Cholesterol Depletion Disrupts Caveolae and Differentially Impairs Agonist-Induced Arterial Contraction
Objective— This study assessed the role of cholesterol-rich membrane regions, including caveolae, in the regulation of arterial contractility.
Methods and Results— Rat tail artery devoid of endothelium was treated with the cholesterol acceptor methyl-β-cyclodextrin, and the effects on force and Ca2+ handling were evaluated. In cholesterol-depleted preparations, the force responses to α1-adrenergic receptors, membrane depolarization, inhibition of myosin light chain phosphatase, and activation of G proteins with a mixture of 20 mmol/L NaF and 60 μmol/L AlCl3 were unaffected. In contrast, responses to 5-hydroxytryptamine (5-HT), vasopressin, and endothelin were reduced by >50%. The rise in global intracellular free Ca2+ concentration in response to 5-HT was attenuated, as was the generation of Ca2+ waves at the cellular level. By electron microscopy, cholesterol depletion was found to disrupt caveolae. The 5-HT response could be restored by exogenous cholesterol, which also restored caveolae. Western blots showed that the levels of 5-HT2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT2A receptors, but not α1-adrenergic receptors, in the caveolin-1–containing fractions, suggesting localization of the former to caveolae.
Conclusions— These results show that a subset of signaling pathways that regulate smooth muscle contraction depends specifically on cholesterol. Furthermore, the cholesterol-dependent step in serotonergic signaling occurs early in the pathway and depends on the integrity of caveolae.
Cellular cholesterol, of which most (up to 90%)1 resides in the plasma membrane, is crucial for normal membrane permeability and fluidity and also plays a role in cellular signaling, via several proposed mechanisms that fall into at least 4 categories. First, cellular cholesterol may influence gene transcription in the nucleus through sterol regulatory element binding proteins.2 Second, the activity of membrane receptors, ion channels, and transporters may depend on the membrane fluidity, per se.3 Third, membrane protein function may be regulated through specific cholesterol-protein interactions.3,4⇓ Fourth, cholesterol stabilizes the structure of caveolae and lipid rafts.
Caveolae, which are 50- to 100-nm membrane invaginations that are abundant in vascular endothelium and smooth muscle cells, are defined by their characteristic morphology and contents of caveolin and cav-p60.5,6⇓ No definitive definition of rafts has appeared because they do not exhibit a characteristic structure, but the term is used for planar aggregations of specific lipids and proteins. Caveolae and lipid rafts are envisaged to serve as platforms for a dynamic association of signaling proteins and for the initiation or modulation of signaling.5,7,8⇓⇓
Some agonists causing contraction of vascular smooth muscle act on receptors that are believed to be located in caveolae (eg, endothelin A receptors)9 or to aggregate in caveolae on ligand binding (eg, angiotensin type 1 or bradykinin B2 receptors).10,11⇓ Cholesterol-rich membrane domains may be considered as possible physical platforms for the coding of intracellular signals. To investigate this, intracellular Ca2+ and contractile responses to agonist stimulation were studied in arterial muscle after depletion of cholesterol by methyl-β-cyclodextrin (mβcd).12 This disrupts caveolar structure, whereas the general morphology of the tissue is preserved. Because the present study was performed on whole tissue, the contractile phenotype and cell-surface contacts were preserved; thus, contractile responses and normal ion homeostasis were maintained. The results suggest that although cholesterol concentration and integrity of caveolae are not critical for contraction or responses to α1-adrenergic agonists, they profoundly influence a specific set of signaling pathways, including those for serotonin, vasopressin, and endothelin.
Preparation of the Tail Artery
Female Sprague-Dawley rats were euthanized by cervical dislocation, as approved by the regional ethics committee. The tail artery was dissected free in nominally Ca2+-free HEPES-buffered Krebs solution (135.5 mmol/L NaCl, 5.9 mmol/L KCl, 1.2 mmol/L MgCl2, 11.6 mmol/L glucose, and 11.6 mmol/L HEPES, pH 7.35) and slid over an insect needle to remove the endothelium. Endothelial denudation by this procedure was verified by confocal and electron microscopy. Additionally, in toluidine blue–stained paraffin sections examined by light microscopy, the endothelium appeared to be absent (data not shown).
Ring segments (1 mm) were mounted for force registration and equilibrated as described.13 Preparations were contracted with agonist for 7 minutes, interspersed by 25-minute relaxation periods, until stable contractions were attained. They were then treated with 10 mmol/L mβcd for 1 hour at 37°C. Extraction was temperature sensitive, and incubations at room temperature were completely ineffective. Longer treatment (3 hours) at 37°C reduced high K+ responses and appeared to affect membrane integrity, as judged by increased leakage of fura 2 and enhanced basal Mn2+ quench rate (data not shown). Additionally, all caveolae had disappeared, and in approximately one third of the cell profiles, signs of cellular degeneration had appeared, including swollen mitochondria and membrane-bound inclusions in the cytoplasm and the nucleus.
Fixation and Microscopy
For electron microscopy, arterial segments were fixed in sodium cacodylate (100 mmol/L), sucrose (170 mmol/L), CaCl2 (1.5 mmol/L), and glutaraldehyde (2%), with pH adjusted to 7.4. After 1 to 2 hours, the preparations were transferred to the same solution devoid of glutaraldehyde and stored at 4°C. The arteries were postfixed in 2% osmium (in 0.1 mol/L phosphate) for 12 hours, dehydrated, and embedded in epoxy resin (Epon) as described.14 Sections (50 nm) were contrasted by lead citrate and bismuth subnitrate. Images were obtained with a Philips 210 transmission electron microscope.
Segments for immunofluorescence microscopy were fixed in 96% ethanol, embedded in paraffin, and sectioned for indirect immunofluorescence. Control sections omitting the primary antibody were devoid of specific fluorescence signal.15
Segments (2 mm) of tail artery were mounted and incubated with fura 2-AM as described.13 They were then incubated for 1 hour with or without 10 mmol/L mβcd at 37°C in HEPES-buffered Krebs solution. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by epifluorescence at 37°C.13 For subcellular [Ca2+]i measurements, segments were incubated with fluo 4-AM for 90 minutes after exposure to mβcd or control solution for 1 hour. Ca2+ wave activity was measured by using a Zeiss 510 laser scanning confocal microscope.16
Proteins were extracted and separated on 12% SDS-PAGE gels, and Western blotting on nitrocellulose membranes was then performed essentially as described,16 with the use of antibodies to α1A-adrenergic receptor (1:50), caveolin-1 (6B6, 1 μg/mL), 5-hydroxytryptamine (5-HT)2A receptor (1:500), or Cav-p60 (1 μg/mL). Peroxidase-conjugated secondary antibodies were used, and peroxidase activity was detected by chemiluminescence (Pierce).
Preparation of Caveolin-Enriched Fractions
Three to 5 freshly dissected tail arteries were transferred to 1 mL of 500 mmol/L sodium carbonate, at pH 11.0, cut manually, homogenized with a Polytron (Janke & Kunkel) tissue grinder (three 10-second bursts), and sonicated (three 20-second bursts). Five percent to 45% discontinuous sucrose gradient centrifugation was performed as described by Song et al.17 Ten fractions were recovered, and the 5%-35% interface was expected in fraction 5, which also contained the majority of caveolin-1.
Chemicals and Reagents
The primary antibodies 6B6 and 2F11 are protein-G–purified mouse monoclonal antibodies (Mabs) against caveolin-1 and cav-p60.6 Anti–caveolin-1 polyclonal antibody (catalog No. c13630), anti–caveolin-2 Mab (clone 65, catalog No. C57820), and anti–caveolin-3 Mab (clone 26, catalog No. C38320) were all from Transduction Laboratories. FITC-conjugated secondary antibodies were from DAKO A/S (catalog No. F205) and Jackson ImmunoResearch Laboratories Inc (No. 115-095-146). The 5-HT2A receptor antibody was a kind gift from Dr Bryan Roth (Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio). Anti–α1A-adrenergic receptor (No. sc-1475, 1:50) and the secondary peroxidase-conjugated mouse anti-goat antibody (1:10 000) were obtained from Santa Cruz. AlF4− (30 μmol/L) was obtained by mixing 20 mmol/L NaF with 60 μmol/L AlCl3. Cirazoline hydrochloride and calyculin A were from ICN. Fura 2-AM and fluo 4-AM were from Molecular Probes. All other reagents were from Sigma Chemical Co.
After equilibration, segmented arteries were incubated with or without 10 mmol/L mβcd for 1 hour at 37°C. Pelleted segments were frozen in liquid nitrogen. Freeze-dried pellets were weighed, and total cholesterol was measured fluorometrically.18
Summarized data are presented as mean±SEM. The Student t test or Wilcoxon matched pairs test was used for statistical comparisons. A value of P<0.05 was considered significant; n denotes the number of preparations. A minimum of 3 different animals was used in all sets of experiments.
Endothelium-denuded rings of rat tail artery were contracted with high K+ (60 mmol/L), the α1-adrenergic–selective agonist cirazoline19 (0.3 μmol/L), 5-HT (1 μmol/L), or arginine vasopressin (AVP, 0.1 μmol/L). These responses, taken as control (Figure 1), were 90% to 100% of those to a maximally active concentration (Emax) of the respective agonist and were stationary for 3 hours (data not shown). After relaxation (25 minutes), preparations were treated for 1 hour with mβcd (10 mmol/L), which reduced their cholesterol contents by 21% (0.56±0.024 versus 0.71±0.026 [control] μg/mg dry tissue, n=12, P<0.001). Contraction was thereafter elicited again (Figure 1A and 1B). Contractions by endothelin-1 (10 nmol/L, giving ≈50% of Emax response), AlF4− (30 μmol/L, giving ≈90% of Emax), or the myosin phosphatase inhibitor calyculin A (3 μmol/L, giving ≈100% of Emax) were poorly reversible; thus, force after cholesterol extraction was compared with controls run in parallel. Summarized data from at least 8 individual experiments (Figure 1C) show that mβcd treatment selectively reduces 5-HT, AVP, and endothelin-1 responses.
Cumulative dose-response relations were determined in mβcd-treated and control preparations. For 5-HT, the dose-response relation was shifted ≈10-fold to the right after extraction of cholesterol, and force at saturating concentrations of 5-HT was reduced by 50% (please see online Figure IA, available at http://atvb.ahajournals.org). Neither of these effects was observed with the α1-agonist or with AlF4−. Responsiveness to 5-HT was restored after cholesterol had been added back in complex with mβcd (please see online Figure IB). This recovery was not spontaneous, inasmuch as extracted preparations run in parallel but not receiving cholesterol continued to weaken (please see online Figure IC). After recovery, force was maintained for at least 1 further contraction without continuing incubation with cholesterol.
[Ca2+]i was determined by the fura 2 technique (please see online Figure IIA, available at http://atvb.ahajournals.org). Basal [Ca2+]i was significantly increased in cholesterol-depleted preparations (121±18 [mβcd] versus 37±5 [control] nmol/L, n=14, P<0.001). This was paralleled by an increased Ca2+-dependent basal tone (7.1±1.1% [mβcd] versus 0.5±0.5% [control] at 1 hour, n=34), which was insensitive to inhibitors of L-type Ca2+ channels (1 or 10 μmol/L verapamil), store-operated Ca2+ channels (10 μmol/L SKF 96365 and 1 mmol/L Ni+), and nonselective cation channels (1 μmol/L LOE 908). Furthermore, Ca2+ extrusion mechanisms were apparently not affected by the treatment, because the rate of decline in [Ca2+]i on repolarization from high K+ in Ca2+-free solution was equal in mβcd-treated and control arteries (half-time 28.5±1.2 versus 23.5±2.6 seconds, n=10).
Average [Ca2+]i during depolarization with high K+ (328±21 [mβcd] versus 267±19 [control] nmol/L, n=12, P=NS) or 0.3 μmol/L cirazoline (212±25 [mβcd] versus 219±30 [control] nmol/L, n=4, P=NS) was not affected by cholesterol depletion, whereas the response to 1 μmol/L 5-HT was drastically reduced (175±17 [mβcd] versus 333±40 [control] nmol/L, n=10, P<0.01; please see online Figure IIA). Contractile responses to 5-HT are critically dependent on extracellular Ca2+ and L-type voltage-activated Ca2+ channels, inasmuch as verapamil (1 μmol/L) completely blocks 5-HT–induced force (not shown). Experiments in Ca2+-free extracellular solution (0.5 mmol/L EGTA) were used to examine the effect of cholesterol depletion on Ca2+ release from intracellular stores. After depletion, the Ca2+-release response with α1-adrenergic stimulation (3 μmol/L cirazoline) increased (expressed as increase over baseline, from 48±8 to 129±3 nmol/L, n=4, P<0.05). Similarly, the increase in [Ca2+]i induced by caffeine (20 mmol/L) was significantly larger in depleted preparations (210±47 versus 47±2 nmol/L, n=4, P<0.05). These data suggest a higher Ca2+ loading of the sarcoplasmic reticulum (SR) in cholesterol-depleted preparations. Interestingly, Ca2+ release by 5-HT (10 μmol/L) was unchanged after cholesterol extraction (68±12 versus 60±9 nmol/L, n=9). To exclude the possibility that an increase in stored Ca2+ would mask an effect of mβcd on α1-adrenergic responses, tissues were treated with thapsigargin and caffeine to deplete Ca2+ from intracellular stores. This did not reveal any difference (control versus mβcd treatment) in α1-adrenergic responses in the presence of extracellular Ca2+ (n=8, data not shown).
Individual smooth muscle cells in the arterial wall display asynchronous Ca2+ oscillations on agonist stimulation, which allow for spatial and temporal coding of [Ca2+]i and most likely contribute to the control of force.20,21⇓ Their frequency and amplitude, together with the fraction of oscillating cells and interwave [Ca2+]i, form the basis for the global [Ca2+]i signal measured over the vascular wall. Ca2+ wave activity was determined by use of confocal laser scanning microscopy of fluo 4–loaded mβcd-treated and control arteries. The number of cells exhibiting waves during stimulation with either cirazoline (0.1 μmol/L) or 5-HT (0.3 μmol/L) was little affected by mβcd treatment, whereas the mean wave frequency in treated vessels was 37% lower during 5-HT stimulation and marginally (14%) lower during activation with cirazoline (please see online Figure IIB and IIC). The wave amplitude was decreased by 23% with 5-HT, whereas no effect was observed with cirazoline. This suggests that the capacity for Ca2+ wave generation is largely maintained after mβcd treatment and that the decrease in activity during 5-HT stimulation is due to an upstream defect.
Light microscopic images of the tail artery appeared normal; this was also the case after force registration and cholesterol extraction (not shown). Immunofluorescence signals from 2 caveola-specific proteins, caveolin-1 and cav-p60,15 were found along the smooth muscle cell surface (please see online Figure IIIA and IIIC, available at http://atvb.ahajournals.org). Distribution of caveolin-2 was similar, but labeling was weaker. Caveolin-3 was not detected. Cholesterol extraction had no detectable effect on the distribution of any of these proteins (please see online Figure IIIB and IIID).
By electron microscopy, we ensured that no tissue damage was induced during force registration, apart from the luminal surface where the endothelium had been removed (not shown). In the nondepleted preparations, the smooth muscle plasma membrane contained numerous, often clustered, caveolae (Figure 2A). Cholesterol depletion (1 hour) caused an overall change in the appearance of caveolae. After depletion, the individual caveolae had either disappeared (had been flattened) or were shallow with wide openings (Figure 2B and 2C). Furthermore, the Golgi complex appeared disorganized but was the only other organelle affected. Traditional signs of tissue damage were not induced by the limited 1-hour cholesterol extraction. After cholesterol reloading of the depleted preparations, the number and morphology of caveolae appeared similar to that of muscle cells in control preparations (Figure 2D).
The levels of the 5-HT2A receptor and caveolin-1 were found to be unaffected by cholesterol extraction in Western blot experiments (Figure 3A and 3B). In silver-stained gels, the protein pattern of depleted and control preparations appeared to be similar (not shown). By subcellular fractionation on a sucrose density gradient, we found that the 5-HT2A receptor cofractionated with caveolin-1 and Cav-p60 in fractions 5 and 6 (Figure 3C). On the other hand, the α1A-adrenergic receptor was present in fractions 9 and 10 (Figure 3C).
A large number of signaling molecules are copurified with caveolin in subcellular fractions prepared on sucrose gradients, suggesting that caveolae are involved in cellular signaling.7,8,22⇓⇓ Additional data, such as coimmunoprecipitation or double immunolabeling with caveolin, have firmly established the presence of several signaling proteins in caveolae. Those of relevance for vascular physiology include endothelial NO synthase, Ca2+-ATPase, the endothelin receptor, and the platelet-derived growth factor receptor.22 Impaired endothelin-induced vascular contractility in caveolin-1 knockout mice23 provides further independent support for a role of caveolae in G-protein–coupled receptor signaling. In the present study, we demonstrate that cholesterol extraction impairs the function of 3 receptors, including that for endothelin. Because caveolin-1 contents and localization appeared normal, these results imply that receptor functionality does not depend on caveolin-1, per se, but rather on a unique membrane organization governed by the interaction between cholesterol and other constituents of the membrane, such as caveolin-1. Although a rigorous morphometric quantification was not performed, the morphology of caveolae was clearly and reversibly altered by cholesterol extraction, with different degrees of flattening, indicating disruption of the caveolar structure. Although the present study did not distinguish the role of caveolae from that of other cholesterol-dependent structures in the plasma membrane, such as rafts, the combination of the present findings with those from caveolin-1–deficient mice suggests a causal relationship between caveolar structure and endothelin responses.
The contractile responses to endothelin-1, 5-HT, and vasopressin in the rat tail artery are mediated by ETA, 5-HT2A, and V1 receptors, respectively.24–26⇓⇓ The ETA receptor was reported to be localized in caveolae,9 but the ultrastructural localization of 5-HT2A or V1 receptors has, to our knowledge, not been described in caveolin-1–expressing cells. However, the membrane localization and dynamics of internalization of the 5-HT2A receptor was elegantly visualized in HEK-293 cells, which express little caveolin-1.27 Thus, caveolae are not required for membrane targeting of the receptor, whereas its function in intact arterial smooth muscle seems to require cholesterol, correlating with intact caveolar structure.
α1A-Adrenergic receptors were found to be present primarily in fractions 9 and 10. This presumably explains the persistence of cirazoline-induced responses after cholesterol extraction and is consistent with findings of unaltered contraction in response to phenylephrine in rabbit aortas after cholesterol extraction28 and in aortas of caveolin-1–deficient mice.23,29⇓ In cultured rat aortic cells, radioligand binding to α1-adrenergic receptors was found in caveolin-3–containing fractions.30 In our preparations, caveolin-3 was not detectable by immunofluorescence staining, and receptors were detected by using a subtype-specific α1A-adrenergic antibody. This, as well as the marked phenotypic effects of culture on smooth muscle cells, potentially explains the discordant results, which suggest that the localization of receptors may be tissue and subtype specific.
The present results allow several conclusions regarding the steps in signaling affected by cholesterol depletion. Because adrenergic contractions are unaffected, it is unlikely that the contractile mechanism is involved. Furthermore, as also reported by Löhn et al,31 L-type channel activation by membrane depolarization seems to function normally in cholesterol-depleted preparations, because responses to depolarization by high K+ were unaffected. Thus, the impaired 5-HT–induced elevation of [Ca2+]i after extraction of cholesterol seems to be due to a defect upstream from Ca2+ channel activation, presumably leading to reduced membrane depolarization after agonist stimulation. Moreover, because direct G-protein activation with the use of AlF4− caused normal responses after cholesterol extraction, signaling downstream from G-protein activation was not affected.
Agonist-stimulated Ca2+ wave activity, which depends on recurring inositol trisphosphate (InsP3)-mediated Ca2+ release from the SR,16,20⇓ was selectively affected by cholesterol extraction, with a larger decrease for 5-HT than for cirazoline stimulation, consistent with fura 2 measurements of global [Ca2+]i. This suggests that InsP3 formation by 5-HT stimulation may have been compromised by cholesterol depletion, as may also be inferred from a comparison of agonist-induced Ca2+ release responses in Ca2+-free medium. The mβcd treatment causes increased basal [Ca2+]i and, probably as a consequence, increased Ca2+ loading of the SR. This is evidenced by the increased response to caffeine, an agent that acts directly at ryanodine receptors on the SR. Because responses to cirazoline were also increased after cholesterol depletion but those to 5-HT were not, it may be inferred that InsP3 production by the latter agonist had been reduced.
Treatment with mβcd might have caused a slight increase of membrane permeability, which causes increased basal [Ca2+]i and contractile tone, but otherwise, it does not seem to impair tissue function. Because Ca2+ efflux during repolarization was unaffected by cholesterol depletion, Ca2+ pumps were not inhibited. The basal tone, although sensitive to extracellular Ca2+, was resistant to a variety of Ca2+ channel blockers and, thus, presumably not associated with decreased activity of Ca2+ sparks and spontaneous transient outward currents, as reported for mβcd-treated cells and after caveolin-1 knockout.23,31⇓
Our results suggest that the signaling step impaired by cholesterol depletion is located either to ligand-receptor or to receptor–second-messenger interaction. Both steps could potentially be influenced by local variations in membrane fluidity3 imposed by caveolin-cholesterol interactions. Because the amount of receptor protein was not affected by cholesterol extraction, the localization of the receptor in cholesterol-rich regions, such as caveolae, seems to be necessary for proper function.
The study was supported by the Swedish Research Council (71X-28), by Magnus Bergvalls Stiftelse, and by postdoctoral fellowships from Svenska Sällskapet för Medicinsk Forskning (to K.S.) and the Danish Medical Research Council (to M.V.). We thank M. Danielsen and L. Immerdal for cholesterol measurements, H. Hadberg, M. Søberg, and P.S. Thomsen for microscopic preparations, and B. Risto for skillful photographic work.
Received January 11, 2002; revision accepted April 24, 2002.
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