Impaired cytoprotective function of muscle in human gallbladders with cholesterol stones

Acute cholecystitis develops in gallbladders (GB) with excessive bile cholesterol (Ch). Increased membrane Ch content affects membrane function and may affect PGE(2) receptors involved in the cytoprotection against acute inflammation. This study was aimed at determining whether the cytoprotective response to PGE(2) is affected by lithogenic bile with Ch. Muscle cells from human GB with cholesterol stones (ChS) or pigment stones (PS) were obtained by enzymatic digestion. PGE(2) levels were measured by radioimmunoassay, and activities of superoxide dismutase (SOD) and catalase were assayed by spectrophotometry. The contraction in response to H(2)O(2) in muscle cells from PS was 14 +/- 0.3%, not different from normal controls, and decreased after the cells were incubated with Ch-rich liposomes (P < 0.05), which increase the Ch content in the plasma membranes. In muscle cells from GB with ChS, H(2)O(2)-induced contraction was only 9.2 +/- 1.3% and increased to 14 +/- 0.2% after Ch-free liposome treatment to remove Ch from the plasma membranes (P < 0.01). H(2)O(2) caused a similar increase in the levels of lipid peroxidation and PGE(2) content in muscle cells from GBs with ChS and PS. However, the activities of SOD and catalase were significantly lower in muscle cells from GBs with ChS compared with those with PS. The binding capacity of PGE(2) receptors was also significantly lower in muscle cells from GBs with ChS compared with those with PS. In conclusion, the cytoprotective response to reactive oxygen species is reduced in muscle cells from GBs with ChS despite a normal increase in the cellular levels of PGE(2). This impaired cytoprotective response may be due to a dysfunction of PGE(2) receptors with decreased binding capacity resulting from excessive Ch levels in the plasma membrane.     

Defect of receptor-G protein coupling in human gallbladder with cholesterol stones

Human gallbladders with cholesterol stones (ChS) exhibit an impaired muscle contraction and relaxation and a lower CCK receptor-binding capacity compared with those with pigment stones (PS). This study was designed to determine whether there is an abnormal receptor-G protein coupling in human gallbladders with ChS using (35)S-labeled guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, (125)I-labeled CCK-8 autoradiography, immunoblotting, and G protein quantitation. CCK and vasoactive intestinal peptide caused significant increases in [(35)S]GTPgammaS binding to Galpha(i-3) and G(s)alpha, respectively. The binding was lower in ChS than in PS (P < 0.01). The reduced [(35)S]GTPgammaS binding in ChS was normalized after the muscles were treated with cholesterol-free liposomes (P < 0.01). Autoradiography and immunoblots showed a decreased optical density (OD) for CCK receptors, an even lower OD value for receptor-G protein coupling, and a higher OD for uncoupled receptors or Galpha(i-3) protein in ChS compared with PS (P < 0.001). G protein quantitation also showed that there were no significant differences in the Galpha(i-3) and G(s)alpha content in ChS and PS. We conclude that, in addition to an impaired CCK receptor-binding capacity, there is a defect in receptor-G protein coupling in muscle cells from gallbladder with ChS. These changes may be normalized after removal of excess cholesterol from the plasma membrane.     

Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors

5-Hydroxytryptamine 2A (5-HT(2A)) serotonin receptors are important for a variety of functions including vascular smooth muscle contraction, platelet aggregation, and the modulation of perception, cognition, and emotion. In a search for 5-HT(2A) receptor-interacting proteins, we discovered that caveolin-1 (Cav-1), a scaffolding protein enriched in caveolae, complexes with 5-HT(2A) receptors in a number of cell types including C6 glioma cells, transfected HEK-293 cells, and rat brain synaptic membrane preparations. To address the functional significance of this interaction, we performed RNA interference-mediated knockdown of Cav-1 in C6 glioma cells, a cell type that endogenously expresses both 5-HT(2A) receptors and Cav-1. We discovered that the in vitro knockdown of Cav-1 in C6 glioma cells nearly abolished 5-HT(2A) receptor-mediated signal transduction as measured by calcium flux assays. RNA interference-mediated knockdown of Cav-1 also greatly attenuated endogenous Galpha(q)-coupled P2Y purinergic receptor-mediated signaling without altering the signaling of PAR-1 thrombin receptors. Cav-1 appeared to modulate 5-HT(2A) signaling by facilitating the interaction of 5-HT(2A) receptors with Galpha(q). These studies provide compelling evidence for a prominent role of Cav-1 in regulating the functional activity of not only 5-HT(2A) serotonin receptors but also selected Galpha(q)-coupled receptors.     

G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes

This study tests the hypothesis that G-protein-coupled receptor (GPCR) signaling components involved in the regulation of adenylyl cyclase (AC) localize with caveolin (Cav), a protein marker for caveolae, in both cell-surface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav-3 in both buoyant membrane fractions (BF) and heavy/non-buoyant fractions (HF); beta2-adrenergic receptors (AR) in BF; and AC5/6, beta1-AR, M4-muscarinic acetylcholine receptors (mAChR), mu-opioid receptors, and Galpha(s) in both BF and HF. In contrast, M2-mAChR, Galpha(i3), and Galpha(i2) were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, Galpha(s), beta2-AR, and mu-opioid receptors in both sarcolemmal and intracellular membranes, whereas M2-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav-3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav-3 co-localized with AC5/6 and Galpha(s) in sarcolemmal and intracellular vesicles, the latter closely allied with T-tubules. Cav-3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist-promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G-proteins, and AC with Cav-3 in both sarcolemmal and intracellular T-tubule-associated regions indicates the existence of multiple Cav-3-localized cellular microdomains for signaling by hormones and drugs in the heart.     

Effect of caveolin-1 scaffolding peptide and 17beta-estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER)alpha or ERbeta. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17beta-estradiol. One important system related to the activity of ERalpha and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82-101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ERalpha and ANG II type 1 and 2 (AT(1) and AT(2)) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca(2+) kinetics and the effect of 17beta-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca(2+) concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca(2+) concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT(2) receptors associate with Cav-1. Our results show that there is a close association of AT(1), AT(2), and ERalpha with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling.     

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca²⁺ sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPS was increased in KO compared with WT as shown using a pull-down assay. Following -toxin permeabilization, no difference in Ca²⁺ sensitivity or in Ca²⁺ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased ₁-adrenergic contraction. Following inhibition of PKC, ₁-adrenergic contraction was normalized. PDBu-induced Ca²⁺ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca²⁺ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca²⁺ sensitivity. Ca²⁺ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor     

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and delta-opioid receptors

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of delta-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 +/- 3 vs. 59 +/- 3% dead cells in the presence and absence of 1 muM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 +/- 4 vs. 62 +/- 3% dead cells, P < 0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.     

Effect of caveolin-1 scaffolding peptide and 17 -estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER) or ERβ. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17β-estradiol. One important system related to the activity of ER and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82–101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ER and ANG II type 1 and 2 (AT₁ and AT₂) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca²⁺ kinetics and the effect of 17β-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca²⁺ concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca²⁺ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT₂ receptors associate with Cav-1. Our results show that there is a close association of AT₁, AT₂, and ER with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling. estrogen receptors; angiotensin type 1 and 2 receptors; phosphatidylinositol 3-kinase; intracellular signaling; tissue culture; angiotensin receptors     

Heterologous desensitization mediated by G protein-specific binding to caveolin

We examined the notion that sequestration of G protein subunits by binding to caveolin impedes G protein reassociation and leads to transient, G protein-specific desensitization of response in dispersed smooth muscle cells. Cholecystokinin octapeptide (CCK-8) and substance P (SP) were used to activate G(q/11), cyclopentyl adenosine (CPA) was used to activate G(i3), and acetylcholine (ACh) was used to activate both G(q/11) and G(i3) via m3 and m2 receptors, respectively. CCK-8 and SP increased only Galpha(q/11), and CPA increased only Galpha(i3) in caveolin immunoprecipitates; caveolin and other G proteins were not increased. ACh increased both Galpha(q/11) and Galpha(i3) in a time- and concentration-dependent fashion: only Galpha(q/11) was increased in the presence of an m2 antagonist, and only Galpha(i3) was increased in the presence of an m3 antagonist. To determine whether transient G protein binding to caveolin affected subsequent responses mediated by the same G protein, PLC-beta activity was measured in cells stimulated sequentially with two different agonists that activate either the same or a different G protein. After treatment of the cells with ACh and an m2 antagonist, the phospholipase C-beta (PLC-beta) response to CCK-8 and SP, but not CPA, was decreased; conversely, after treatment of the cells with ACh and an m3 antagonist, the PLC-beta response to CPA, but not CCK-8 or SP, was decreased. Similarly, after treatment with CCK-8 or SP, the PLC-beta response mediated by G(q/11) only was decreased, whereas after treatment with CPA, the PLC-beta response mediated by G(i3) only was decreased. A caveolin-binding Galpha(q/11) fragment blocked the binding of activated Galpha(q/11) but not Galpha(i3) to caveolin-3 and prevented desensitization of the PLC-beta response mediated only by other G(q/11)-coupled receptors. A caveolin-binding Galpha(i3) fragment had the reverse effect. Thus, transient binding of receptor-activated G protein subunits to caveolin impedes reassociation of the heterotrimeric species and leads to desensitization of response mediated by other receptors coupled to the same G protein.     

Role of PGE2 on gallbladder muscle cytoprotection of guinea pigs

H2O2 and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2 and damage functions of membrane proteins except for PGE2 receptors. The aim of this study was to examine whether the preserved PGE2 actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2 prevented the inhibition of H2O2 or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2 and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2 pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2 and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2 before H2O2 and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2 prevents the muscle cell damage caused by H2O2 and TCDC due to the resistance of PGE2 receptors to agonist-induced desensitization. The preservation of PGE2 receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.     

Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 muM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 muM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 muM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.     

NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1(-/-) mouse ileum

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1(-/-) mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1(-/-) mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1(-/-) mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1-knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1-associated proteins.     

The caveolin proteins

The caveolin gene family has three members in vertebrates: caveolin-1, caveolin-2, and caveolin-3. So far, most caveolin-related research has been conducted in mammals, but the proteins have also been found in other animals, including Xenopus laevis, Fugu rubripes, and Caenorhabditis elegans. Caveolins can serve as protein markers of caveolae ('little caves'), invaginations in the plasma membrane 50-100 nanometers in diameter. Caveolins are found predominantly at the plasma membrane but also in the Golgi, the endoplasmic reticulum, in vesicles, and at cytosolic locations. They are expressed ubiquitously in mammals, but their expression levels vary considerably between tissues. The highest levels of caveolin-1 (also called caveolin, Cav-1 and VIP2I) are found in terminally-differentiated cell types, such as adipocytes, endothelia, smooth muscle cells, and type I pneumocytes. Caveolin-2 (Cav-2) is colocalized and coexpressed with Cav-1 and requires Cav-1 for proper membrane targeting; the Cav-2 gene also maps to the same chromosomal region as Cav-1 (7q31.1 in humans). Caveolin-3 (Cav-3) has greater protein-sequence similarity to Cav-1 than to Cav-2, but it is expressed mainly in muscle cells, including smooth, skeletal, and cardiac myocytes. Caveolins participate in many important cellular processes, including vesicular transport, cholesterol homeostasis, signal transduction, and tumor suppression.     

Caveolins in rhabdomyosarcoma

Caveolins are scaffolding proteins that play a pivotal role in numerous processes, including caveolae biogenesis, vesicular transport, cholesterol homeostasis and regulation of signal transduction. There are three different isoforms (Cav-1, -2 and -3) that form homo- and hetero-aggregates at the plasma membrane and modulate the activity of a number of intracellular binding proteins. Cav-1 and Cav-3, in particular, are respectively expressed in the reserve elements (e.g. satellite cells) and in mature myofibres of skeletal muscle and their expression interplay characterizes the switch from muscle precursors to differentiated elements. Recent findings have shown that caveolins are also expressed in rhabdomyosarcoma, a group of heterogeneous childhood soft-tissue sarcomas in which the cancer cells seem to derive from progenitors that resemble myogenic cells. In this review, we will focus on the role of caveolins in rhabdomyosarcomas and on their potential use as markers of the degree of differentiation in these paediatric tumours. Given that the function of Cav-1 as tumour conditional gene in cancer has been well-established, we will also discuss the relationship between Cav-1 and the progression of rhabdomyosarcoma.     

Caveolin-1 assembles type 1 inositol 1,4,5-trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (I(Cat)) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP(3)R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP(3)R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-β-cyclodextrin (MβCD), which disassembles caveolae, reduced IP(3)-induced I(Cat) activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP(3)-induced vasoconstriction, but did not alter TRPC3 and IP(3)R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82-101) also attenuated IP(3)-induced I(Cat) activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP(3)R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP(3)R1 are spatially co-localized in arterial smooth muscle cells. IP(3)R1 and TRPC3 channel spatial localization was disrupted by MβCD and a CSD peptide. Cholesterol replenishment re-established IP(3)R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP(3)R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP(3)-induced physical coupling of these proteins, leading to I(Cat) generation and vasoconstriction.     

The biology of caveolae: lessons from caveolin knockout mice and implications for human disease

Caveolae, plasma membrane invaginations that serve as membrane organizing centers, are found in most cell types, but are enriched in adipocytes, endothelial cells, and myocytes. Three members of the caveolin family (Cav-1, -2, and -3) are essential for the formation of caveolae. Specialized motifs in the caveolin proteins function to recruit lipids and proteins to caveolae for participation in intracellular trafficking of cellular components and operation in signal transduction. Mutations in the gene encoding CAV-1 are associated with the development and progression of breast cancers, whereas mutations in the CAV-3 gene result in Rippling Muscle Disease and a form of Limb-Girdle Muscular Dystrophy. The generation of caveolin-null mice has confirmed the essential role of these proteins in caveolae biogenesis and in the pathophysiology of diverse tissues. Caveolin-null mice provide new animal models for studying the pathogenesis of a number of human diseases, including cancer, diabetes, atherosclerosis, restrictive lung disease and pulmonary fibrosis, cardiomyopathy, muscular dystrophy, and bladder dysfunction.     

Loss of stromal caveolin-1 expression in malignant melanoma metastases predicts poor survival

Caveolins are the principal protein component of caveolae, plasma membrane invaginations found in most cell types. Caveolin-1 (Cav-1) plays a major role in oncogenesis through its various functions in lipid transport, membrane trafficking, and signal transduction. Increased expression of Cav-1 in tumor cells has been associated with aggressiveness and poor survival. More recently, loss of stromal Cav-1 expression was linked to poor survival and increased metastatic potential in breast and prostate cancer. To date, there is no study addressing the clinical significance of Cav-1 expression in malignant melanoma (MM). Our study consisted of 44 cases of MM: 12 MM lymph node metastases from patients with short survival, 12 MM lymph node metastases from patients with long survival and 20 primary MM. All cases were stained with Cav-1 antibodies. Cav-1 expression in melanoma and stromal cells was quantified using a 3 point scale: 0=no staining, 1=diffuse weak staining or strong staining in     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

Glut-4 is translocated to both caveolae and non-caveolar lipid rafts, but is partially internalized through caveolae in insulin-stimulated adipocytes

Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.