pH recovery from a proton load in rat cardiomyocytes: effects of chronic exercise

The ability of cardiomyocytes to recover from a proton load was examined in the hearts of exercise-trained and sedentary control rats in CO2/[Formula: see text]-free media. Acidosis was created by the NH4Cl prepulse technique, and intracellular pH (pHi) was determined using fluorescence microscopy on carboxy-SNARF-1 AM-loaded isolated cardiomyocytes. CO2-independent pHi buffering capacity (βi) was measured by incrementally reducing the extracellular NH4Cl concentration in steps of 50% from 20 to 1.25 mM. βi increased as pHi decreased in both exercise-trained and sedentary control groups. However, the magnitude of increase in βi as a function of pHi was found to be significantly ( P < 0.001) greater in the exercise-trained group compared with the sedentary control group. The rate of pHi recovery from an imposed proton load was found to not be different between the exercise-trained and control groups. The Na+/H+ exchanger-dependent H+ extrusion rate during the recovery from an imposed proton load, however, was found to be significantly greater in the exercise-trained group compared with the control group. By increasing βi and subsequently the Na+/H+ exchanger-dependent H+ extrusion rate, exercise training may provide cardiomyocytes with the ability to better handle an intracellular excess of H+ generated during hypoxia/ischemic insults and may serve in a cardioprotective role. These data may be predictive of two positive outcomes: 1) increased exercise tolerance by the heart and 2) a protective mechanism that limits the degree of myocardial acidosis and subsequent damage that accompanies ischemia-reperfusion stress. NEW & NOTEWORTHY The enhanced ability to deal with acidosis conferred by exercise training is likely to improve exercise tolerance and outcomes in response to myocardial ischemia-reperfusion injury.

Changes in air quality in different phases of forest management process in a sub-mountain beech ecosystem (West Carpathian Mts.)

We studied air quality in a sub-mountain beech ecosystem in the Kremnické vrchy Mts., Central Slovakia. We chose the method of passive sampling. The amounts of airborne pollutants (H⁺ and O₃) were determined at regular time intervals, covering the whole vegetation period, on four plots with different stocking. The original stand was subjected to two cuts with a purpose to simulate the phases of a common silvicultural process. The first research period (1999–2003) started 10 years after the first cutting, the second (2004–2006) was launched immediately after the second cut. Ten years after applying the first cut, the differences in the proton load input were getting smaller – with the dynamically changing crown canopy. The largest difference in proton load (H⁺ was found between plots C and I after the second intervention, when the correlation coefficient value was 0.15. The differences in proton load input between the plots were influenced by the cut, especially in the first three years after its application. No significant differences in ground level ozone concentrations between plots I (intensive cut), Me (medium intensive), Mo (moderate) and C (control) were revealed either after the first or after the second cutting intervention. Differences in ozone concentrations are not significant, and they indicate that the stocking density does not play an important role in association with ozone affecting the stands. The increase in ozone concentrations after the second intervention was evident on all plots – indicating the absence of connection with the individual phases of forest management process, but at the same time indicating the presence of climate change. In the studied sub-mountain beech ecosystem in the Kremnické vrchy Mts., an important role of episodes with high ozone concentrations is evident.

Calculation of the equilibrium pH in a multiple-buffered aqueous solution based on partitioning of proton buffering: a new predictive formula

Upon the addition of protons to an aqueous solution containing multiple buffers, the final H+concentration ([H+]) at equilibrium is determined by the partitioning of added H+among the various buffer components. In the analysis of acid-base chemistry, the Henderson-Hasselbalch equation and the Stewart strong ion formulation can only describe (rather than predict) the equilibrium pH following a proton load since these formulas calculate the equilibrium pH only when the reactant concentrations at equilibrium11 The term “equilibrium” refers to the steady state proton and reactant concentrations when the buffering of excess protons by the various buffers is complete. are already known. In this regard, it is simpler to directly measure the equilibrium pH rather than measure the equilibrium reactant concentrations to calculate the equilibrium pH. As these formulas cannot predict the final equilibrium [H+] following a proton load to a multiple-buffered aqueous solution, we developed a new quantitative approach for predicting the equilibrium [H+] that is based on the preequilibrium22 The term “preequilibrium” refers to the initial proton and reactant concentrations immediately upon addition of protons and before the buffering of excess protons by the various buffers. concentrations of all buffers in an aqueous solution. The mathematical model used to derive our equation is based on proton transfer buffer equilibria without requiring the incorporation of electroneutrality considerations. The model consists of a quartic polynomial equation that is derived based solely on the partitioning of H+among the various buffer components. We tested the accuracy of the model using aqueous solutions with various buffers and measured the equilibrium pH values following the addition of HCl. Our results confirmed the accuracy of our new equation ( r2= 1; measured pH vs. predicted pH), indicating that it quantitatively accounts for the underlying acid-base phenomenology.

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 ± 1,200, n = 6; with DCA, RPP = 13,700 ± 1,800, n = 9). Intracellular pH, from 31P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6.00 ± 0.08, lactate + glucose + DCA; pH = 6.27 ± 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.

The effect of acid–base balance on fatigue of skeletal muscle

H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20–25 μmol∙g−1, resulting in a fall in intracellular pH of 0.3–0.5 units in mammalian muscle and 0.6–0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70–80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10-fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation–contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell.

The response of the kidney of the freshwater rainbow trout to true metabolic acidosis

Infusion of lactic acid into the bloodstream of trout produced a short-lived depression of blood pH and a long-lasting elevation of blood lactate. The lactate injected was distributed in a volume of 198 ml/kg. Renal excretion of lactate anion and total acid increased by approximately equal amounts during the period of high blood lactate levels, but total renal loss over 72 h accounted for only 2% of the lactate load and 6% of the proton load. Comparable differences in the time courses of blood lactate and pH changes occurred when lactacidosis was induced endogenously by normocapnic hypoxia. The immediate response of the kidney was similar to that with lactic acid infusion, but there was a long-lasting (12–72 + h) elevation of urinary acid efflux that was not associated with lactate excretion. Following hypoxia, renal excretion over 72 h accounted for 1% of the estimated lactate load and 12–25% of the proton load. A renal lactate threshold of 4–10 muequiv/ml prevents significant urinary lactate excretion. The response of the trout kidney to true metabolic acidosis is similar to that of the mammalian kidney.