Reliability of the quantitation of intramitochondrial pH and pH gradient of heart mitochondria

Addanki, Somasundaram; Cahill, Fergal; Sotos, Juan F.

doi:10.1016/0003-2697(68)90076-6

Cited by 51 publications

(50 citation statements)

References 16 publications

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“…Table 2 shows the ratio of [14C]dimethyl-oxazolidinedione count in the pellet and the supernatant after 5 min of incubation, and the external pH a t this time. From these two values together with the extramitochondrial water, and assuming a p K of 6.31 for the dimethyl-oxazolidinedione, ApH is calculated [8,19]. It is seen that although in all cases there is a positive ApH (that is the pH inside is higher than outside) this difference is very small, sometimes within the experimental error of the technique.…”

Section: And Discussionmentioning

confidence: 93%

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ATP Synthesis and Electrical Membrane Potential in Mitochondria

Rottenberg

1970

Eur J Biochem

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The difference of the proton's electrochemical potential ( A j i~) across the membranes of rat liver mitochondria was compared with the rate of phosphorylation. It was found that in high potassium concentration (100 DIM), A/i= was approx. zero and there is no phosphorylation. This finding is predicted by the chemiosmotic model. I n low potassium concentrations phosphorylation increases in parallel with the increase in Ah=. In all the experiments ApH was found to be negligibly small, thus A j i~ was effectively due to the membrane potential ( A y ) . dy was calculated from the difference between the mitochondrial potassium concentration and the external concentration in the presence of valinomycin. The mitochondrial potassium concentration was determined from the pellet potassium content (by flame photometry) and from the mitochondrial water content (corrected for extramitochondrial water). A pH was calculated from the distribution of 5,5-[14C]dimethyl-2,4-oxazolidinedione and the mitochondrial water. The extent of potassium uptake in high potassium concentrations was lower than the uptake in low potassium concentration. This finding and other considerations are not compatible with an explanation of the valinomycin uncoupling effect which is based on an assumed competition between the phosphorylation and a potassium pump. Thus, it is concluded that the valinomycin induced uncoupling is due to the lowering of A y as postulated by the chemiosmotic model. However, a comparison of the free energy of the phosphorylation with the free energy of A/iH shows that the postulated H/ N P ratio of 2 is not sufficient t o support phosphorylation.The mechanism of the coupling between oxidation and phosphorylation in mitochondria is still unknown, despite two decades of intensive investigation. The models and theories that have been proposed in order to explain oxidative phosphorylation fall into two categories: the "chemical" hypothesis and the "chemiosmotic" hypothesis.I n all the models that belong to the "chemical" hypothesis it is postulated that the coupling between oxidation and phosphorylation is a direct chemical coupling that involves the formation of a high energy compound that drives the phosphorylation reaction [l]. The recent suggestion [2] that conformationd changes are the intermediate energy conserving steps is classified here as a "chemical" hypothesis. I n this version the energy is passed t o the phosphorylation reaction through a group of weak bonds (hydrogen bonds, hydrophobic bonds, etc.), while in the older models the energy was conserved in a single covalent bond.Mitchell suggests in a recent version of the "chemiosmotic" hypothesis [3] that the oxidation is coupled to a "pump" that ejects protons from the mitochondria. The electrochemical potential gradient of hydrogen ions that is formed and maintained by Emym. Hexokmaae (EC 2.7.1.1).this pump drives the phosphorylation of ADP in the membrane by a reversal of an ATPase hydrogen P-P.The main difference between the two categories is in the role of the pr...

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Section: And Discussionmentioning

confidence: 93%

“…The pellets that were collected in cytocrit tubes [l8] were squeezed and the water content was determined gravimetrically. The intramitochondrial pH was calculated from the distribution of [14C]dimethyl-oxazolidinedione [8]. The potassium content of the pellet and supernatant were determined by flame photometry.…”

Section: Methodsmentioning

confidence: 99%

ATP Synthesis and Electrical Membrane Potential in Mitochondria

Rottenberg

1970

Eur J Biochem

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“…The internal volume of the cells was determined in parallel experiments as the difference in volume occupied by ['4C]sucrose (0.2 pCi/ ml) and ,HzO (0.5 pCi/ml). The apparent pHi was calculated as described previously [23].…”

Section: The Measurement Of P H I In Hepatocyte Monolayer Culturesmentioning

confidence: 99%

Epidermal growth factor and cyclic AMP stimulate Na⁺/H⁺ exchange in isolated rat hepatocytes

Moule

McGivan

1990

European Journal of Biochemistry

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Na+/H+ exchange in acid‐loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl‐carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride‐sensitive Na+‐dependent recovery from cytoplasmic‐acid‐loading was found to be increased in cells treated with epidermal growth factor (EGF), 8‐(4‐chlorophenylthio)adenosine 3′,5′‐monophosphate (ClPhScAMP) or phorbol 12‐myristate 13‐acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid‐loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride‐inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.

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“…For example, high concentrations of particular molecules can be accumulated into liposomes upon their addition into the external medium in the presence of a pH gradient between the inside and outside of the liposome [1][2][3]. Similar distributions of various fluorescent probes have been used to measure the internal pH of various cells and organelles and results were explained in terms of the Henderson-Hasselbach equation [1][2][3]5,6].…”

Section: Introductionmentioning

confidence: 99%

Gelation of liposome interior A novel method for drug encapsulation

Lasiç¹,

Frederik²,

Stuart³

et al. 1992

FEBS Letters

241

119

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Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X‐Ray diffraction, electron microscopy and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel‐like phase

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Reliability of the quantitation of intramitochondrial pH and pH gradient of heart mitochondria

Cited by 51 publications

References 16 publications

ATP Synthesis and Electrical Membrane Potential in Mitochondria

ATP Synthesis and Electrical Membrane Potential in Mitochondria

Epidermal growth factor and cyclic AMP stimulate Na⁺/H⁺ exchange in isolated rat hepatocytes

Gelation of liposome interior A novel method for drug encapsulation

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Reliability of the quantitation of intramitochondrial pH and pH gradient of heart mitochondria

Cited by 51 publications

References 16 publications

ATP Synthesis and Electrical Membrane Potential in Mitochondria

ATP Synthesis and Electrical Membrane Potential in Mitochondria

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Gelation of liposome interior A novel method for drug encapsulation

Contact Info

Product

Resources

About

Epidermal growth factor and cyclic AMP stimulate Na⁺/H⁺ exchange in isolated rat hepatocytes