gene (120a). Both coagulase-positive and coagulase-negative staphylococci may produce other hemolysins, designated beta-, gamma-, and delta-toxins, that are molecularly distinct from alpha-toxin and whose roles as virulence factors 733

Streptolysin-O (SLO) is a thiol-activated, membrane-damaging protein toxin of Mr 69,000 that is produced by most strains of I-hemolytic group A streptococci. Native, primarily water-soluble toxin molecules bind to cholesterol-containing target membranes to assemble into supramolecular curved rod structures (25 to 100 nm long by ca. 7.5 nm wide), forming rings and arcs that penetrate into the apolar domain of the bilayer. Electron microscopic analyses of toxin polymers in their native and reconstituted membrane-bound form indicate that the convex surface of the rod structures is a hydrophobic, lipid-binding domain, whereas the concave surfaces appear to be hydrophilic. The embedment of the rings and arcs generates large transmembrane slits or pores of up to 30-nm diameter that can be directly visualized by negative staining and freeze-fracture electron microscopy. SLO oligomers were isolated in extensively delipidated form in detergent solution, and cholesterol was found not to detectably contribute to the observed rod structures. The rods are stable structures that resist prolonged exposure to trypsin and chymotrypsin. They can be reincorporated into cholesterol-free phosphatidylcholine liposomes to generate lesions identical to those observed on erythrocytes lysed by native SLO. Thus, although cholesterol plays a key role in the initial binding of SLO to the membrane, it does not directly participate in the formation of the membrane-penetrating toxin channels. Membrane damage by SLO is basically analogous to that mediated by previously studied channel formers, namely, the C5b-9 complement complex and staphylococcal ot-toxin. Lancefield group A P-hemolytic streptococci are the ma-* Corresponding author.

Rabbit or human erythrocytes lysed with Staphylococcus aureus a-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and a2-macroglobulin in gel chromatography . A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE . In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter . An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membranederived form of a-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of -3-nm effective diameter in toxin-treated membranes, the possibility is raised that native a-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel .

Objective-This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility. Methods and Results-Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-␤-cyclodextrin, and the effects on force and Ca 2ϩ handling were evaluated. In cholesterol-depleted preparations, the force responses to ␣ 1 -adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 mol/L AlCl 3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by Ͼ50%. The rise in global intracellular free Ca 2ϩ concentration in response to 5-HT was attenuated, as was the generation of Ca 2ϩ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT 2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT 2A receptors, but not ␣ 1 -adrenergic receptors, in the caveolin-1-containing fractions, suggesting localization of the former to caveolae. Conclusions-These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae. Key Words: smooth muscle Ⅲ caveolae Ⅲ 5-hydroxytryptamine Ⅲ endothelin Ⅲ intracellular calcium C ellular cholesterol, of which most (up to 90%) 1 resides in the plasma membrane, is crucial for normal membrane permeability and fluidity and also plays a role in cellular signaling, via several proposed mechanisms that fall into at least 4 categories. First, cellular cholesterol may influence gene transcription in the nucleus through sterol regulatory element binding proteins. 2 Second, the activity of membrane receptors, ion channels, and transporters may depend on the membrane fluidity, per se. 3 Third, membrane protein function may be regulated through specific cholesterol-protein interactions. 3,4 Fourth, cholesterol stabilizes the structure of caveolae and lipid rafts.Caveolae, which are 50-to 100-nm membrane invaginations that are abundant in vascular endothelium and smooth muscle cells, are defined by their characteristic morphology and contents of caveolin and cav-p60. 5,6 No definitive definition of rafts has appeared because they do not exhibit a characteristic structure, but the term is used for planar aggregations of specific lipids and proteins. Caveolae and lipid rafts are envisaged to serve as platforms for a dynamic association of signaling proteins and for the initiation or modulation of signaling. 5,7,8 Some agonists causing contraction of vascular smooth muscle act on receptors that are believed to be located in caveo...