine levels were determined on an amino acid AutoAnalyzer. Fast-twitch fiber percentage was determined by staining intensity of myosin adenosinetriphosphatase. High-intensity running performance was assessed on an inclined treadmill run to fatigue (20% incline; 3.5 m X s-1). Significantly (P less than 0.01) elevated buffer capacities, carnosine levels, and high-intensity running performances were demonstrated by the sprinters and rowers, but no significant differences existed between these variables for the marathoners vs. untrained subjects. Low but significant (P less than 0.05) interrelationships were demonstrated between buffer capacity, carnosine levels, and fast-twitch fiber composition. These findings indicate that the sprinters and rowers possess elevated buffering capabilities and carnosine levels compared with marathon runners and untrained subjects.

Adult↗
PubMed2005

Pyruvate in the correction of intracellular acidosis: a metabolic basis as a novel superior buffer.

The review focuses on biochemical metabolisms of conventional buffers and emphasizes advantages of sodium pyruvate (Pyr) in the correction of intracellular acidosis. Exogenous lactate (Lac) as an alternative of natural buffer, bicarbonate, consumes intracellular protons on an equimolar basis, regenerating bicarbonate anions in plasma while the completion of gluconeogenesis and/or oxidation occurs via tricarboxylic-acid cycle in mitochondria mainly in liver and kidney, or heart. The general assumption that Lac is 'metabolized to bicarbonate' in liver to serve as a buffer has been questioned. Pyr as a novel buffer would be superior to conventional ones in the correction of metabolic acidosis. Several likely biochemical mechanisms of Pyr action are discussed. Experimental evidence, in vivo, strongly suggested that Pyr would be particularly efficient in the correction of severe acidemia: type A lactic acidosis, hypercapnia with cardiac arrest, and diabetic and alcoholic ketoacidosis in animal experiments and clinic settings. Because of its multi-cytoprotection, Pyrs not only correct acidosis, but also benefit theunderlying dysfunction of vital organs. In addition, Pyr is also a potential buffer component of dialysis solutions. However, the instability of Pyr in aqueous solutions restricts its clinical applications as a therapeutic agent. Attempts to create a stable Pyr preparation are needed.

Acidosis↗
PubMed1987

The influence of incorporated and adsorbed fluoride on the dissolution of powdered and pelletized hydroxyapatite in fluoridated and non-fluoridated acid buffers.

The effects of fluoride (F) incorporated into hydroxyapatite (HA), adsorbed onto the HA surface, and fluoride in solution were studied during HA dissolution. Dissolution rates at pH 5.0 and 25 degrees C were determined in fluoridated (0.1, 5.0 micrograms/mL) and non-fluoridated buffers for the following powdered and pelletized synthesized HAs: non-fluoridated HA, partially-fluoridated (100, 1000, 37,000 micrograms/g) HA, and surface-adsorbed fluoridated (100, 1000 micrograms/g) HA. The dissolution rate curves were used to derive two components we have called 'diffusion' and 'surface chemical reactivity'. With powders, the rate-determining factor was surface chemical reactivity during the first 60 min. Diffusion was reduced by increasing the F levels in the apatite or buffer. In non-fluoridated buffers, diffusion was reduced 29% for apatite with 100 micrograms/g incorporated F and 99% with 37,000 micrograms/g. Diffusion was reduced by 37% and 81% by 0.1 and 5.0 micrograms/mL F, respectively, in the buffer. With pelletized HA, the rate-determining factor during the first 10 min was surface chemical reactivity. Diffusion then became relatively more important, but the diffusion rate was independent of F content. During the dissolution of powdered apatites, F was taken up by the remaining apatite solid, as evidenced by the lower F concentration in solution than that calculated to be released during dissolution. There was also a decrease in the F concentration of the 0.1 microgram/mL buffer as reaction progressed. Pelletized apatites did not release enough F to influence dissolution significantly. Surface-adsorbed F was more effective than incorporated F in reducing HA dissolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed2003

Effect of microenvironment pH of swellable and erodable buffered matrices on the release characteristics of diclofenac sodium.

The aim of this work is to design pH-dependent swellable and erodable-buffered matrices and to study the effect of the microenvironment pH on the release pattern of diclofenac sodium. Buffered matrix tablets containing diclofenac sodium, physically mixed with hydrophilic polymer (hydroxypropyl methylcellulose [HPMC]) and pH-dependent solubility polymer (Eudragit L100-55) were prepared with different microenvironment pHs. The release of diclofenac sodium from the buffer matrices was studied in phosphate buffer solutions of pH 5.9 and 7.4. The swelling and erosion matrices containing only HPMC and Eudragit L100-55 were studied in phosphate buffer solution of pH similar to the microenvironment pHs of the matrices. Drug release from matrices was found to be linear as a function of time. Amount of drug released was found to be higher in the medium of pH 7.4 than that of pH 5.9. The rate of drug release increased with the increase of the microenvironment pH of the matrices as determined from the slope. The pattern of drug release did not change with the change of microenvironment pH. The swelling and erosion occurred simultaneously from matrices made up of HPMC and Eudragit L100-55. Both extent of swelling and erosion increased with increase of the medium pH. It was concluded from this study that changing the pH within the matrix influenced the rate of release of the drug without affecting the release pattern.

Buffers↗
PubMed2005

The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens.

To evaluate the role of mineralized tissues of the leopard frog in buffering acid, we analyzed the composition of femur and auditory capsule, the latter of which encloses a portion of the endolymphatic lime sacs, and investigated the extent to which these tissues are involved in buffering lactic acid after 2.5 h of anoxia and 10-19 min of strenuous exercise at 15 degrees C. We analyzed the following tissues for lactate: plasma, heart, liver, gastrocnemius muscle, femur, auditory capsule and carcass. Plasma [Ca(2+)], [Mg(2+)], [inorganic phosphate (P(i))], [Na(+)] and [K(+)] were also measured. Femur Ca(2+), P(i) and CO(3)(2-) compositions were similar to bone in other vertebrates. Auditory capsule had significantly more CaCO(3) than femur. Lactate was significantly elevated in all tissues after anoxia and exercise, including femur and auditory capsule. Anoxia increased plasma [Ca(2+)], [Mg(2+)], [P(i)] and [K(+)] and had no effect on plasma [Na(+)]. Exercise increased plasma [Mg(2+)], [P(i)] and [K(+)] and had no effect on plasma [Ca(2+)] or [Na(+)]. The skeleton and endolymphatic lime sacs buffered 21% of the total lactate load during anoxia, and 9% after exercise. The exact contribution of the entire endolymphatic sac system to lactate buffering could not be determined in the present study. We conclude that the mineralized tissues function as buffers during anoxia and exercised induced lactic acidosis in amphibians.

Analysis of Variance↗
PubMed2005

Oxidation of delta-ALA-D and DTT mediated by ascorbic acid: modulation by buffers depends on free iron.

Ascorbic acid (AA) is one of the most important endogenous reducing agents and can participate in a variety of cellular events. In vitro, AA can act as a potent oxidant agent in the presence of free metals, promote modifications in protein structures and form reactive oxygen species during its oxidation. We have observed that AA (above 6 mmol/l) inactivates delta-aminolevulinate dehidratase (delta-ALA-D), a sulfhydryl-containing enzyme and that the inhibitory action was considerably decreased when 3-morpholinepropanesulfonic acid buffer (MOPS - pH: 6.8; 100 mmol/l) was used in the delta-ALA-D activity assay instead of potassium phosphate buffer (PB - pH: 6.8; 100 mmol/l). delta-ALA-D inhibition, probably, is mediated by the oxidation of -SH groups caused by the auto-oxidation of AA promoted by metals or another oxidizing system present in liver supernatants. This hypothesis was confirmed by studying dithiothreitol (DTT - 400 micromol/l) oxidation, as a model of enzyme thiols, where we observed that the mechanism underlying DTT and delta-ALA-D oxidation caused by ascorbate is the same. The difference observed between different buffers may be related to the oxidation of Fe(II) to Fe(III) that was more accentuated in PB than in MOPS buffer. The presence of ethilenediamintetraacetic acid (EDTA - 100 micromol/l) and Fe(III) (5 micromol/l) stimulated DTT oxidation more in PB than in MOPS buffer. Deferroxamine (DF - 100 micromol/l) considerably decreased DTT oxidation. Catalase (0.4 mg/ml) and Superoxide dismutase (SOD - 300 U/ml) had only a modest effect on DTT oxidation. The present results suggest that delta-ALA-D inhibition by AA is mediated primarily by the oxidized form of AA and reactive oxygen species play only a modest role in the process.

Ascorbic Acid↗
PubMed1993

Attenuation of myocardial stunning by an increase in the H+ buffering capacity of the perfusate and that by hypoxic preperfusion are affected differently by the free [Ca2+] of the perfusate.

OBJECTIVES: Protons produced during ischemia may increase intracellular Na+ (Na+i) through Na+/H+ exchange, and may lead to Ca2+ overload through Na+/Ca2+ exchange to cause myocardial stunning. This study investigated whether an increase in the H+ buffering capacity of the perfusate or a reduction of H+ production by a brief hypoxic preperfusion before ischemia would reduce myocardial stunning. We also investigated whether the protective effect of these maneuvers depends on the free [Ca2+] of the perfusate. METHODS: Isolated rat hearts were preperfused with oxygenated or hypoxic buffer (pH 7.4) containing 100 mM of either sucrose or HEPES for 10 min, followed by 15 min of total ischemia and 30 min of reperfusion. To investigate the dependence of the effects of HEPES or a brief hypoxic preperfusion, the free Ca2+ concentration in the buffer was changed from 1.25 mM to 2.5 mM in some hearts. RESULTS: Oxygenated preperfusion with buffer containing HEPES and 1.25 or 2.5 mM Ca2+ improved the metabolic and functional recovery with a decrease in the accumulation of Na+i during ischemia and in 45Ca2+ uptake during reperfusion. A brief hypoxic preperfusion with 1.25 mM Ca2+ provided a similar protective effect whereas no protective effect was observed when the [Ca2+] was raised to 2.5 mM. CONCLUSIONS: An increase in the H+ buffering capacity or a brief hypoxic preperfusion reduced myocardial stunning with improved metabolic recovery, and reduced Ca2+ uptake. However, the effects of these interventions were affected differently by the free [Ca2+] of the perfusate, which suggests that they work, at least in part, through some different mechanism(s).

Animals↗
PubMed1981

Artificial buffers do not inhibit contractile responses in the smooth muscle of rat portal vein and guinea pig taenia coli.

Effects of substitution for NaHCO3 (and 5% CO2) in the physiological solution with equimolar N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or morpholinopropane sulfonic acid (MOPS) (and 100% O2) at various pH levels on the contractility of smooth muscle were examined. At pH 7.4, spontaneous contraction in rat portal vein was not inhibited by the artificial buffer solutions, as compared to findings in the case of bicarbonate buffer solution. Norepinephrine-dose response curve in the case of rat portal vein and the histamine-dose response curve in cases of guinea pig taenia coli remained unchanged in the artificial buffer solutions. At pH 7.2 and 7.0, the spontaneous contraction in portal vein was reversibly inhibited either in artificial or in bicarbonate buffer solutions. The norepinephrine- and histamine-dose response curve shifted downwards and/or to the right in these low pH solutions. Thus, HEPES and MOPS had no inhibitory effect on the smooth muscle contractility. Since low pH strongly inhibited the contractility, attention should be directed to the temperature-dependent decreases in pKa of the artificial buffers (i.e., if pH of the solution is adjusted at room temperature and then warmed, pH decreases).

Animals↗