Plasma membrane vesicles, using

Primarily using isolated plasma membrane vesicles as an experimental preparation, the functional properties of Na /H exchangers have been elucidated. The important kinetic properties include (1) stoichiometry (one-for-one) (2) reversibility (3) substrate specificity (monovalent cations Na, H, Li, NH4, but not K, Rb, Cs, choline) (4) modes of operation (Na -for-H, Na -for-Na Li " -for-Na, Na -for-NH4 ) (5) existence of an internal site for allosteric activation by (6) reversible inhibition by amiloride (Af-amidino-5-amino-6-chloropyr-azine carboxamide) and its 5-amino-substituted analogs and (7) competitive nature... 

The first studies of specificity were carried out using cholate, the glycine and taurine conjugates and taurine conjugates of the dihydroxy bile acids cheno-deoxycholate and ursodeoxycholate. Kramer and colleagues prepared plasma membrane vesicles from rat liver and compared bile-acid transport with values from CHO cells stably expressing NTCP. This work established that transport by the liver enzyme was maximal when 2 hydroxyls were present,... 

LIVER Use of isolated perfused liver in studies of biological transport processes, 192, 485 measurement of unidirectional calcium ion fluxes in liver, 192, 495 preparation and specific applications of isolated hepatocyte couplets, 192, 501 characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles, 192, 517 preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes, 192, 534. 

Rat liver canalicular membrane vesicles (CMV) have been used to examine the mechanisms of uptake of P-gp substrates such as daunomycin, daunorubicin, and vinblastine, whose biliary excretion is extensive (47,137, 408,409). Early work with plasma membrane vesicles, partially purified from MDR human KB carcinoma cells that accumulated [3H]vinblastine in an ATP-dependent manner, definitively showed how P-gp can act to efflux substrates from cancer cells (410). Additionally, these vesicles have been used to study microscopic aspects of P-gp-mediated efflux, such as the relationship of P-gp function to the membrane fluidity (137). 

The first reported instances using isolated plasma membrane vesicles to study Na+-coupled transport were derived from brush borders of the small intestine (Murer and Hopfer, 1974 Sigrist-Nelson et al., 1975) and Ehrlich cells (Colombini and Johnstone, 1974). In rapid succession a number of other systems were established to study translocation of many solutes in many animal cell systems (Schuld-iner and Kaback, 1975 Lever, 1977 Hammerman and Sacktor, 1978 Wright et al., 1983 Saieret al., 1988 see also Table 1). 

The preparation of plasma membrane vesicles from liver canalicular membrane is highly enriched with the canalicular (apical) isoform MRP2 (Buchler et al. 1996). Methods for the isolation of hepatocyte canalicular membranes from liver tissue have been described in detail (Bohme et al. 1994 and Boyer and Meier et al. 1990). The percentage of inside-out-oriented vesicles in these preparations amounts to 32%. Alternatively, transfected I ILK and MDCK cells are often used to study ATP-dependent transport into inside-out vesicles (Cui et al. 1999 Leier et al. 2000). 

By use of the vesicular transport assay, the direct transport of ABC substrates into inside-out plasma membrane vesicles can be detected. These vesicles can be derived from several different cell lines, such as drug-selected cells, transfected cells, and baculovirus-infected insect cells [80]. Similar to the cytotoxicity assay, this assay can be executed using a direct or indirect setup. 

In the 1960s, Mitchell showed how the energy released in electron transport is used to pump protons from the matrix side of the inner mitochondrial membrane to the cytoplasm side. The subsequent dissipation of the proton gradient, via gates in the stalks of the knoblike projections of the inner membrane, activate ATP synthetase. Recently ascorbic acid has been implicated in such membrane potentiation by the establishment of proton gradients in the plasma membrane vesicles extracted from the soybean (Glycine max). 

A frequently used model for studying the role of ions in the 5-HT transport process employs plasma membrane vesicles prepared from porcine blood platelets [12-17]. 

A similar mechanism of binding and cotranslocation of 5-HT", Na and Cl was demonstrated by using plasma membrane vesicles prepared from a synaptosomal fraction of mouse cerebral cortex [18,19]. The stoichiometry of transport in this case however was 5-HT Na Cr=l 2 l. 

Asard et al. (1992) showed that sealed plasma membrane vesicles (isolated from Phaseolus vulgaris) containing a particular / -type cytochrome with a midpoint reduction potential between +120 and +160 mV were able to transfer electrons to external impermeable electron acceptors, such as ferricyanide and cytochrome c, when loaded with ascorbate. The cytochrome b was reduced by internal ascorbate but not by NADH. Generation of AFR in the external medium using a mixture of ascorbate and ascorbate oxidase resulted in the reoxidation of the 6-cytochrome, suggesting that AFR may be the natural acceptor to cytochrome 6-mediated transmembrane electron transport (Horemans et al., 1994). 

In our laboratory, studies of lipid transfer in leek seedlings in vivo, have already shown the existence of a vesicular process for the transfer of phospholipids and particularly of very long chain fatty acid-containing lipids [6]. This process follows the vesicular endoplasmic reticulum- Golgi apparatus- plasma membrane pathway. Using the cell-free system developed by Morre and coworkers, we have reconstituted in vitro the vesicular transfer of some phospholipids between the ER and the GA. This transfer is ATP and cytosol-dependent, is N Ethyl Maleimide and temperature sensitive and specific for the ER as donor and the GA as acceptor. The phospholipids transferred via an ATP-dependent manner in vitro between the ER and the GA were phosphatidylcholine (PC +79%), phosphatidylethanolamine (PE +67%) and phosphatidylserine (PS +123%) [7]. All those results are in favour of a vesicular transport of phospholipids between the ER and the GA of leek seedlings, and brought us to purify these transition vesicles issued from the ER. 

Plasma membrane vesicles (oriented predominantly right side-out) were isolated from immature fruit of yellow summer squash CucurUta pepo L. cv Early prolific straight neck) using an aqueous pol)maer two-phase system similar to that described previously [10]. More... 

Palmgren MG, Askerlund P, Fredrikson K, Widell S, Sommarin M, Larsson C. Sealed inside-out and right-side-out plasma membrane vesicles. PI Physiol 1990 92 871-880. Askerlund P, Larsson C, Widell S. Localization of donor and acceptor sites of NADH dehydrogenase activities using inside-out and right-side-out plasma membrane vesicles from plants. FEBS Lett 1988 239 23-28. 

Although several allelochemicals (primarily phenolic acids and flavonoids) have been shown to inhibit mineral absorption, only the phenolic acids have been studied at the physiological and biochemical levels to attempt to determine if mineral transport across cellular membranes can be affected directly rather than indirectly. Similar and even more definitive experiments need to be conducted with other allelochemicals that are suspected of inhibiting mineral absorption. Membrane vesicles isolated from plant cells are now being used to elucidate the mechanism of mineral transport across the plasma membrane and tonoplast (67, 68). Such vesicle systems actively transport mineral ions and thus can serve as simplified systems to directly test the ability of allelochemicals to inhibit mineral absorption by plant cells. 

Attempts to study the entry of ES products into cells using markers of fluid phase endocytosis yielded unexpected results. When larvae browse resistant IEC-6 cells in the presence of extracellular fluorescent dextran, dextran enters the cytoplasm of a significant proportion of the cells in the mono-layer (Butcher et al., 2000). The parameters of dextran entry are most compatible with the conclusion that larvae wound the plasma membranes of IEC-6 cells that is, they create transient breaches in the membrane that allow impermeant markers to enter the cell (McNeil and Ito, 1989). Wounding is considered to be a common occurrence in intestinal epithelia (McNeil and Ito, 1989). Injured cells are able to heal their wounds by recruiting vesicles to seal the breach (Steinhardt et al., 1994). In an experimental system, healing allows the injured cell to retain cytoplasmic dextran. In epithelial cell cultures inoculated with T. spiralis larvae, the relationship between glycoprotein delivery and injury of plasma membranes is not clear, i.e. dextran-laden cells do not always stain with Tyv-specific antibodies and... 

Lipids are transported between membranes. As indicated above, lipids are often biosynthesized in one intracellular membrane and must be transported to other intracellular compartments for membrane biogenesis. Because lipids are insoluble in water, special mechanisms must exist for the inter- and intracellular transport of membrane lipids. Vesicular trafficking, cytoplasmic transfer-exchange proteins and direct transfer across membrane contacts can transport lipids from one membrane to another. The best understood of such mechanisms is vesicular transport, wherein the lipid molecules are sorted into membrane vesicles that bud out from the donor membrane and travel to and then fuse with the recipient membrane. The well characterized transport of plasma cholesterol into cells via receptor-mediated endocytosis is a useful model of this type of lipid transport. [9, 20]. A brain specific transporter for cholesterol has been identified (see Chapter 5). It is believed that transport of cholesterol from the endoplasmic reticulum to other membranes and of glycolipids from the Golgi bodies to the plasma membrane is mediated by similar mechanisms. The transport of phosphoglycerides is less clearly understood. Recent evidence suggests that net phospholipid movement between subcellular membranes may occur via specialized zones of apposition, as characterized for transfer of PtdSer between mitochondria and the endoplasmic reticulum [21]. 

---