Studies of the effects of trimethyltin, tripropyltin, tributyltin and triphenyltin chlorides on the permeability of the inner mitochondrial membrane by the osmotic swelling technique in NaCl and NH,C1 media show that these compounds mediate transport of chloride ions. Studies in KC1 media show that these compounds mediate a strictly coupled chloride-hydroxide antiporter transport. This exchange allows rapid titration of mitochondrial contents by pulses of HCl. Similar results on erythrocytes and smectic mesophases indicate that this is a direct action of the tin compounds and is not due to modification ofa pre-existing anion carrier in the membrane.The highly toxic trialkyltin compounds have been shown to be potent inhibitors of mitochondrial oxidative phosphorylation by Aldridge and Cremer[1], and Sone and Hagihara [2]. Aldridge et al. [3--51 have shown that these compounds also produce several other effects on mitochondria, for example, uncoupling of oxidative phosphorylation, inhibition of the 2,4 dinitrophenol-stimulated ATPase but stimulat'ion of the ATPase in the absence of 2,4 dinitrophenol, a time-dependent inhibition of 2,4 dinitrophenolstimulated respiration and of state 4 respiration. Unlike inhibitors of oxidative phosphorylation such as oligomycin and aurovertin, the trialkyltin compounds are of known structure and chemical investigation of their mode of action may be possible.A preliminary communication by Selwyn, Stockdale and Dawson [6] outlined investigations intended to define the mode of action of trialkyltin compounds on mitochondria and reported that these compounds produced a rapid chloride-hydroxide exchange across the mitochondrial membrane. This paper describes the effect of trialkyltin compounds on the permeability properties of mitochondrial membranes, erythrocytes and smectic mesophase (liposome) artificial membrane systems.Unusual Abbreviations. Carbonylcyanide p-trifluoromethoxy phenylhydrazone, FCCP ; ethylene glycol-bis-(aminoethy1)-tetraacetic acid, EGTA; N-2-hydroxymethylpiperazine-N'-2-ethanesulphonic acid, HEPES.Enzymes. Carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1). MATERIALS AND METHODS MitochondriaMitochondria prepared from the livers of adult (150 to 250 g) Wistar rats were used throughout this investigation. The rats were killed by decapitation and the mitochondria prepared by the method of Hogeboom et al. [7] with the following modifications. The isolation medium was 0.25 M sucrose, deionised by passage through a column of Permutit "Deminrelit" mixed bed resin, and contained 5 mM HEPES adjusted to pH 7.5 with sodium hydroxide. The final centrifugation was performed a t 14 500 x g for 10 min. A light fluffy layer on top of the mitochondrial pellet was poured off with the supernatant fluid and the tightly packed mitochondrial pellet resuspended in the same medium t o give a final concentration of about 80 mg/ml mitochondrial protein. ErythrocytesErythrocytes were prepared from human blood (containing acid-citr ate dextrose anti-coagulant) obtained from the r...

Starch that escapes digestion in the small intestine increases the elimination of chenodeoxycholate and its metabolites in the faeces of both mice and hamsters. In contrast, the elimination of cholate and its metabolites is not increased. In vitro, the affinity of starch for chenodeoxycholate is about 90-fold greater than for cholate. beta-Cyclodextrin, which approximates to one turn of the helical structures formed by the 1,4-linked glucose units of starch, shares these properties. It is proposed that these helical structures in starch act as binding sites for bile salts.

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.

Aqueous dispersions of phosphatidic acid and mixtures of phosphatidic acid with other phospholipids vesiculate when the pH is transiently increased to a pH near or above the second pK of the phosphatidic acid. Both small unilamellar vesicles (SUV) of a narrow size distribution (average diameter 25-30 nm) and large unilamellar vesicles (LUV) of a wide size distribution are formed. The fraction of SUV increases linearly with increasing pH from 6 to 12 and was also increased by increasing the rate of the pH change from 2 min to 1 s. For mixed phospholipid dispersions, the fraction of SUV appears to be linearly related to the phosphatidic acid content. Unilamellar vesicles formed by the transient pH increase are subsequently relatively stable to changes in pH and ionic strength. If, after vesiculation is induced, the dispersion (pH 7-8) is acidified to pH 3, the fraction of SUV decreases. However, the multilamellar structures present in the original dispersion do not re-form, and most of the reduction in the SUV fraction is reversible when the pH is returned to 7-8. The addition of NaCl to the dispersion after vesiculation has no effect on the fraction of SUV up to physiological NaCl concentrations. Subsequent addition of NaCl to a concentration in excess of 0.2 M reduces the SUV fraction; this disappears above 1 M NaCl due to aggregation or fusion. SUV of phosphatidic acid or mixed phospholipids containing phosphatidic acid can be stored at 4 degrees C for 14 days without detectable aggregation and/or fusion.