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A permeability barrier in mitochondria in ;high-energy states' against positively charged disulphides.

ABSTRACT: 1. In rat liver mitochondria in state 1 or 4 there is a permeability barrier against cystamine, probably in the inner membrane. 2. The permeability barrier was broken (a) when oxidative phosphorylation was uncoupled, (b) when the respiratory chain was inhibited or in anaerobiosis, or (c) when phosphate was added in the absence of exogenous substrate. Under these conditions increased amounts of [(35)S]cystamine residues were bound to matrix proteins. 3. It appears that the permeability barrier against cystamine in mitochondria reflects a ;high-energy state'. A gradual increase in the permeability for cystamine strikingly coincided with the loss of respiratory control induced by increasing concentrations of different uncoupling agents. 4. Cystamine caused uncoupling of oxidative phosphorylation in state 2 or 5, but not in state 1, 3 or 4. The uncoupling effect of cystamine was dependent on the phosphorylation potential. ATP counteracted, whereas ADP potentiated, the uncoupling by cystamine. 5. The variable penetration of cystamine appears to depend on its positive charge, since a dication derivative, NNN'N'-tetramethylcystamine, has a similar pattern of penetration, whereas an uncharged derivative, NN'-diacetylcystamine, penetrates rapidly into mitochondria irrespective of their metabolic state. 6. It is suggested that a charge barrier is present in or across the inner mitochondrial membrane in ;high-energy states'.

SUBMITTER: Skrede S

PROVIDER: S-EPMC1198716 | biostudies-other | 1968 May

REPOSITORIES: biostudies-other

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Project description:Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-?) and interferon gamma (IFN-?). While TNF-?/IFN-? treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

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A permeability barrier in mitochondria in ;high-energy states' against positively charged disulphides.

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