Osmotic homeostasis in the brain involves movement of water through aquaporin-4 (AQP4) membrane channels. Perivascular astrocyte end-feet contain distinctive orthogonal lattices (square arrays) assembled from 4-to 6-nm intramembrane particles (IMPs) corresponding to individual AQP4 tetramers. Two isoforms of AQP4 result from translation initiation at methionine residues M1 and M23, but no functional differences are known. In this study, Chinese hamster ovary cells were transfected with M1, M23, or M1؉M23 isoforms, and AQP4 expression was confirmed by immunoblotting, immunocytochemistry, and immunogold labeling. Square array organization was examined by freeze-fracture electron microscopy. In astrocyte end-feet, >90% of 4-to 6-nm IMPs were found in square arrays, with 65% in arrays of 13-30 IMPs. In cells transfected with M23, 95% of 4-to 6-nm IMPs were in large assemblies (rafts), 85% of which contained >100 IMPs. However, in M1 cells, >95% of 4-to 6-nm IMPs were present as singlets, with <5% in incipient arrays of 2-12 IMPs. In A quaporins are specialized water transport channels in plasma membranes of water-permeable tissues (1). Aquaporins 1 and 4 (AQP1 and AQP4) are most important to fluid movements in mammalian brain. AQP4 exists as two isoforms, differing at their N termini, because of translation initiation at the first methionine (M1, 323 aa) or the second methionine (M23, 301 aa) (2, 3). Both isoforms are present in brain, but M23 is at least 3-fold more abundant (4, 5). Endogenous AQP4 is a tetramer usually containing M1 and M23 subunits. The water permeabilities of M1 and M23 are similar, and functional differences are not known (3, 4).Fluid movements are precisely orchestrated within the rigid cranium to prevent physical damage from swelling or shrinkage. Interfaces between brain parenchyma and cerebrospinal fluid occur around the ventricles, surrounding blood vessels, and at the brain surface. AQP1 is expressed in rat choroid plexus, the site of cerebrospinal fluid secretion (6), whereas AQP4 is enriched in rat astrocyte end-feet surrounding brain capillaries (7,8). Astrocyte processes forming the glia limitans at brain surfaces, ependymal cells lining brain ventricles, and Müller cells facing the vitreous body and retinal blood vessels all have abundant AQP4 (9). AQP4 in perivascular membranes of astrocyte end-feet has been implicated in neurological disorders, including acute hyponatremic edema, postischemic injury, and epileptic seizures (10-13).Perivascular membranes of astrocyte end-feet contain numerous strikingly regular arrays of intramembrane particles (IMPs) in freeze-fracture electron micrographs. These IMP arrays have been referred to as square arrays, assemblies, or orthogonally arranged particles (OAPs) (14). In early freeze-fracture images of astrocyte end-feet (15), square arrays were resolved as 6-nm IMP protrusions in P-face images (protoplasmic leaflets) or as smaller pits in E-faces (extraplasmic leaflets). The sizes and shapes of square arrays vary, but the IMPs and pits have un...

Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin-(Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive, spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43. Conversely, Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32͞Cx43 single-and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43. Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF, Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata. On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.

The treatment of ischemic strokes is limited to the prevention of cerebrovascular risk factors and to the modulation of the coagulation cascade during the acute phase. A new therapeutic strategy could be to preventively protect the brain against noxious biological reactions induced by cerebral ischemia such as oxidative stress and inflammation to minimize their neurological consequences. Here, we show that a peroxisome proliferator-activated receptor (PPAR-alpha) activator, fenofibrate, protects against cerebral injury by anti-oxidant and anti-inflammatory mechanisms. A 14 d preventive treatment with fenofibrate reduces susceptibility to stroke in apolipoprotein E-deficient mice as well as decreases cerebral infarct volume in C57BL/6 wild-type mice. The neuroprotective effect of fenofibrate is completely absent in PPAR-alpha-deficient mice, suggesting that PPAR-alpha activation is involved as a mechanism of the protection against cerebral injury. Furthermore, this neuroprotective effect appears independently of any improvement in plasma lipids or glycemia and is associated with (1) an improvement in middle cerebral artery sensitivity to endothelium-dependent relaxation unrelated to an increase in nitric oxide synthase (NOS) type III expression, (2) a decrease in cerebral oxidative stress depending on the increase in numerous antioxidant enzyme activities, and (3) the prevention of ischemia-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in cerebral vessels without any change in NOS II expression. These data demonstrate that PPAR-alpha could be a new pharmacological target to preventively reduce the deleterious neurological consequences of stroke in mice and suggest that PPAR-alpha activators could preventively decrease the severity of stroke in humans.

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructuraly-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocytc gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytcs, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in Ieptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.

Objective-Uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors results in lipid droplets accumulation and foam cell formation. Excess lipid deposition in macrophages has been reported to modulate expression of several genes including adipophilin. In this study, we investigated the function of adipophilin in lipid accumulation and cholesterol efflux in THP-1 macrophages. Methods and Results-Adipophilin mRNA expression was 3.5-fold higher in human atherosclerotic plaques compared with healthy areas of the same arteries. Moreover, in the presence of acetylated LDL (AcLDL), triglycerides and cholesteryl esters were increased in macrophages overexpressing adipophilin by 40% and 67%, respectively, whereas their accumulation was reduced when endogenous cellular adipophilin was depleted using siRNA approach. In addition, neither overexpression nor downregulation of adipophilin altered expression of genes involved in lipid efflux. However, the affinity and the number of AcLDL receptors were not affected. After 24-hour incubation of lipid-loaded macrophages with apolipoprotein A-I, cholesterol efflux was reduced by 47% in adipophilin transfected cells versus control cells. Key Words: adipophilin Ⅲ macrophage Ⅲ acetylated LDL Ⅲ cholesterol efflux Ⅲ atherosclerosis A therosclerosis, a complex disease process, is initiated by an infiltration of low-density lipoproteins (LDL) into the subendothelial space where they accumulate and become modified mainly by oxidation. In parallel, chemokines elaborated by endothelial cells attract circulating monocytes into the intima, where they differentiate into macrophages. 1 Intimal macrophages contribute to the formation of arterial lesions by accumulating large amounts of cholesteryl ester (CE) through the uptake of modified lipoproteins such as oxidized LDL (oxLDL) by a variety of mechanisms, including the scavenger pathway. 2,3 Several studies indicated that oxLDL has a number of diverse effects on macrophage function including growth stimulation 4 proinflammatory effects such as expression of inflammatory cytokines, 5 increase in cytotoxicity and expression of metalloproteinases, 6 inhibition of expression of inducible nitric oxide synthase, 7 and effects on lipid metabolism and accumulation. 8,9 Using either a DNA array approach 10 or a subtractive approach, 11 numerous genes have been shown to be regulated on exposure of THP-1 cells to modified LDL. Adipophilin, or adipose differentiation-related protein (ADRP), a 50-kDa protein initially described in adipocytes, 12 which is considered a marker of lipid accumulation, is among those genes upregulated by modified LDL. Conclusion-OurAdipophilin is found associated with intracellular lipid droplets in a variety of cells and tissues that store or synthesize lipids. 13,14 In addition to oxLDL, 15 acetylated LDL (AcLDL) 11 or enzymatically modified LDL, 16 the nuclear receptor PPAR␦, is involved in lipid accumulation in human macrophages and THP-1-derived macrophages, and also increases adipophilin e...

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Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the wide dispersion of connexons in string and ribbon gap junctions suggests unique structural features of gap junctional coupling in the OFF vs. ON sublamina.

The peroxisome proliferator-activated receptor K K (PPARK K) is a transcription factor belonging to the PPAR subfamily of nuclear receptors. Fatty acids and eicosanoids are natural PPARK K ligands. Here, we show using transient transfection assays that oxidized (oxLDL) but not native lowdensity lipoproteins (LDL) dose-dependently activate PPARK K in endothelial cells without affecting PPARK K protein expression. Fractioning of oxLDL lipids followed by transactivation experiments demonstrated that the oxidized phospholipid component in oxLDL is responsible for PPARK K activation. Using specific inhibitors, it is shown that oxLDL-mediated PPARK K activation requires phospholipase A2 activity and that the oxidized fatty acids 9-and 13-HODE activate PPARK K directly. Finally, we found that, similar to the synthetic PPARK K ligand Wy-14643, oxLDL induced expression of the fatty acid transport protein-1 in human primary endothelial cells. Our findings define a novel group of PPARK K activators and provide a molecular basis for certain effects of these biologically active phospholipids on gene transcription.z 2000 Federation of European Biochemical Societies.