Changes in the surface charge properties of isolated cardiac sarcolemmal vesicles measured by light scattering. I. Characteristics of rat and canine preparations.

The cation-binding characteristics of isolated sarcolemmal vesicles from rat and canine cardiac muscle cells were investigated. To help elucidate the molecular properties involved in these interactions the cation-induced aggregation behavior of rat and canine cardiac sarcolemmal vesicles, sonicated unilamellar vesicles (SUVs) made from sarcolemmal lipid extracts, and SUVs generated from combinations of synthetic lipids similar to those found in the sarcolemmal membrane, as well as mitochondrial and sarcoplasmic reticulum enriched membrane fractions were examined. Our results indicate that cations, such as Ca2+, to indeed bind to the sarcolemmal membrane surface. They also suggest that two (or more) interacting sites are involved in the Ca2+-induced aggregation of the isolated sarcolemmal vesicles, and that sarcolemmal lipid components could be the primary binding sites. The modulating (secondary) sites on the other hand may be protein or carbohydrate in nature, or require specific lipid organizational properties. Finally, the results indicate that the interactions of cations, such as Ca2+, with the sarcolemmal surface are species specific, with the sarcolemmal membranes of both rat and canine preparations having different physico-chemical properties.

Changes in the surface charge properties of isolated cardiac sarcolemmal vesicles measured by light scattering. II. Characteristics of rabbit preparations.

Numerous studies suggest that cation-sarcolemmal interactions play an essential role in the excitation/contraction/relaxation cycles of cardiac muscle cells. To help elucidate the molecular mechanisms involved in these processes the cation binding characteristics of isolated rabbit cardiac sarcolemmal vesicles were investigated. Cation-membrane interactions were studied by examining the cation-induced aggregation properties of the vesicles. The results obtained were qualitatively very similar to those previously reported for rat and canine cardiac sarcolemmal vesicle preparations (Leonards, K.S. (1988) Biochim. Biophys. Acta 938, 293-309), indicating that all three species have a shared set of basic membrane characteristics. Specifically the results indicate that cations, such as Ca2+, bind to the sarcolemmal surface, and suggest that two (or more) interacting sites are involved in the process. The selectivity series for the cation-induced aggregation of the sarcolemmal vesicles was: La3+ greater than or equal to Cd2+ much greater than Mn2+ greater than Ca2+ greater than Ba2+ = Sr2+ = Mg2+. Protons (H+) could also induce massive vesicle aggregation at pH 5.60-5.75. However, the results obtained also show that the interactions of cations with the rabbit cardiac sarcolemmal membrane surface are quantitatively distinct from those obtained in either rat or canine sarcolemmal vesicle preparations, thereby confirming the species specific nature of cation-sarcolemmal interactions in cardiac cells.

Effects of cations of the plasma membrane of Blastocladiella emersonii zoospores An ESR study.

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T parallel, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (TL and TH) were found to be independent of the external K+, Ca2+, or Mg2+ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore liability. In contrast, the middle break point (TM) was markedly influenced by the external Ca2+ concentration. Ca2+ increased TM from 12 degrees C (no Ca2+ added) to 22 degrees C (10 mM Ca2+), i.e., growth temperature. K+ reversed this Ca2+ effect, downshifting TM from 22 degrees C to 10 degrees C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in TM, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggest that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.

Electron spin resonance study of the isolated lipid components from Blastocladiella emersonii zoospores.

The physico-chemical properties of lipid components isolated from zoospores of the aquatic phycomycete, Blastocladiella emersonii, were investigated with electron spin resonance (ESR) spectroscopy using the spin label, 5-nitroxystearate. Lipid dispersions were made from zoospore phospholipids and glycolipids, both singly and in combination with each other and with isolated neutral lipid components. Plots of the hyperfine splitting parameter (2T parallel) vs. temperature indicate that it is the zoospore glycolipids rather than the phospholipids which are responsible for the phase transformations previously observed in aqueous dispersions of the total lipids extracted from zoospores and in zoospores in vivo. The discontinuities observed in the glycolipid dispersions seem to represent the onset and completion of a gel-to-liquid-crystalline phase transition. Over the temperature range tested, Ca2+ increased the rigidity of the glycolipid dispersions, the major component of which is probably a diglucosyldiglyceride, but had no effect on the phospholipid dispersions. The increase in 2T parallel was not affected by inclusion of neutral lipids into the glycolipid dispersion but was eliminated at high (5 : 1, w/w) phospholipid-to-glycolipid ratios. The Ca2+ effect was relatively independent of both the absolute rigidity of the dispersion and its phase (gel or liquid-crystalline), suggesting an interaction with the glycolipid head group rather than the hydrocarbon core. The Ca2+-induced increase in 2T was neither prevented nor reversed by the presence of K+. The presence of two spin label populations co-existing in a dynamic equilibrium was found in glycolipid/neutral lipid dispersions. Plots of the percentage ([HA/(HA + HB)] X 100 of the spin label population, as measured by the peak height of the low-field peaks, corresponding to the more immobilized component (HA) vs. temperature indicated two break points. The temperatures at which these break points occurred are similar to those obtained for the glycolipid dispersions, and match the break points (TL and TH) found in ESR experiments using zoospores in vivo. The importance of the glycolipids in the development of this organism is discussed.

Isolation and characterization of large (0.5 - 1.0 micron) cytoskeleton-free vesicles from human and rabbit erythrocytes.

Large (0.5 - 1.0 micron) cytoskeleton-free vesicles were obtained, by 'budding', from fresh human and rabbit erythrocytes incubated at 45 degrees C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0 - 11.0 and temperature range 4-50 degrees C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.

The in vitro and ex vivo antioxidant properties, and hypolipidemic activity of CGP 2881.

This report describes the in vitro and ex vivo antioxidant properties of a new antioxidant, CGP 2881. This compound is structurally similar to probucol, in that both compounds contain bis-tertiary butyl phenyl groups. However, CGP 2881 consistently inhibited CuSO4 (Cu2+)- and macrophage (MO)-induced oxidation of human low density lipoproteins (LDL) more potently than equimolar concentrations of probucol. CGP 2881 (1 mumol/l) prolonged the lag phase of diene formation during Cu(2+)-induced LDL oxidation by 3.4 versus 1.5-fold prolongation with 1 mumol/l probucol (P < 0.05 vs CGP 2881). The IC50 for inhibiting the formation of Cu(2+)-induced thiobarbituric acid-reactive substances (TBARS) was 0.15 mumol/l for CGP 2881, versus approximately 10 mumol/l for probucol. The IC50 for MO-induced oxidation of LDL (TBARS) was 0.64 mumol/l. In contrast, 1 mumol/l probucol failed to inhibit MO-induced oxidation of LDL. Treatment of cholic acid/cholesterol-fed rats with CGP 2881 (50 mg/kg per day, orally for 5 days) inhibited ex vivo Cu(2+)-induced oxidation (TBARS) of the very low density lipoproteins (VLDL) + LDL lipoprotein fraction by 93% versus vehicle controls (P < 0.0001), and prolonged the lag phase for Cu(2+)-induced diene formation by 3.4-fold over vehicle-treated controls. Five days of orally administered CGP 2881 reduced plasma total cholesterol and LDL cholesterol levels to 55 and 54% of vehicle-treated controls, respectively (P < 0.05). In contrast, probucol had no appreciable effect on plasma total cholesterol or LDL cholesterol levels, unless administered for longer than 5 days. Treatment of hypercholesterolemic rabbits with 50 mg/kg per day orally for 5-12 days delayed the lag phase of diene formation during LDL oxidation by 4.3-fold over controls. However, the relative antioxidant potencies of CGP 2881 and probucol seen with oral administration to hypercholesterolemic rabbits were reversed when the compounds were given intravenously. In addition, the effects of these antioxidants were potentiated when given to normocholesterolemic rabbits compared to hypercholesterolemic animals. These data establish that CGP 2881 demonstrates hypolipidemic activity and is a substantially more potent antioxidant than probucol (in vitro and ex vivo). CGP 2881 may be useful as a new antioxidant tool in the effort to better understand the atherogenicity of oxidized LDL (oxLDL).

Demonstration of potent lipid-lowering activity by a thyromimetic agent devoid of cardiovascular and thermogenic effects.

A potent lipid-lowering thyromimetic (CGS 26214) devoid of cardiac and thermogenic activity was identified based on its ability to preferentially access and bind the nuclear fraction of hepatocytes over that of myocytes in culture. The difference in access achieved with CGS 26214 was at least 100-fold better for hepatocytes than for myocytes. This in vitro hepatoselectivity resulted in a compound with unprecedented in vivo lipid-lowering potency with a minimal effective dose of 1 microgram/kg in rats and dogs (approximately 25x that of L-T3). At the same time, CGS 26214 was free of any cardiovascular effects up to the highest dose tested of 25 mg/kg and 100 micrograms/kg in rats and dogs, respectively.

Roles of proteins in cation/membrane interactions of isolated rat cardiac sarcolemmal vesicles.

To ascertain the roles of the membrane proteins in cation/sarcolemmal membrane binding, isolated rat cardiac sarcolemmal vesicles were extensively treated with Protease (S. aureus strain V.8). SDS-gel electrophoresis, protein and phosphate analysis confirmed that at least 20-22% of the protein, but none of the phospholipid, was solubilized by this procedure, and that the remaining membrane proteins were extensively hydrolyzed into small fragments. The cation binding properties of the treated vesicles were then examined by analyzing their aggregation behavior. The results demonstrate that this procedure had no effect on the selectivity series for di- and trivalent cation binding, or the divalent cation-induced aggregation behavior of the sarcolemmal vesicles at different pHs, indicating that proteins are probably not involved in these interactions and cannot be the low affinity cation binding sites previously observed. It did, however, change the pH at which protons induced sarcolemmal vesicle aggregation, suggesting a possible role for proteins in these processes. Protease treatment also modified the effects of fluorescamine labelling on divalent cation-induced vesicle aggregation, indicating that the NH2 groups being labelled with fluorescamine are located on the sarcolemmal proteins. Together, these results support the hypothesis that di- and trivalent cation binding to the sarcolemmal membrane is largely determined by lipid/lipid and/or lipid/carbohydrate interactions within the plane of the sarcolemmal membrane, and that membrane proteins may exert an influence on these interactions, but only under very specialized conditions.

A possible role of cholesterol in membrane adhesion.

Calcium phosphate induced membrane aggregation was studied for erythrocyte vesicles and lipid membrane vesicles. The later lipid membrane components were similar in composition to those of erythrocyte membranes. The presence of an appropriate amount of cholesterol is an important factor in the production of the calcium phosphate dependent membrane aggregation.

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein.

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.

Roles of lipids and proteins in the Ca2+-PO4-induced aggregation of cytoskeleton-free erythrocyte vesicle membranes.

The roles of lipids and proteins in Ca2+-PO4-induced membrane aggregation were investigated. Cytoskeleton-free vesicles derived from intact human and rabbit erythrocytes (HEves and REves, respectively) were employed as a model system. The HEves and REves have a simplified membrane protein composition [band 3 proteins and glycoproteins PAS-1, -2, and -3 (HEves)] and normal lipid composition. Optimal experimental conditions for pH, [PO4], and [CaCl2] were determined for quantitatively examining the dynamics and extent of HEves and REves aggregation, measured turbidimetrically. The aggregation process was found to be quite sensitive to small changes in pH and [PO4] and much less sensitive to [CaCl2]. The roles of membrane proteins in vesicle aggregation were examined by selectively modifying the proteins enzymatically. The roles of lipids were studied by using sonicated lipid vesicles [small unilamellar vesicles (SUVs)] made from Dodge ghost lipid extracts. Enzymatic treatment with trypsin, chymotrypsin, or Pronase had no effect on either the rates or the extent of vesicle aggregation (2-min incubation period). Neuraminidase treatment reduced both factors by approximately 20%. SUVs aggregated with Ca2+-PO4 in a way which depended on the PO4/lipid ratio. Together the results suggest the following: (1) PO4 is associated with the vesicle surface, involving the membrane lipids; (2) the vesicle + PO4 incubation time component of the PO4 effect is eliminated by enzymatically modifying the vesicle membrane proteins; (3) qualitative, rather than quantitative, properties of sialic acid containing molecules affect vesicle aggregation; and (4) with the exception of the incubation time effect, membrane proteins seem neither to promote nor to inhibit Ca2+-PO4-induced HEves or REves aggregation.

Potassium- and calcium-induced alterations in lipid interactions of isolated plasma membranes from blastocladiella emersonii. Evidence for an adenosine 5'-triphosphate requirement.

The physical-chemical properties of the isolated plasma membranes from zoospores of the chytridiomycete Blastocladiella emersonii were investigated, with electron spin resonance (ESR) spectroscopy, using the spin-label 5-nitroxystearate (5-NS). Both isolated plasma membranes and aqueous dispersion of the lipids extracted from the plasma membranes were spin-labeled and analyzed. Plots of the hyperfine splitting parameter (2T) vs. temperature indicated that the middle break point, TM, initially observed in experiments with spin-labeled zoospores in vivo [Leonards, K. S., & Haug, A. (1980) Biochim. Biophys. Acta 600, 805-816], was the result of a lipid-lipid interaction (glycolipid-glycolipid or glycolipid-neutral lipid) rather than a lipid-protein interaction. This interaction was markedly affected by Ca2+ ions, which interacted directly with the lipid components, increasing TM from 11 +/- 1 (Ca2+ removed by EDTA) to 21 +/- 1 degree C (10 mM Ca2+) in the lipid dispersions and from 12 +/- 1 to 23 +/- 1 degree C in the plasma membrane preparations. The initial ESR studies on spin-labeled zoospores in vivo had also demonstrated that the addition of K+ ions could reverse the Ca2+ ion effect, downshifting TM from 22 +/- 1 to 10 +/- 1 degree C. The addition of of K+ ions to the isolated plasma membrane had no affect on TM, indicating that K+ ions do not simply replace Ca2+ ions but exert their effect indirectly on the membrane. However, after the inclusion of ATP, K+ ions could reverse the Ca2+ ion effect. it was determined that the ATp generated an "energized membrane" state which permitted the K+ ions to reverse the Ca2+ effect. Since K+ ions have been shown to depolarize the membrane potential in both zoospores and isolated zoospore plasma membrane preparations (generated by ATP), were suggest that the K+ ion induced reversal of the Ca2+ ion effect, and therefore the change in the lipid-lipid interactions responsible for TM, is a consequence of the K+ ion induced depolarization of the membrane potential.

Effects of charged amphiphiles on cardiac cell contractility are mediated via effects on Ca2+ current.

Exposure of isolated adult rabbit myocytes to the negatively charged amphiphile dodecylsulfate (DDS; 10 microM) increased the contraction amplitude to 185% of control. The positively charged amphiphile dodecyltrimethylammonium (DDTMA; 10 microM) decreased the amplitude to 58%. DDS increased Ca2+ uptake by the same cells, but this uptake was partially prevented by nifedipine. DDTMA had no effect on Ca2+ uptake. Ca2+ binding to isolated sarcolemma of neonatal heart cells was increased by 10 microM DDS and, at higher concentrations, reduced by DDTMA. Single-cell voltage-clamp studies, using isolated rabbit myocytes, showed that DDS enhanced L-type Ca2+ currents (ICa,L), whereas DDTMA depressed ICa,L. DDS shifted current-voltage (I-V) and isochronal inactivation curves of ICa,L in the negative direction, whereas DDTMA shifted them in positive direction. Furthermore, DDS depressed T-type Ca2+ currents (ICa,T), and DDTMA enhanced ICa,T. The inotropic effects of the amphiphiles are therefore mediated to a significant degree by ICa,L. The shifts in the I-V and inactivation curves of ICa,L and the effect on ICa,T can be explained by changes in the actual membrane potential (Em), induced by the insertion of the amphiphiles in the outer monolayer of the sarcolemma. However, the changes in the Em do not explain the effect on the maximal current, indicating effects on the channel per se, possibly by an alteration of the lipid environment.