Large numbers of thin-walled vesicles, 0.5 to 10 p in diameter, can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distilled water or an aqueous solution of nonelectrolytes. Electron micrographs and phospholipid analyses indicate that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium, making them a useful system for studying permeability. The osmolarity of the solution in the interior of the vesicles can be estimated by immersion refractometry. The osmolarity of the internal aqueous phase is linearly related to the osmolarity of the external medium.Membranes are a basic structural element of living systems; they form the boundaries of the cells and of their internal organelles, divide their interiors into compartments, and provide a framework for many metabolic and physiological processes. Recently, much attention has been devoted to phospholipid membranes in the hope that studies utilizing these simple models will provide insight into the organization, permeability and electrical properties of natural membranes.One type of model system consists of a phospholipid bilayer formed across an aperture in a thin plastic diaphragm which separates two aqueous phases (Mueller et al., '63; Hanai et al., '64; Huang et al., '64). The composition of such membranes cannot be readily controlled or determined. Furthermore, the surface areas are small, which limits their use for permeability studies to rapidly penetrating substances. Bangham et al. ('65) described a membrane system consisting of a large number of multilamellar phospholipid spherules. The spherules are fragments of tubular myelin figures which form spontaneously when dried lipids swell in an aqueous medium. The lipid composition of the spherules can be controlled and the total surface area may exceed several hundred square centimeters. However, it is difficult to describe the diffusion of substances within the particle itself, owing to the series of diffusion barriers created by the multiple phospholipid lamellae. J. CELL. PHYSIOL., 73: 49-60.This paper describes the preparation and properties of phospholipid vesicles of predetermined composition several microns in diameter bounded by walls one or a few bilayers thick. The thinness of the vesicle walls and the large available surface area make this system particularly well-suited for studying the permeability of lipid membr anes . MATERIALS AND METHODS Vesicle formation. Approximately 5 1iMof pure phospholipids (or mixed brain phospholipids) dissolved in 0.5 ml of 1 : 2 chloroform-methanol was spread evenly over the bottom of a flat-bottomed 2 1 erlenmeyer flask at room temperature. The solvent was evaporated slowly by passing nitrogen through the flask, taking care not to disturb or agitate the solution. Watersaturated nitrogen was then circulated through the flask for 15 minutes or more. The cloudy appearance of the phospholipid layer on the bottom of the flask gradually disappeared as water was ...

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.

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Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+4loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.Na+-Ca2+ exchange is a process whereby transmembrane movements of Ca2+ are coupled directly to reciprocal movements of Na+. This process has been demonstrated in a variety of excitable tissues (1-3), as well as in dog erythrocytes (4). In cardiac muscle, attention has been focused on Na+-Ca2+ exchange as a possible entry mechanism for contractile Ca2+ on a beat-to-beat basis (5), as a mechanism for extrusion of Ca2+ from the cell (6), and as a mediator of the inotropic effects of cardiac glycoside administration and changes in stimulation frequency (1,5,(7)(8)(9).Thus far, the study of Na+-Ca2+ exchange in cardiac tissue has been based on mechanical responses (10-12) or isotopic flux data (3, 6, 9, 13) obtained with intact muscle preparations. The pioneering work of Kaback and colleagues (14,15) showed that transport phenomena could be studied by using sealed membrane vesicles, a system in which the conditions on either side of the membrane can be manipulated at will. Vesicles derived from cardiac sarcolemma have been used to study the enzymatic activity of transport ATPases (16)(17)(18)(19), and several investigators have recently reported that sarcolemmal membrane vesicles exhibit ATP-dependent transport of Ca2+ (20)(21)(22)(23)(24).The present report shows that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. The data demonstrate that transmembrane Ca2+ movements in either direction are markedly stimulated by generating an oppositely directed gradient of Na+. These Na+-Ca2+ interactions appear to be associated with the sarcolemmal membranes within the preparation. MATERIALS AND METHODSPreparation of Membrane Vesicles. Ventricular tissue from male New Zealand rabbits (1.4-2.0 kg) was finely minced with scissors in 3-4 vol of ice-cold 0.3 M sucrose/5 mM MgSO4/10 mM imidazole-HCI (pH 7.0). Several different homogenization procedu...

The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation. We assumed that the rate of Ca2+ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca2+ ([Ca2+]i) at which the rate of Ca2+ influx was half-maximal (Kh). After correcting for the effects of mitochondrial Ca2+ uptake and fura-2 buffering, Kh values of ∼300 nM were obtained. After an increase in [Ca2+]i, the activated state of the exchanger persisted following a subsequent reduction in [Ca2+]i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca2+]i, exchange-mediated Ca2+ entry began without a lag period and displayed a linear rate of Ca2+ uptake in most cells; a sigmoidal time course of Ca2+ uptake returned 60–90 s after the transient increase in [Ca2+]i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca2+ pump, suggesting that local Ca2+ gradients contribute to maintaining exchanger activation after the return of global [Ca2+]i to low values.