Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

Presynaptic inhibition in the cercal-afferent giant-interneurone synapses of the cockroach, Periplaneta americana L.

The occurrence of presynaptic control of synaptic transmission in the cercal-afferent giant-interneurone system of the cockroach was investigated. Reduction in amplitude (up to 50%, lasting about 200–250 ms) of the compound evoked EPSP followed repetitive (300 to 500 Hz) supra-threshold stimulation of cercal nerves XI. A similar but weaker depressive effect was detected on the unitary EPSP resulting from stimulation of an ipsilateral cercal mechanoreceptor.This inhibition is attributed to multisynaptic inhibitory pathways impinging upon presynaptic excitatory neurones. The involvement of chloride ions is suggested by the observation that both picrotoxin and chloride-deficient salines abolished the inhibitory phenomenon. Presynaptic mannitolgap recording from cercal nerve XI revealed a chloride-dependent hyperpolarization in response to repetitive conditioning stimulation. The time course of this response was similar to that of presynaptic inhibition. Bath-application of GABA (20 mM) produced a chloride-dependent hyperpolarization followed by a depolarization of the intraganglionic part of the cerca-afferents. GABA-induced hyperpolarization and electrically-induced presynaptic hyperpolarization were both reversed in low chloride saline (166 mM chloride). It is proposed that presynaptic modulation of acetyl-choline release occurs at the cercal-afferent giant-interneurone synapses. The role played by GABA is duscussed.     

Clinical assay of the human erythrocyte lactate transporter. II. Analysis and display of normal human data

We have applied a lactate efflux assay to human red cells at two temperatures and with initial lactic acid loads up to 8 mM, metabolically generated. Efflux was about 1.5 times faster at external pH of 8.5 than at 7.5; the latter was the standard pH used thereafter. Multiple lactate loads in a single blood specimen demonstrated clear evidence of saturation kinetics at both pH levels, since the efflux rate did not increase proportionally with the lactate load. Best-fitting rectangular hyperboles were determined for 129-131 assays from 43 volunteers at 20 degrees and 30 degrees. In most cases high and low lactate loads permitted a two-point evaluation of saturation kinetics, and a positive indication was obtained in 88 of 89 tests. The apparent efflux Km and Vm values may be influenced by pH as well as by lactate levels and cannot be taken as rigorous, although they agree reasonably well with literature data on influx and exchange velocities. The data displayed a Hill constant of 1, a 30 degrees/20 degrees velocity ratio of 2.7, and no significant clustering by sex or age. A single assay with initial lactate level above 5 mM at 30 degrees should be sufficient to identify cases with a defective transporter, using the 95% tolerance limits developed in this report.     

Reflex cardiovascular and ventilatory responses to increasing H+ activity in cat hindlimb muscle

Static exercise increases arterial pressure, heart rate, and ventilation, effects which are believed in part to arise reflexly from a metabolic stimulus in the working muscle. In anesthetized cats, we tested the hypothesis that intra-arterial injections of lactic and hydrochloric acid, which created levels of these substances in muscle similar to those seen during contraction, reflexly increased cardiovascular and ventilatory function. Hydrochloric acid (32 and 57 mM; 1 ml) injected into the arterial supply of the triceps surae decreased intramuscular pH from 7.26 +/- 0.05 to 7.17 +/- 0.05 (P less than 0.01) and reflexly increased arterial pressure (23 +/- 7 mmHg; P less than 0.01), heart rate (11 +/- 2 beats/min; P less than 0.001), and ventilation (187 +/- 72 ml/min; P less than 0.05). Static contraction of the triceps surae decreased intramuscular pH from 7.28 +/- 0.06 to 7.13 +/- 0.06 (P less than 0.01). Lactic acid was more potent in causing reflexes than was equimolar HCl. For example, lactic acid containing 4 mM lactate and 0.87 mM H+ reflexly increased arterial pressure, heart rate, and ventilation, whereas 0.87 mM HCl did not. Intra-arterial sodium lactate (13 and 33 mM) at a neutral pH had no effect on these variables. We conclude that contraction-induced accumulation of H+, especially that arising from lactic acid, might provide a metabolic stimulus to evoke reflex autonomic effects.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1. The accumulation ratio measured with propionate increased with decreasing pH from ca. 24-fold at pH 6.0 to ca. 1,400-fold at pH 3.0. The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM). The lactate system was inducible and was subject to glucose repression. Undissociated lactic acid entered the cells by simple diffusion. The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.     

The mechanism of block of glutamate synapses by dipicolinic acid

The effect of dipicolinic acid (2,6-pyridine dicarboxylic acid) on the mealworm neuromuscular junction was studied using conventional microelectrode recording techniques. Dipicolinic acid (10^?5-10^?3 M) added to the bathing solution reversibly blocked neuromuscular transmission. The depolarization in response to iontophoretically applied L-glutamate (glutamate potential) was not affected by dipicolinic acid even when the neurally evoked excitatory postsynaptic potential (EPSP) was totally abolished. Focal extracellular recordings from single synaptic sites revealed that in the presence of 1 x 10^?4 M dipicolinic acid the presynaptic spike was unchanged, but the quantal content for evoked transmitter release was reduced. The calcium-dependent action potential elicited by direct stimulation of the muscle fiber was not impaired by dipicolinic acid. These results suggest that dipicolinic acid interferes with the transmitter-releasing mechanism from the presynaptic terminal.     

Influence of lactic acid on the proliferation,metabolism, and differentiation of rabbit mesenchymal stem cells

Lactic acid, originated from degradation of biomaterials, cell cultures, and so on, would be a toxic compound in acute states. The present study was undertaken to ascertain whether the proliferation, metabolism, and differentiation of rabbit mesenchymal stem cells (rMSCs) were affected by additional lactic acid. Furthermore, this study aimed to determine whether this influence was due to decreasing pH, increasing osmotic pressure, or chemical action of lactate ion. It was shown that the proliferation and metabolism of MSCs were inhibited by decreasing pH or increasing lactate. However, when osmolarity was adjusted to the same level as that of sodium lactate using sodium chloride, cell proliferation was little affected by osmotic pressure. We also concluded that colony-forming potential and osteogenic differentiation capacity were significantly depressed by decreasing pH or increasing lactate. As was shown, this inhibition of lactate was not only due to osmotic pressure, but also mainly due to chemical action of lactate ion. However, we observed that acidifying extracellular medium and lactate ion promoted the retention of adipogenic differentiation potential of MSCs during in vitro expansion, which suggested that growth arrest and the decrease of osteogenic differentiation potential did not affect the adipogenic conversion of MSCs.     

Neurotoxins from venoms of the Hymenoptera--twenty-five years of research in Amsterdam

1. In co-operation with colleagues in Europe, Japan and the U.S.A., 25 years of research in Amsterdam have provided new views on the way some hymenopteran insects incapacitate their prey by a diversity of neurotoxins, resulting in block of synaptic transmission in CNS or neuromuscular junctions, or affecting voltage dependent phenomena in nerve and muscle fibers. 2. Nicotinic synaptic transmission in the insect CNS is irreversibly blocked at the presynaptic side by kinins, or reversibly and postsynaptically blocked by philanthotoxins. 3. Glutamatergic neuromuscular transmission is reversibly blocked by philanthotoxins at the pre- and/or postsynaptic side. 4. A presynaptic block of neuromuscular transmission was found with the Microbracon toxins. 5. An irreversible deactivation, without paralysis, of cockroaches is caused by a sting of Ampulex compressa into the suboesophageal ganglion. 6. Poneratoxin, a 25 amino acid residue polypeptide, isolated from an ant venom, is the first described hymenopteran neurotoxin affecting excitability of nerve and muscle fibres by changing the kinetics of the voltage-dependent sodium channel.     

Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Role of GABA<Subscript>B</Subscript> receptor in the regulation of excitatory synaptic transmission in trigeminal motoneurons

The aim of the present study was to determine if excitatory synaptic transmission onto trigeminal motoneurons is subject to a presynaptic modulation by gamma-aminobutyric acid (GABA) via GABA(B) receptor in this system. Whole cell recordings were made from trigeminal motoneurons in longitudinal brain stem slices taken from 8-day-old rats. Monosynaptic excitatory postsynaptic potential (EPSP) activity was evoked by placing bipolar stainless steel electrodes dorsal-caudal to the trigeminal motor nucleus. Bath application of the GABA(B) receptor agonist, baclofen, produced a marked reduction in the mean amplitude and variance of evoked EPSPs and also increased the portion of transmission failures. It also produced a decrease in the frequency, but not in the mean amplitude, of spontaneous miniature EPSPs. Bath application of GABA(B) receptor antagonists 6-hydroxy-saclofen and CGP35348 increased both the amplitude and frequency of miniature EPSP activity. Taken together the above results suggest that the excitatory synaptic inputs onto trigeminal motoneurons are controlled by tonic presynaptic modulation by GABA(B) receptor.     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Effect of acid-base status on plasma phosphorus response to lactate

The mechanism(s) underlying the hyperphosphatemia of lactic acidosis is uncertain. We assessed the interacting influence of the acid anion and acid-base status on plasma phosphorus concentration by administering lactic acid alone, lactic acid plus sodium bicarbonate, sodium bicarbonate alone, and sodium lactate alone to four different groups of dogs. The findings of (1) no increase in plasma phosphorus concentration with lactic acid plus sodium bicarbonate versus a marked increment with lactic acid alone, and (2) no difference in the plasma phosphorus response to sodium lactate versus sodium bicarbonate indicate that acidemia is necessary for the expression of lactate-induced hyperphosphatemia. The apparent greater propensity for marked hyperphosphatemia in lactic acidosis than in other types of metabolic acidosis remains unexplained, but conceivably might relate to differences in intracellular pH and in the rate of glycolysis.     

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts.

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate. Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells. The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2--16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.     

The monosynaptic connection: the modulating effect of opioid peptides on the plasticity of presynaptic neurons and identified synapses]

Study of opioid peptides (leucine-enkephalin and methionine-enkephalin) action on plastic properties of the system of monosynaptically connected neurones LPa7--LPa3, PPa3 and LPa8--LPa3, PPa3 was conducted in the snail brain. It has been shown that all three links in the system studied (presynaptic neurone, postsynaptic neurone and synapse) manifest one and the same type of plasticity--habituation to rhythmic stimulation. Enkephalins have a modulating action on plastic properties of the presynaptic neurone and synapse: they retard the habituation of the presynaptic neurone to intracellular stimulation and retard the development of habituation at synaptic level. However, changes in the character of postsynaptic response in the presence of enkephalins are not a direct consequence of their influence on plastic properties of the presynaptic neurone. Besides, enkephalines reduce the effectiveness of synaptic transmission in the given system: they reduce EPSP duration in the postsynaptic neurone.