Caveolar Membrane Research Articles

The modulation of caveolin‐1 expression controls satellite cell activation during muscle repair

We have previously shown that caveolin-1, the principal structural protein component of caveolar membrane domains, inhibits cellular proliferation and induces cell cycle arrest. We demonstrate here for the first time that caveolin-1 is expressed in satellite cells but not in mature muscle fibers. Satellite cells are quiescent myogenic precursors that, after muscle injury, become mitotically active, proliferate, and fuse together or, to existing myofibers, to form new muscle fibers. We show that down-regulation of caveolin-1 expression occurs in satellite cells/myogenic precursor cells (MPCs) during muscle regeneration and that hepatocyte growth factor, which is produced after muscle injury, down-regulates caveolin-1. We also demonstrate that down-regulation of endogenous caveolin-1 expression activates ERK and that activation of the p42/44 MAP kinase pathway is necessary to promote muscle regeneration. Finally, we show that overexpression of caveolin-1 inhibits muscle repair mechanisms both in vitro and in vivo. Taken together, these results propose caveolin-1 as a novel regulator of satellite cell functions and suggest that the following signaling pathway modulates satellite cell activation during muscle repair: injured fibers release HGF --> HGF down-regulates caveolin-1 protein expression --> down-regulation of caveolin-1 activates ERK --> activation of ERK promotes muscle repair by stimulating the proliferation and migration of MPCs toward the wounded area.

Colocalization of eNOS and the catalytic subunit of PKA in endothelial cell junctions: a clue for regulated NO production.

Localization and coordinate phosphorylation/dephosphorylation of endothelial nitric oxide synthase (eNOS) are critical determinants for the basal and stimulated production of nitric oxide. Several phosphorylation sites in eNOS have been identified as targets of the cAMP-dependent protein kinase A (PKA). Basal eNOS activity is also regulated by interaction with caveolin-1, the major coat protein of caveolae. In the present study we have examined in rat aorta endothelium the subcellular steady-state distribution of eNOS, the catalytic subunit of PKA (PKA-c), and caveolin-1. Basal eNOS expression was found in two distinct locations, the endothelial cell surface and the Golgi complex. Cell surface eNOS was equally distributed over caveolar and non-caveolar membranes but was 2.5-fold enriched on luminal lamellipodia located at endothelial cell contacts. PKA-c colocalized with eNOS in the lamellipodia, whereas caveolin-1 was absent from these membrane domains. PKA-c was also found associated with cell surface caveolae and with tubulovesicular membranes of Golgi complex and endosomes. The topological proximity of eNOS with the catalytic subunit of PKA in restricted intracellular locations may provide mechanisms for differential PKA-mediated eNOS regulation.

Glycosynapses: microdomains controlling carbohydrate-dependent cell adhesion and signaling.

The concept of microdomains in plasma membranes was developed over two decades, following observation of polarity of membrane based on clustering of specific membrane components. Microdomains involved in carbohydrate-dependent cell adhesion with concurrent signal transduction that affect cellular phenotype are termed "glycosynapse". Three types of glycosynapse have been distinguished: "type 1" having glycosphingolipid associated with signal transducers (small G-proteins, cSrc, Src family kinases) and proteolipids; "type 2" having O-linked mucin-type glycoprotein associated with Src family kinases; and "type 3" having N-linked integrin receptor complexed with tetraspanin and ganglioside. Different cell types are characterized by presence of specific types of glycosynapse or their combinations, whose adhesion induces signal transduction to either facilitate or inhibit signaling. E.g., signaling through type 3 glycosynapse inhibits cell motility and differentiation. Glycosynapses are distinct from classically-known microdomains termed "caveolae", "caveolar membrane", or more recently "lipid raft", which are not involved in carbohydrate-dependent cell adhesion. Type 1 and type 3 glycosynapses are resistant to cholesterol-binding reagents, whereas structure and function of "caveolar membrane" or "lipid raft" are disrupted by these reagents. Various data indicate a functional role of glycosynapses during differentiation, development, and oncogenic transformation.

Caveolin-1 expression is critical for vascular endothelial growth factor-induced ischemic hindlimb collateralization and nitric oxide-mediated angiogenesis.

Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor L-NAME administered to operated Cav+/+ mice. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav+/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology.

A Pump and a Signaling Receptor All in One

Evidence is mounting that the Na + ,K + -ATPase is not just a regulator of ionic balance, but also a signal-transducing receptor for such ligands as ouabain, which were originally identified as inhibitors of the ion pump activity of Na + ,K + -ATPase. Wang et al. revisited the question of whether Na + ,K + -ATPase was located in caveolar membranes by using a detergent-free isolation method and found that Na + ,K + -ATPase, caveolin-1, and the nonreceptor tyrosine kinase Src were abundant in these membrane microdomains from LLC-PK1 cells. Na + ,K + -ATPase interacted with caveolin-1 in glutathione S -transferase pull-down experiments. Ouabain treatment of LLC-PK1 cells stimulated the association of Na + ,K + -ATPase and caveolin-1 based on coimmunoprecipitation. In the response to ouabain, a Na + ,K + -ATPase-caveolin-1-Src complex was detected by coimmunoprecipitation, and the tyrosine phosphorylation of caveolin-1, along with that of multiple other proteins, was stimulated in isolated caveolar membrane fractions. Exposure of cells to cholesterol-depleting agents promoted the redistribution of Na + ,K + -ATPase and Src out of caveolin-1-positive membranes. Cholesterol depletion or depletion of caveolin-1 by RNA interference inhibited ouabain-stimulated increase in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Thus, Na + ,K + -ATPase appears to serve a dual role as an ion pump and a signaling receptor, with ouabain both inhibiting ion pumping activity and stimulating downstream signaling cascades mediated by protein kinases. H. Wang, M. Haas, M. Liang, T. Cai, J. Tian, S. Li, Z. Xie, Ouabain assembles signaling cascades through the caveolar Na + /K + -ATPase. J. Biol. Chem. 279 , 17250-17259 (2004). [Abstract] [Full Text]

Contribution of cholesterol and phospholipids to inhibitory effect of dimethyl-beta-cyclodextrin on efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in vinblastine-resistant Caco-2 cell monolayers.

The purpose of this study is to reveal the contribution of membrane components to the inhibitory effect of 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) function in vinblastine-resistant Caco-2 (Caco-2R) cell monolayers. The transport of rhodamine-123 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was studied in Caco-2R cell monolayers. P-gp and MRP2 residing in the monolayers and releasing in cell supernatants were detected by Western blotting. The mRNA levels of MDR1 and MRP2 were detected by reverse transcription-polymerase chain reaction (RT-PCR) method. Cholesterol, phospholipids, and proteins were mainly determined by each assay kit. Of various beta-cyclodextrin derivatives (beta-CyDs), DM-beta-CyD most significantly impaired the efflux function of P-gp and MRP2 without changing cell viability and membrane integrity. The treatment with CyDs did not change the mRNA levels of MDR1 and MRP2. DM-beta-CyD lowered cholesterol content and P-gp level in caveolar membranes. In addition, DM-beta-CyD released not only cholesterol and phospholipids but also proteins including P-gp and MRP2 from apical membranes of the monolayers. DM-beta-CyD may impair P-gp and MRP2 function in Caco-2R cell monolayers, probably, at least in part, through the release of these transporters from the apical membranes of monolayers, and the exertion of the inhibitory effect of DM-beta-CyD may require the extraction of not only cholesterol but also phospholipids from the monolayers.

Co-secretion of soluble caveolin-1 and pepsinogen in the oesophagus of the red-legged frog Rana aurora aurora

Caveolin-1, an integral membrane protein, is the principal component of caveolae, which are specialised vesicular microdomains of the plasma membrane. Caveolae are found in most cell types, but they are most abundant in adipocytes, endothelial cells, fibroblasts, and muscle cells. Functionally, they have been implicated in endothelial transcytosis, potocytosis, and signal transduction. Recently, caveolin-1 has been found unexpectedly in the cytoplasm, mitochondria and elements of the secretory pathways of exocrine secretory cells. We have co-localised caveolin-1 and pepsinogen immunohistochemically in serous cells of oesophageal glands of the red-legged frog, Rana aurora aurora. Thus, according to its intracellular localisation pattern, caveolin-1 may be either a soluble protein, located in secretory droplets, or a protein that is inserted in caveolar membranes. Soluble caveolin-1, which is probably embedded in a lipid particle surrounded by a phospholipid shell, may be involved in intracellular and extracellular lipid transport. In the gut, caveolin-1-rich lipid particles can act as donor particles to facilitate (protein-mediated) intestinal uptake of cholesterol and phospholipids. Our findings strengthen the hypothesis that caveolin-1 has a physiological autocrine/paracrine function and demonstrate that secretion of this protein also occurs in vertebrates other than mammals, such as amphibians, which may be a useful alternative animal model to study caveolin-1.

Phosphofructokinase Muscle-Specific Isoform Requires Caveolin-3 Expression for Plasma Membrane Recruitment and Caveolar Targeting: Implications for the Pathogenesis of Caveolin-Related Muscle Diseases

Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [DeltaTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(-/-) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene.

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.

Free Radical Biology and Medicine

Nitric oxide and various by-products including nitrite contribute to tissue injury by forming novel intermediates via redox-mediated nitration reactions. Nitration of unsaturated fatty acids generates electrophilic nitrofatty acids such as 9-nitrooleic acid (9-NO) and 10-nitrooleic acid (10-NO), which are known to initiate intracellular signaling pathways. In these studies, we characterized nitrofatty acid- induced signaling and stress protein expression in mouse keratinocytes. Treatment of keratinocytes with 5-25 μM 9-NO or 10-NO for 6 h upregulated mRNA expression of heat shock proteins (hsp's) 27 and 70; primary antioxidants heme oxygenase-1 (HO-1) and catalase; secondary antioxidants glutathione S-transferase (GST) A1/2, GSTA3, and GSTA4; and Cox-2, a key enzyme in prostaglandin biosynthesis. The greatest responses were evident with HO-1, hsp27, and hsp70. In keratinocytes, 9-NO activated JNK and p38 MAP kinases. JNK inhibition suppressed 9-NO-induced HO-1, hsp27, and hsp70 mRNA and protein expression, whereas p38 MAP kinase inhibition suppressed HO-1. In contrast, inhibition of constitutive expression of Erk1/2 suppressed only hsp70, indicating that 9-NO modulates expression of stress proteins by distinct mechanisms. 9-NO and 10-NO also upregulated expression of caveolin-1, the major structural component of caveolae. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation revealed that HO-1, hsp27, and hsp70 were localized within caveolae after nitrofatty acid treatment of keratinocytes, suggesting a link between induction of stress response proteins and caveolin-1 expression. These data indicate that nitrofatty acids are effective signaling molecules in keratinocytes. Moreover, caveolae seem to be important in the localization of stress proteins in response to nitrofatty acids.

Caveolin-1 regulates contractility in differentiated vascular smooth muscle.

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.

Transgenic overexpression of caveolin-3 in the heart induces a cardiomyopathic phenotype.

Caveolins are structural protein components of caveolar membrane domains. Caveolin-3, a muscle-specific member of the caveolin family, is expressed in skeletal muscle tissue and in the heart. The multiple roles that caveolin-3 plays in cellular physiology are becoming more apparent. We have shown that lack of caveolin-3 expression in skeletal muscle resembles limb-girdle muscular dystrophy-1C. In contrast, we have demonstrated that overexpression of caveolin-3 in skeletal muscle tissue promotes defects similar to those seen in Duchenne muscular dystrophy (DMD). Thus, a tight regulation of caveolin-3 expression is fundamental for normal muscle functions. Since caveolin-3 is also endogenously expressed in cardiac myocytes, and cardiomyopathies are observed in DMD patients, we looked at the effects of overexpression of caveolin-3 on cardiac structure and function by characterizing caveolin-3 transgenic mice. Our results indicate that overexpression of caveolin-3 causes severe cardiac tissue degeneration, fibrosis and a reduction in cardiac functions. We also show that dystrophin and its associated glycoproteins are down-regulated in caveolin-3 transgenic heart. In addition, we demonstrate that the activity of nitric oxide synthase (NOS) is down-regulated by high levels of caveolin-3 in the heart. Taken together, these results indicate that overexpression of caveolin-3 is sufficient to induce severe cardiomyopathy. In addition, these findings suggest that caveolin-3 transgenic mice may represent a valid mouse model for studying the molecular mechanisms underlying cardiomyopathies associated with Duchenne muscular dystrophy.

Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton.

Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 microm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call 'cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.

Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae.

Caveolin-2 is a member of the caveolin gene family with no known function. Although caveolin-2 is coexpressed and heterooligomerizes with caveolin-1 in many cell types (most notably adipocytes and endothelial cells), caveolin-2 has traditionally been considered the dispensable structural partner of the widely studied caveolin-1. We now directly address the functional significance of caveolin-2 by genetically targeting the caveolin-2 locus (Cav-2) in mice. In the absence of caveolin-2 protein expression, caveolae still form and caveolin-1 maintains its localization in plasma membrane caveolae, although in certain tissues caveolin-1 is partially destabilized and shows modestly diminished protein levels. Despite an intact caveolar membrane system, the Cav-2-null lung parenchyma shows hypercellularity, with thickened alveolar septa and an increase in the number of endothelial cells. As a result of these pathological changes, these Cav-2-null mice are markedly exercise intolerant. Interestingly, these Cav-2-null phenotypes are identical to the ones we and others have recently reported for Cav-1-null mice. As caveolin-2 expression is also severely reduced in Cav-1-null mice, we conclude that caveolin-2 deficiency is the clear culprit in this lung disorder. Our analysis of several different phenotypes observed in caveolin-1-deficient mice (i.e., abnormal vascular responses and altered lipid homeostasis) reveals that Cav-2-null mice do not show any of these other phenotypes, indicating a selective role for caveolin-2 in lung function. Taken together, our data show for the first time a specific role for caveolin-2 in mammalian physiology independent of caveolin-1.

Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude.

This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the alpha subunit of the stimulatory heterotrimeric G-protein, Galpha(s), via stimulation of the cell surface beta-adrenergic receptor. Stimulation of beta-adrenergic receptors with 10 micromol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of beta-adrenergic receptors, and thereby Galpha(s), promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.

Impairment of Caveolae Formation and T-System Disorganization in Human Muscular Dystrophy with Caveolin-3 Deficiency

Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle.

Membrane Microdomains Defining Cell Adhesion and Signaling

Following a retrospective view on development of the present concept of microdomain, current views of multiple microdomains are briefly presented. Based on physical properties of membrane components, some lipids are clustered, segregated from other lipids, and organized with cytoplasmic lipophilic signal transducer molecules and with transmembrane receptors with hydrophobic domain, and their associated partners. Typical examples are: (i) glycosphingolipids (GSLs) and sphingomyelin organized with cSrc, Src family kinases, and small G-proteins, collectively termed “glycosignaling domain (GSD)” and involved in GSL-dependent cell adhesion and signaling; (ii) caveolae and caveolar membrane, characterized by high content of cholesterol and caveolin, organized with GPI-anchors and signal transducers, and involved in endocytosis and signal transduction; (iii) integrins and tetraspan membrane proteins (TMP) complexed with some type of GSL; (iv) growth factor receptors with tyrosine kinases complexed with cholesterol-enriched domain, with or without some type of GSL. The defined composition and association of signal transducer molecules in microdomain iii and iv is yet unclear. Relative quantity and proportion of these types of microdomain differs and is characteristic for different types of cells.

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.

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.

Pre-paired Receptors

A fundamental biological question that remains largely unresolved concerns the mechanism by which binding of ligands to receptors on the cell surface causes transmission of a signal through the plasma membrane. One appealing explanation has been that ligand binding brings receptors together into multimeric complexes. Three reports describe cases in which the opposite approach is taken, and the receptors are bound and lie in wait for the ligand. Siegel et al . and Chan et al . have examined how the Fas and tumor necrosis factor (TNF) receptors signal. They define a protein interaction domain in these receptors that mediates assembly of the receptors into complexes in the absence of ligand. Such association is shown to be necessary for ligand binding and subsequent signaling. The results also explain how abnormal forms of Fas can dominantly prevent Fas-induced signaling in the human disease known as autoimmune lymphoproliferative syndrome. When bound to their cognate receptors, interferons (IFNs) induce cellular resistance to viral infection. Takaoka et al . show that certain cells, if they are to maximize this response when exposed to the cytokine IFN-γ, must express not only a functional IFN-γ receptor but a functional IFN-α/β receptor as well. Before these cells are even stimulated with IFN-γ, the IFN-γ receptor is preassociated with the IFN-α/β receptor. The IFN-α/β receptor brings a transcription activator to the complex that is poised to become activated upon stimulation with IFN-γ. Cross talk between two IFN receptor subtypes appears to increase the efficiency and strength of a cell's antiviral response to IFN-γ. A Perspective by Golstein discusses pre-clustering of receptors before activation. Siegel, R.M., Frederiksen, J.K., Zacharias, D.A.,Chan, F.K.-M., Johnson, M., Lynch, D., Tsien, R.Y., and Lenardo, M.J. (2000) Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288 : 2354-2357. [Abstract] [Full Text] Chan, F.K.-M., Chun, H.J., Zheng, L., Siegel, R.M., Bui, K.L., and Lenardo, M.J. (2000) A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 288 : 2351-2354. [Abstract] [Full Text] Takaoka, A., Mitani, Y., Suemori, H., Sato, M., Yokochi, T., Noguchi, S., Tanaka, N., and Taniguchi, T. (2000) Cross talk between interferon-γ and -α/β signaling components in caveolar membrane domains. Science 288 : 2357-2360. [Abstract] [Full Text] Golstein, P. (2000) Signal Transduction: FasL binds preassembled Fas. Science 288 : 2328-2329. [Full Text]