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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1267.)
© 2002 American Heart Association, Inc.

Vascular Biology

Cholesterol Depletion Disrupts Caveolae and Differentially Impairs Agonist-Induced Arterial Contraction

Karl Dreja; Marianne Voldstedlund; Jørgen Vinten; Jørgen Tranum-Jensen; Per Hellstrand; Karl Swärd

From the Department of Physiological Sciences (K.D., P.H., K.S.), Lund University, Lund, Sweden, and the Department of Medical Physiology (M.V., J.V.) and the Department of Medical Anatomy (J.T.-J.), Panum Institute, University of Copenhagen, Copenhagen, Denmark.

Correspondence to Dr Karl Swärd, Department of Physiological Sciences, Lund University, BMC F12, S-221 84 Lund, Sweden. E-mail karl.sward{at}mphy.lu.se

	Abstract

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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-ß-cyclodextrin, and the effects on force and Ca²⁺ handling were evaluated. In cholesterol-depleted preparations, the force responses to ₁-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-HT_2A receptor and of caveolin-1 were unaffected by cholesterol extraction. Sucrose gradient centrifugation showed enrichment of 5-HT_2A receptors, but not ₁-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.

Key Words: smooth muscle • caveolae • 5-hydroxytryptamine • endothelin • intracellular calcium

	Introduction

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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-ß-cyclodextrin (mßcd).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 ₁-adrenergic agonists, they profoundly influence a specific set of signaling pathways, including those for serotonin, vasopressin, and endothelin.

	Methods

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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).

Force Measurements
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 mßcd 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

[Ca²⁺]_i Measurements
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 mßcd 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 mßcd or control solution for 1 hour. Ca²⁺ wave activity was measured by using a Zeiss 510 laser scanning confocal microscope.¹⁶

Western Blotting
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 _1A-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-HT_2A receptor antibody was a kind gift from Dr Bryan Roth (Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio). Anti–_1A-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.

Cholesterol Analysis
After equilibration, segmented arteries were incubated with or without 10 mmol/L mßcd 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

Statistical Analysis
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.

	Results

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Endothelium-denuded rings of rat tail artery were contracted with high K⁺ (60 mmol/L), the ₁-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 (E_max) of the respective agonist and were stationary for 3 hours (data not shown). After relaxation (25 minutes), preparations were treated for 1 hour with mßcd (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 E_max response), AlF₄^- (30 µmol/L, giving 90% of E_max), or the myosin phosphatase inhibitor calyculin A (3 µmol/L, giving 100% of E_max) 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 mßcd treatment selectively reduces 5-HT, AVP, and endothelin-1 responses.

View larger version (24K): [in this window] [in a new window]   Figure 1. Cholesterol depletion selectively reduces agonist responses in the rat tail artery. A and B, Original recordings of force in deendothelialized segments of the rat tail artery contracted with 60 mmol/L K+ (A) or 1 µmol/L 5-HT (B). After stable contractions had been attained, cholesterol was extracted by treatment with 10 mmol/L mßcd for 1 hour at 37°C. Thereafter, the same agonist was again used to elicit contraction. C, Summarized data on force elicited by the phosphatase inhibitor calyculin A (3 µmol/L), membrane depolarization by high K+ (60 mmol/L), the 1-adrenergic selective agonist cirazoline (0.3 µmol/L), 5-HT (1 µmol/L), AVP (0.1 µmol/L), endothelin-1 (10 nmol/L), and AlF4- (30 µmol/L) before (solid bars) and after (open bars) cholesterol depletion. ***P<0.001 by Student t test (n8).

Cumulative dose-response relations were determined in mßcd-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 ₁-agonist or with AlF₄^-. Responsiveness to 5-HT was restored after cholesterol had been added back in complex with mßcd (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 [mßcd] 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% [mßcd] 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 mßcd-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 [mßcd] versus 267±19 [control] nmol/L, n=12, P=NS) or 0.3 µmol/L cirazoline (212±25 [mßcd] 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 [mßcd] 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 ₁-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 mßcd on ₁-adrenergic responses, tissues were treated with thapsigargin and caffeine to deplete Ca²⁺ from intracellular stores. This did not reveal any difference (control versus mßcd treatment) in ₁-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 mßcd-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 mßcd 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 mßcd 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).

View larger version (100K): [in this window] [in a new window]   Figure 2. Micrographs (by electron microscopy [EM]) of the medial smooth muscle cells (SMCs) in tail arteries. Plasma membrane areas of SMCs from different preparations are shown. A, Normal -shaped caveolae (arrows) are shown in an SMC from a control preparation. Arrowheads indicate Golgi apparatus; M, mitochondrion; N, nucleus; and F, filaments. B and C, Cholesterol-depleted SMCs are shown. Caveolae are less numerous and, when present, are opened to a variable degree. The Golgi apparatus is disorganized. D, Normal caveolae are seen after reloading with cholesterol.

The levels of the 5-HT_2A 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-HT_2A receptor cofractionated with caveolin-1 and Cav-p60 in fractions 5 and 6 (Figure 3C). On the other hand, the _1A-adrenergic receptor was present in fractions 9 and 10 (Figure 3C).

View larger version (32K): [in this window] [in a new window]   Figure 3. A and B, 5-HT2A receptor and caveolin-1 content in control (solid bars) and cholesterol-extracted (open bars) tail artery. Contents are given relative to the optical densityxmm2 of the control band (100%) on the same blot (n=8, P>0.05 for both comparisons). Identical amounts of tissue dry weight (60 µg) were loaded in each lane. C, Coenrichment of 5-HT2A receptors, but not 1A-adrenergic receptors, with caveolin-1 and cavP-60 in sucrose density fractionations of nontreated tail artery. Proteins in a homogenate of tail artery (4 rats per experiment) were separated on a discontinuous (5%, 35%, 45%) sucrose gradient, and equal volumes of each fraction (1 to 10) were loaded on the gel. The Western blots shown are representative of 3 independent fractionations.

	Discussion

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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 ET_A, 5-HT_2A, and V₁ receptors, respectively.^24–26 The ET_A receptor was reported to be localized in caveolae,⁹ but the ultrastructural localization of 5-HT_2A 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-HT_2A 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.

_1A-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 ₁-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 _1A-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 mßcd 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 mßcd 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 mßcd-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.

	Acknowledgments

 
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; accepted April 24, 2002.

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