Abstract. Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.

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Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.

Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 g/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.Studies in animal models and in humans have shown that hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective impairment of endothelium-derived relaxation. This impairment is due to a decrease in bioavailable endothelium-derived nitric oxide (NO) 1 (1). In the initial phase of the disease process, there is impaired responsiveness to receptor-dependent stimuli, such as acetylcholine, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. As such, the early pathogenesis involves attenuated endothelial NO production in response to extracellular stimuli, but the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, there is nonspecific inhibition of NO bioavailability. Although this later inhibition is most likely multifactorial in origin, it is at least partly due to enhanced inactivation of NO by superoxide anions (2-4). These processes result in increased neutrophil adherence to the endothelium, thereby promoting a key step in the pathogenesis of atherosclerosis (5). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (6). In vitro investigations have further shown that oxidized LDL (oxLDL) inhibits NO-mediated responses. Antioxidants reduce both the formation of free radicals and the oxidative modification of LDL that lead to impaired NO-related responses (7). Thus, NO is critically involved ...

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