Filipin Treatment Research Articles

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.

Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

Free radicals are believed to play an active role in the bystander response. This study investigated their origin as well as their temporal and spatial impacts in the bystander effect. We employed a precise alpha-particle microbeam to target a small fraction of subconfluent osteoblastic cells (MC3T3-E1). gammaH2AX-53BP1 foci, oxidative metabolism changes, and micronuclei induction in targeted and bystander cells were assessed. Cellular membranes and mitochondria were identified as two distinct reactive oxygen species producers. The global oxidative stress observed after irradiation was significantly attenuated after cells were treated with filipin, evidence for the primal role of membrane in the bystander effect. To determine the membrane's impact at a cellular level, micronuclei yield was measured when various fractions of the cell population were individually targeted while the dose per cell remained constant. Induction of micronuclei increased in bystander cells as well as in targeted cells and was attenuated by filipin treatment, demonstrating a role for bystander signals between irradiated cells in an autocrine/paracrine manner. A complex interaction of direct irradiation and bystander signals leads to a membrane-dependent amplification of cell responses that could influence therapeutic outcomes in tissues exposed to low doses or to environmental exposure.

CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity

The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-β-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca 2+ and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Gαi G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Gαi G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Gαi G-proteins.

Membrane Raft Disruption Promotes Axonogenesis in N2a Neuroblastoma Cells

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

Lipoprotein binding preference of CD36 is altered by filipin treatment

The class B scavenger receptor CD36 binds multiple ligands, including oxidized and native lipoprotein species. CD36 and the related receptor SR-B1 have been localized to caveolae, domains that participate in cell signaling, transcytosis, and regulation of cellular cholesterol homeostasis. Previous work has indicated that the ligand preference of CD36 may depend on the cell type in which it is expressed. To determine if the presence or absence of caveolae is the determining factor for lipoprotein preference, we treated CHO-CD36 and C32 cells with filipin. Filipin treatment rapidly increased the binding capacity of CD36 for the native lipoproteins HDL and LDL, but did not affect the binding capacity of CD36 for oxidized LDL. Filipin treatment affected the distribution of caveolin and CD36 suggesting that the presence caveolae may modulate the ligand preference of CD36. However, its molecular mechanism how CD36 and caveolin interaction in regulating lipoprotein transport remains to be further studied.

Apical Sterol-rich Membranes Are Essential for Localizing Cell End Markers That Determine Growth Directionality in the Filamentous FungusAspergillus nidulans

In filamentous fungi, hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules, and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell end factor destinations and thereby polarized growth.

Functional domains of the mouse β3-adrenoceptor associated with differential G-protein coupling

Localization of G-protein-coupled receptors within membrane microdomains is associated with differential signalling pathway activation. We have shown that two mouse β3-AR (β3-adrenoceptor) isoforms encoded by alternatively spliced mRNAs differ in their signalling properties; the β3a-AR couples only with Gs, whereas the β3b-AR couples with both Gs and Gi. Our previous studies indicated that the β3a-AR is restrained from coupling with Gi due to the interaction of residues in the C-terminus with other protein(s). We have investigated the hypothesis that the β3a-AR interacts with caveolin. Disruption of caveolae in CHO (Chinese-hamster ovary)-K1 cells expressing wild-type β3a-ARs with filipin III, or mutation of a putative caveolin-binding site in the β3a-AR, causes cAMP accumulation to become PTX (pertussis toxin)-sensitive. Likewise, filipin treatment of mouse brown adipocytes that express endogenous β3a-ARs produces a substantial reduction in agonist-stimulated cAMP production that is rescued by pre-treatment with PTX. These studies suggest that β3a-ARs may be restricted to caveolae and that localization of the receptor may play a specific role in G-protein-mediated signalling.

Functional domains of the mouse β3-adrenoceptor associated with differential G-protein coupling

Localization of G-protein-coupled receptors within membrane microdomains is associated with differential signalling pathway activation. We have shown that two mouse beta(3)-AR (beta(3)-adrenoceptor) isoforms encoded by alternatively spliced mRNAs differ in their signalling properties; the beta(3a)-AR couples only with G(s), whereas the beta(3b)-AR couples with both G(s) and G(i). Our previous studies indicated that the beta(3a)-AR is restrained from coupling with G(i) due to the interaction of residues in the C-terminus with other protein(s). We have investigated the hypothesis that the beta(3a)-AR interacts with caveolin. Disruption of caveolae in CHO (Chinese-hamster ovary)-K1 cells expressing wild-type beta(3a)-ARs with filipin III, or mutation of a putative caveolin-binding site in the beta(3a)-AR, causes cAMP accumulation to become PTX (pertussis toxin)-sensitive. Likewise, filipin treatment of mouse brown adipocytes that express endogenous beta(3a)-ARs produces a substantial reduction in agonist-stimulated cAMP production that is rescued by pre-treatment with PTX. These studies suggest that beta(3a)-ARs may be restricted to caveolae and that localization of the receptor may play a specific role in G-protein-mediated signalling.

Influence of surface charge and inner composition of porous nanoparticles to cross blood–brain barrier in vitro

The aim of these studies was to evaluate the binding, uptake and transcytosis of 60 nm porous nanoparticles (NPs) that differed in their surface charge and inner composition on the blood–brain barrier (BBB). They were prepared from maltodextrins derived with or without a cationic ligand. In the cationic NPs an anionic lipid was inserted in their core to give DPPG-NPs. The data showed that at 4 °C the three NPs bind in different areas on endothelial cells: cationic NPs were found mainly around the paracellular area, while neutral NPs were mainly on the cell surface and DPPG-NPs binding was found at both paracellular areas and on the surface of the cells. At 37 °C neutral and cationic NPs had similar degrees of binding and uptake and were transcytosed. Filipin treatment increased their binding and uptake suggesting that sterols are implied in their efflux. Neutral NPs transcytosis was also inhibited by filipin. This inhibition shows that neutral NPs, like LDL in this model, use the caveolae pathway. Neutral and cationic 60 nm porous NPs are potential candidates for drug delivery to the brain.

Disruption of endothelial caveolae is associated with impairment of both NO- as well as EDHF in acetylcholine-induced relaxation depending on their relative contribution in different vascular beds

Caveolae represent an important structural element involved in endothelial signal-transduction. The present study was designed to investigate the role of caveolae in endothelium-dependent relaxation of different vascular beds. Caveolae were disrupted by cholesterol depletion with filipin (4 × 10 − 6 g L − 1 ) or methyl-β-cyclodextrin (MCD; 1 × 10 − 3 mol L − 1 ) and the effect on endothelium-dependent relaxation was studied in rat aorta, small renal arteries and mesenteric arteries in the absence and presence of L-NMMA. The contribution of NO and EDHF, respectively, to total relaxation in response to acetylcholine (ACh) gradually changed from aorta (71.2 ± 6.1% and 28.8 ± 6.1%), to renal arteries (48.6 ± 6.4% and 51.4 ± 6.4%) and to mesenteric arteries (9.1 ± 4.0% and 90.9 ± 4.1%). Electron microscopy confirmed filipin to decrease the number of endothelial caveolae in all vessels studied. Incubation with filipin inhibited endothelium-dependent relaxation induced by cumulative doses of ACh (3 × 10 − 9 –10 − 4 mol L − 1 ) in all three vascular beds. In aorta, treatment with either filipin or MCD only inhibited the NO component, whereas in renal artery both NO and EDHF formation were affected. In contrast, in mesenteric arteries, filipin treatment only reduced EDHF formation. Disruption of endothelial caveolae is associated with the impairment of both NO and EDHF in acetylcholine-induced relaxation.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Modulation of Tissue Factor-Factor VIIa Coagulant and Signaling Functions by Membrane Cholesterol and Caveolae.

Cholesterol in the membrane not only regulates flexibility and mechanical stability of the membrane but also plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin, form caveolae, flask-shaped invaginations in the plasma membrane. Lipid rafts and caveolae are shown to regulate various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors and signaling pathways. In the present study, we investigated the role of membrane cholesterol and caveolae in modulating coagulant and signaling functions of tissue factor (TF)-factor VIIa (VIIa) complexes on tumor cells. Breast carcinoma cells, MDA-MB-231, were treated with ß-methyl cyclodextrin (CD), which depletes membrane cholesterol and thereby disrupts caveolae, or with filipin, which disrupts caveolae without depleting the membrane cholesterol. TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in IP3 hydrolysis and IL-8 gene induction. As expected, CD (10 mM) treatment of tumor cells depleted cellular cholesterol level by more than 70% whereas filipin (5 μg/ml) treatment did not reduce the cellular cholesterol level. Both the treatments had no effect on the cell viability as measured in trypan blue exclusion method and MTT assay. CD treatment, in a dose-pendent manner (1 to 10 mM CD), impaired both TF-VIIa coagulant function and the signaling function (both IP3 hydrolysis and IL-8 gene expression). In contrast, varying effects were observed with filipin treatment. Filipin treatment (5 μg/ml) increased TF-VIIa coagulant function, reduced the TF-VIIa-induced IP3 hydrolysis and no effect on TF-VIIa-induced IL-8 gene expression. Detergent (Triton X-100) extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF was distributed both in lipid rafts and soluble fractions. CD and filipin treatments slightly reduced TF association with lipid rafts. Overall these data suggest that the membrane cholesterol modulates TF-VIIa proteolytic function and hence the TF-VIIa protease-induced signaling. In contrast, disruption of caveolae uncouples selective signaling pathways activated by TF-VIIa without impairing TF-VIIa protease activity.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8 + T cells and induce cytolytic effector T cells in vitro.

Dual Mechanisms for Shedding of the Cellular Prion Protein

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.

Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells.

IL-6 is an inflammatory cytokine that has been linked to aggressive prostate cancer (PCa). Previous studies have demonstrated that IL-6 can enhance the differentiation of PCa cells toward a neuroendocrine (NE) phenotype, a possible indicator of hormone-refractory disease. In this report, we present evidence that the mechanism of IL-6-stimulated NE differentiation employs a detergent-resistant (lipid raft) membrane compartment for signal transduction in LNCaP PCa cells. Signal transducer and activator of transcription (STAT)3, a mediator of IL-6 signaling, was rapidly phosphorylated and translocated to the nucleus in LNCaP cells treated with IL-6. Both processes were inhibited by filipin, a cholesterol-binding compound that disrupts plasma membrane lipid rafts. Isolation of Triton X-100-insoluble raft fractions from LNCaP cells by discontinuous sucrose gradient centrifugation demonstrated that the 80-kDa IL-6 receptor localized almost exclusively to the raft compartment. Although STAT3 was located predominantly in the Triton X-100-soluble subcellular fraction in exponentially growing cells, abundant phosphorylated STAT3 was detected in the raft fraction after stimulation with IL-6. Increases in expression of the NE marker, neuron-specific enolase, and neuron-specific enolase promoter activity after IL-6 treatment were reduced after membrane rafts were disrupted by filipin treatment. LNCaP cells expressed the raft-resident proteins flotillin-2 and G(ialpha2), but notably not caveolins, the predominant structural protein present in caveolar membrane rafts in many tissues and tumor cells. These results are the first to define a role for lipid raft membrane microdomains in signal transduction mechanisms capable of promoting the NE phenotype in PCa cells, and they demonstrate that the raft compartment is capable of mediating such signals in the absence of caveolins. Our results also suggest a mechanistic role for membrane cholesterol in cell signaling events relevant to PCa progression.

Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity

AbstractMany surface receptors and signaling molecules are thought to associate with unique membrane microdomains termed lipid rafts. We examined the involvement of lipid rafts in the activation of leukocyte function–associated antigen-1 (LFA-1). Depletion or sequestration of cholesterol with methyl-β-cyclodextrin (MCD) or filipin, respectively, strongly inhibited LFA-1–mediated adhesion of T-cell lines and primary T cells. This inhibition was reversed by cholesterol reconstitution. LFA-1 on T-cell lines was detected in cold Triton X-100–insoluble lipid rafts, which were disrupted by MCD or filipin treatment. However, no LFA-1 on primary T cells was detected in lipid rafts isolated by the same procedures, and these rafts were resistant to cholesterol depletion or sequestration. Association of LFA-1 with lipid rafts of primary T cells could be detected only when they were isolated with another nonionic detergent, Brij 35. Upon treatment with MCD, LFA-1 in Brij 35–insoluble lipid rafts partially shifted to nonraft fractions. T-cell lines were found to have a high level of cholesterol and a low level of ganglioside GM1, a common marker for lipid rafts, whereas primary T cells have a much lower level of cholesterol and a very high amount of GM1. Cross-linking of LFA-1 on primary T cells induced cocapping of cholesterol but not GM1. These results suggest that lipid rafts of T cells are heterogenous, and LFA-1 associates with a subset of lipid rafts containing a high level of cholesterol. This association seems to regulate LFA-1 functions, possibly by facilitating LFA-1 clustering. (Blood. 2003;102: 215-222)

The ectodomain shedding of angiotensin-converting enzyme is independent of its localisation in lipid rafts.

Angiotensin-converting enzyme (ACE), a type I integral membrane protein that plays a major role in vasoactive peptide metabolism, is shed from the plasma membrane by proteolytic cleavage within the juxtamembrane stalk. To investigate whether this shedding is regulated by lateral segregation in cholesterol-rich lipid rafts, Chinese hamster ovary cells and human neuroblastoma SH-SY5Y cells were transfected with either wild-type ACE (WT-ACE) or a construct with a glycosylphosphatidylinositol (GPI) anchor attachment signal replacing the transmembrane and cytosolic domains (GPI-ACE). In both cell types, GPI-ACE, but not WT-ACE, was sequestered in caveolin or flotillin-enriched lipid rafts and was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C. When cells were treated with activators of the protein kinase C signalling cascade (phorbol myristate acetate or carbachol) the shedding of GPI-ACE was stimulated to a similar extent to that of WT-ACE. The release of WT-ACE and GPI-ACE from the cells was inhibited in an identical manner by a range of hydroxamate-based zinc metalloprotease inhibitors. Disruption of lipid rafts by filipin treatment did not alter the shedding of GPI-ACE, and phorbol ester treatment did not alter the distribution of WT-ACE or GPI-ACE between raft and non-raft membrane compartments. These data clearly show that the protein kinase C-stimulated shedding of ACE does not require the transmembrane or cytosolic regions of the protein, and that sequestration in lipid rafts does not regulate the shedding of the protein.

Membrane Lipid Domains Distinct from Cholesterol/Sphingomyelin-Rich Rafts Are Involved in the ABCA1-mediated Lipid Secretory Pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

PC12 Cells Have Caveolae That Contain TrkA: CAVEOLAE-DISRUPTING DRUGS INHIBIT NERVE GROWTH FACTOR-INDUCED, BUT NOT EPIDERMAL GROWTH FACTOR-INDUCED, MAPK PHOSPHORYLATION

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.

Disruption of Caveolae Results in Attenuated Endothelium Dependent Relaxation

P33 A significant pathology associated with hypertension is endothelial dysfunction caused by reduced availability of endothelial nitric oxide. There are several potential contributing mechanisms to the decreased nitric oxide availability including changes in membrane receptor function, alterations in second messenger systems, or decreases in crucial cofactors associated with enzyme regulation. Recently it has been demonstrated that endothelial nitric oxide synthase is concentrated in the caveolae of endothelial cells, presumably by formation of a heteromeric complex. The current studies hypothesize that disruption of caveolar membranes by the sterol binding agent filipin will result in the attenuation of endothelium dependent vasodilator responses. Aorta from male Sprague-Dawleys rats were removed, cleaned of adhering fat, and cut into rings for the measurement of isometric force development. Rings were incubated in the presence of filipin complex (5 ug/ml) or vehicle for one hour. To determine vasodilation all vessels were precontracted with an EC50 concentration of phenylephrine. Filipin treatment significantly attenuated endothelium dependent relaxation in response to both cholinergic activation with acetylcholine (maximum relaxation, filipin 40±7% vs. vehicle 95±2%; EC50 -5.8±.20 vs. -6.6±.03) and the Ca 2+ ionophore A-23187 (maximum relaxation, filipin 24±5% vs. vehicle 82±3%; EC50 -4.2±.19 vs. -6.5±.09). Maximal relaxation in response to 10 -5 M sodium nitroprusside was unchanged between groups. These results implicate the caveolae in endothelium dependent relaxation and suggest that the damage to the endothelium such as in hypertension may result in diminished caveolar function. This impaired caveolar function may provide a novel mechanism contributing to the reduction in nitric oxide available in hypertension.