Skip Navigation

Cardiovascular Research 2006 69(4):825-835; doi:10.1016/j.cardiores.2005.12.016
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Shaw, L.
Right arrow Articles by Taggart, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shaw, L.
Right arrow Articles by Taggart, M. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: The relevance for Ca2+ oscillations and tone

Linda Shawa, Michele A. Sweeneyb, Stephen C. O'Neilla, Carolyn J.P. Jonesb, Clare Austina and Michael J. Taggarta,b,*

aDivision of Cardiovascular and Endocrine Sciences, University of Manchester, Great Britain, United Kingdom
bDivision of Human Development, University of Manchester, Great Britain, United Kingdom

* Corresponding author. Smooth Muscle Physiology Group, Division of Cardiac and Endocrine Sciences, Department of Medicine, University of Manchester, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, United Kingdom. Tel.: +44 161 276 5469; fax: +44 161 276 8433. Email address: Michael.j.Taggart{at}manchester.ac.uk

Received 12 September 2005; revised 1 December 2005; accepted 20 December 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action.

Methods: Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca2+ oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-β-cyclodextrin (mβcd), examined by light and electron microscopy.

Results: Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale–SR–myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR–caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mβcd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca2+ oscillations.

Conclusion: We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca2+ oscillations and contractile activation.

KEYWORDS Caveolae; Sarcoplasmic reticulum; Resistance arteries; Ca oscillations


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Considerable support has grown for a role of caveolae, {Omega}-shaped invaginations of the plasma membrane, in the regulation of smooth muscle contractile activation [1–3]. The distinct morphological appearance of caveolae arises from the interaction of caveolin proteins with cholesterol and sphingolipids. Caveolins may participate in the co-ordination of signal transduction pathways by their ability to bind to and/or alter the activation of a variety of intracellular signal transduction molecules [4]. The enrichment of particular receptors, channels and proteins in caveolae may facilitate the regulation of cellular excitability from within these microdomains [3–11]. This, and the positioning of caveolae in relation to other intracellular structures, may impart a direct role in the transmission of Ca2+ signals regulating force production. In this context, depletion of cholesterol from smooth muscle has been reported to alter the occurrence of Ca2+ sparks and store-operated Ca2+ entry [8,12].

In recent years, it has become evident that excitatory agonists that tonically constrict smooth muscle, do so by regulating the temporal and/or spatial characteristics of Ca2+ oscillations of individual cells [13–18]. Such oscillations commonly take the form of waves of Ca2+ originating in one point of the cell, propagating a distance of many tens of microns and lasting 2–6 s. These Ca2+ waves may be modulated by Ca2+ entry mechanisms and are stimulated in smooth muscle cells of many tissues by a variety of receptor-mediated agonists (for reviews see Refs. [19,20]). In freshly isolated single smooth muscle cells, the propagated Ca2+ waves have been suggested to involve IP3-induced Ca2+ release from the sarcoplasmic reticulum (SR) either exclusively, or with the involvement of ryanodine-sensitive calcium-induced calcium release [21–23]. In resistance arteries the regulation of vessel diameter will be critically dependent upon this governance of Ca2+ dynamics in individual smooth muscle cells. The dose-dependent constriction of pressurised rat mesenteric arteries is associated with elevations in (i) the number of smooth muscle cells exhibiting Ca2+ oscillations; (ii) the frequency of oscillations per individual cell [18,24]. Here too, the SR appears critical as pharmacological modulators of SR Ca2+ homeostasis alter the dynamics of Ca2+ waves in intact isobaric arteries [25,26]. Therefore, an important determinant of the characteristics of Ca2+ oscillations, and whether they will be able to activate myofilament contraction in smooth muscle cells of resistance arteries, will be the spatial distribution of SR. In many smooth muscles, a close link between caveolae and SR at the cell periphery has been reported (Ref. [19] for review). In particular, a recent structural study of non-vascular bladder smooth muscle suggested that co-localisation of SR proteins and the L-type Ca2+ channel in caveolar domains may contribute to the organisation of functional Ca2+ release sites [3]. However, given this, it is surprising that detailed ultrastructural information on the intracellular distribution of the SR in relation to caveolae in resistance vessels is scant.

In this study, therefore, we use electron microscopy to report the spatial arrangement of caveolae and SR in smooth muscle cells of pressurised rat mesenteric small arteries. Furthermore, we make use of two agents that, by different mechanisms of action, are suggested to disrupt caveolae and/or peripheral SR arrangements: (i) The phosphatase inhibitor calyculin-A has recently been suggested to alter peripheral SR–plasmalemmal couplings in venous smooth muscle [27] and another serine/threonine phosphatase inhibitor results in caveolae internalisation in several cultured cells [28–30]. (ii) The cholesterol sequestering agent methyl-β-cyclodextrin (mβcd) has been reported to disrupt caveolae in many smooth muscle preparations [4,8,9]. Thus, both these agents, although acting by different cellular mechanisms, may alter the structural relationship between caveolae and SR (and myofilaments). We examine this in the present study and relate it to agonist-mediated changes in [Ca2+]i oscillations and vessel diameter. Our data suggests that caveolae and peripheral SR in smooth muscle cells of pressurised resistance arteries are arranged in a manner that serves to facilitate the generation and propagation of Ca2+ waves and subsequent regulation of vasomotor tone.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Rat mesenteric resistance artery isolation and cannulation
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (250–300 g) were killed by stunning followed by cervical dislocation. The mesentery was removed and placed in ice-cold physiological salt solution (PSS) of composition (in mmol/l): NaCl 119, KCl 4.7, MgSO4.7H20 1.2, NaHCO3 25, KH2PO4 1.17, K2EDTA 0.03, glucose 5.5, CaCl2.2H20 1.6 at pH 7.4. A 3rd or 4th order mesenteric artery (2–3 mm in length) was dissected from each animal and placed in PSS within a bath chamber of a pressure myograph (model CH/1 or CH/1/QT, Living Systems Instrumentation, USA), cannulated onto two glass micropipettes (tip diameters 30–50 µm) as described previously [18] and pressurised to 50 mm Hg using a pressure servo-control unit (Living Systems Instrumentation, USA). The arteriograph was placed on top of a Nikon Diaphot 200 microscope and viewed with a x 10 objective. Lumen diameters were measured using a video image analyser and contractile responses to the {alpha}-adrenoceptor agonist phenylephrine (10–9–10–5 mol/l), the thromboxane mimetic U46619 [GenBank] (10–9–10–6 mol/l) or high K+ solution (isosomotically substituted for NaCl) assessed.

2.2 Arterial diameter measurements following treatment with calyculin-A or mβcd
Arteries were treated for 15 min with 2 x 10–6 mol/l of the phosphatase inhibitor calyculin-A, washed for 15 min and then stimulated with 10–5 mol/l phenylephrine or 10–6 mol/l U46619. [GenBank] In a separate set of experiments, arteries were treated with 10–2 mol/l mβcd for 1 h, the agent washed off and, 15 min later, the contractile response to a single dose of 10–5 mol/l phenylephrine assessed. Following washout, a concentration–response curve to phenylephrine (10–9–10–5 mol/l) was performed. The contractile responses of other arteries similarly treated by mβcd to a single dose (10–6 mol/l) and cumulative doses of U46619 [GenBank] (10–9–10–6 mol/l) were also assessed.

In a separate set of experiments, the influence on function and structure of mesenteric arteries of replenishing cholesterol to mβcd-treated tissues was established. Following mβcd treatment, arteries were exposed to a single dose of 10–5 mol/l phenylephrine; after 5 min, this was supplemented with 0.5 mM free cholesterol for 20 min and diameter changes compared to that observed to phenylephrine in the same vessels before mβcd exposure.

2.3 Electron microscopy of pressurised vessels
Vessels were mounted in arteriograph model CH/1/QT if to be prepared for electron microscopy by rapid rotation through 180° and immersion in fixative (~2 s, 2.5% gluteraldehyde in 0.1 M sodium cacodylate pH 7.3) whilst all the while remaining pressurised. Following 30 min equilibration, vessels were stimulated with phenylephrine (10–5 mol/l) to check for contractile viability and returned to PSS. Arteries were fixed following treatment and washout of calyculin-A or mβcd (as above) and compared to fixed arteries that had not been exposed to calyculin-A nor mβcd. The arteries were incubated in this fixative at room temperature for 3 h, washed several times in 10–1 mol/l sodium cacodylate buffer, to which 3 x 10–3 mol/l CaCl2 had been added, and stored at 4 °C. Approximately 0.5 mm thick sections of vessel were post-fixed in the dark (2 h, room temperature) in 2% osmium tetroxide in 10–1 mol/l sodium cacodylate buffer to which was added 0.8% (w/v) potassium ferricyanide [31,32]. Tissues were block stained with 2% aqueous uranyl acetate (90 min), dehydrated in an ascending alcohol series, treated with propylene oxide and embedded in Taab epoxy resin in Beem capsules or rectangular moulds. Ultrathin sections of transverse or longitudinal orientation to the vessel lumen were cut with a diamond knife, mounted on copper grids and stained with lead citrate. Specimens were examined in a Philips EM 301 electron microscope at an accelerating voltage of 60 kV.

2.4 Measurement of arterial smooth muscle cell [Ca2+]i signals
Measurements of global vessel wall [Ca2+]i were performed as described previously in pressurised mesenteric arteries [18]. Arteries were loaded with 20 µM of the membrane-permeant acetoxymethylester form of the ratiometric calcium-sensitive dye Indo-1 for 3 h in Hepes-buffered salt solution (in mM: NaCl 154, KCl 5.4, MgSO4 1.2, glucose 10, CaCl2 10, Hepes 10, pH 7.4 with NaOH) at room temperature. Following washing with PSS, the vessels were mounted on a Nikon Diaphot 200 inverted microscope, viewed with a x 10 Fluor objective (via a 600 nm filter), excited at 340 nm and emissions at 400 and 500 nm recorded as an indicator of [Ca2+]i.

For experiments involving measurement of cellular [Ca2+]i by laser scanning confocal microscopy the arteriograph chamber model CH/1 had cannulae between securable jaws at a slight angle (~10°) to allow positioning of the vessel wall close to the bottom of the microscope slide. Arteries were incubated with the membrane-permeant acetoxymethylester form of the calcium-sensitive dye Fluo-3 (15 x 10–6 mol/l, Molecular Probes) for 3 h in Hepes-buffered salt solution. The arteriograph was placed on a Nikon Diaphot 300 inverted microscope attached to a Bio-Rad MRC 1024 confocal laser scanning head and viewed with a x 60 Fluor water objective (N.A. 1.2). Tissues were constricted with 3 x 10–7–10–5 mol/l phenylephrine or 10–6 mol/l U46619 [GenBank] and changes in individual cellular [Ca2+]i measured as detailed in Ref. [18]. Fluo-3 fluorescence was excited at 488 nm with a Kr/Ar laser source (100 mW) and fluorescence >515 nm emitted in the confocal plane collected by a photomultiplier tube and digitally recorded with Bio-RAD Lasersharp software. Whole field optical scans in the same z-plane of focus were taken every 1 s. Off-line analysis of stored images was performed with Image J software (Version 1.27z). A region of interest (ROI) was described within individual cells in confocal images, positioned such that the ROI always remained within an identified cell. The software calculated the average ROI fluorescence value to follow Ca2+ alterions with time in individual cells. Similarly, in selected individual cells, the linescan mode of laser excitation allowed the time-course of Ca2+ oscillations to be monitored.

2.5 Drugs and chemicals
All drugs and chemicals, with the exception of indo-1(AM) and Fluo-3(AM) (Molecular Probes), gluteraldehyde and osmium tetroxide (Agar Scientific Ltd), potassium ferricyanide (BDH) and epoxy resin (Taab Laboratories Equipment, Aldermarston) were obtained from Sigma. Fluo 3(AM) and indo-1(AM) were dissolved in Pluronic F-127 20% solution in DMSO (obtained from Molecular Probes) to give 10–3 mol/l stock solutions. A 10–2 mol/l stock solution of U46619 [GenBank] was made by dissolving in a mixture of 100% ethanol and 1 mg kg–1 sodium carbonate (1:2). PE was dissolved in PSS. Cholesterol PEG 600 (Sigma UK catalogue number C1145) was a polyoxyethyl–cholesterol sebacate, a water soluble complex containing at least 20% cholesterol (batch-dependent). A stock solution of 60 mg/ml complex was dissolved in PSS and further diluted to a final bath concentration of 0.5 mM cholesterol.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Electron microscopic examination of caveolae and SR in smooth muscle cells of pressurised arteries
The smooth muscle cells of pressurised arteries, 2–4 layers deep, were predominantly circular in orientation wrapping around the vessel wall. Ultrastructural examination of these smooth muscle cells revealed abundant plasmalemmal caveolae appearing singularly or in rows (Figs. 1 and 2Go) in all normal vessels examined (n=18). The sarcoplasmic reticulum (appearing blackened by OsFeCN staining) when evident at the cell periphery was observed in close proximity to caveolar domains in 13 of 18 vessels. However, not all caveolar regions were associated with peripheral SR. Abundant SR staining was also noted in the centre of cells near to mitochondria and the nucleus. The location of the nucleus, central SR and mitochondria in the middle of the cell could be explained by the enveloping of these organelles by the tightly packed myofilaments running parallel to the longitudinal axis of the cell (Fig. 1). These arrangements occurred in both vessels fixed whilst relaxed (n=5, Fig. 2) and in vessels constricted with maximal doses of U46619 [GenBank] (n=7, Fig. 1), phenylephrine (n=3) or high K+ (n=3). Interestingly, even though the central SR was generally enveloped by the myofilament lattice, occasional strands of peripheral SR are seen to course from caveolar regions deeper through the cell forming a direct link to the central SR (indicated by asterisks in Fig. 1).


Figure 1
View larger version (135K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Arrangement of caveolae and sarcoplasmic reticulum in smooth muscle cell of pressurised arteries. (A, B) EM examination (longitudinal sections) of smooth muscle cells of a pressurised artery fixed whilst contracted maximally to phenylephrine. Caveolae denoted by black arrows are in close apposition to peripheral black-stained SR marked by white arrowheads. In the positions marked by white asterisks, peripheral SR strands close to caveolae are seen to connect to centrally positioned SR. White arrows indicate myofilaments. The latter interweaves close to mitochondria. Scale bars=200 nm.

 

Figure 2
View larger version (173K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Caveolae and peripheral SR distributions. (A) A transverse section of pressurised artery indicating prominent peripheral SR in close juxtaposition to caveolae of the plasma membrane as well as central SR close to mitochondria and the nucleus. Scale bar=200 nm. (B) Enlargement of the area denoted by the dotted box in panel A further indicates the close proximity of peripheral SR to caveolae; this peripheral SR is often observed enveloping the caveolae. White arrows indicate myofilaments. Scale bar=200 nm.

 
In magnified sections the peripheral SR could be seen to approach to within 10–20 nm of the plasma membrane; this positioning could be observed in a continuum along lengths of membrane up to 1.5 µm in the same plane of section (Fig. 2). Also, in regions of the plasma membrane enriched in caveolae, the periperal SR was found to encircle these invaginations (Fig. 2). This caveolae–peripheral SR region was also closely arranged next to the myofilament lattice running parallel to the axis of the plasma membrane.

3.2 Bi-directional Ca2+ oscillations in smooth muscle cells of pressurised arteries
Stimulation with the G-protein-coupled receptor agonists (phenylephrine or U46619 [GenBank] ) resulted in vessel diameter reductions that were accompanied by smooth muscle Ca2+ oscillations that were asynchronous between neighbouring cells. Typically, these oscillations took the form of a Ca2+ wave originating in one region of the cell and propagating along the length of the rest of the cell (up to 70 µm length visualised) (Fig. 3). However, this did not indicate a unique directionality to the Ca2+ wavefront. In 11 vessels, a Ca2+ wave could be seen to proceed in one direction along the length of a cell and, in later acquisitions of the same cell, another Ca2+ wave originated in a different region and propagated in the opposite direction along the longitudinal axis of the cell (Fig. 3B–D). In 4 vessels, there was also evidence of elevations in Ca2+ in two distinct parts of the cell that propagated towards each other and ‘collided’ (Fig. 3E). Only in one vessel was a Ca2+ wave observed to originate near the middle of the cell and progress away from the point of origin in both directions along the cell. These findings suggest that the origins of a Ca2+ wave are usually towards the ends of the cell distant from the middle portion. This is further supported by circumstances (5 vessels) where the propagated Ca2+ wave clearly avoided the nucleoplasm (not shown).


Figure 3
View larger version (57K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Smooth muscle cellular [Ca2+]i, changes in response to different agonists. Laser scanning confocal microscopy of pressurised vessels revealed several patterns of [Ca2+]i waves of individual smooth muscle cells to phenylephrine (10 µM). (A) Panels (i)–(vi) illustrate consecutive (1 s between images) changes in smooth muscle cell [Ca2+]i in the form of a wave propagated from one end of the cell (indicated by white dot). (B) Separate [Ca2+]i waves travelling in opposite directions in the same cell. The cell in question is indicated in the first frame by two white boxes marked i and ii. A [Ca2+]i wave began in the top portion of the cell and, in consecutive frames (1 s), travelled progressively downwards to the bottom of the cell. In the same cell (dotted lines indicate a 10 s break in recording) in the continued presence of phenylephrine, a [Ca2+]i wave originated in the bottom portion of the cell and progressed in consecutive frames towards the top of the cell. (C) Continuous line-plots of the [Ca2+]i changes (including the 10 s break of panel A) recorded from the ROI marked by the white boxes in panels A. The solid trace refers to the time-course of the [Ca2+]i wave in box (i) and the dotted line illustrates the time-course of the [Ca2+]i wave in box (ii). The first [Ca2+]i wave clearly passes box (i) before reaching box (ii). In contrast, the second wave of [Ca2+]i passes box (ii) before reaching box (i). (D) Linescan confocal images of a separate smooth muscle cell of an artery also illustrating progression of separate [Ca2+]i waves (initiated at points indicated by white spot) in opposite directions. (E) Illustration of a [Ca2+]i wave originating in two distinct regions of the cell, as indicated by the two white circles at the top and bottom, and progressing towards each other. Panels A–B, D–E scale bars=20 µm.

 
3.3 Functional and ultrastructural effects of calyculin-A or mβcd on pressurised resistance arteries
Calyculin-A treatment of isobaric rat mesenteric small arteries (Fig. 4A) resulted in the development of maximal tonic constrictions that were of even greater magnitude (change in diameter 178.0 ± 8.3 µm) than those to maximal phenylephrine in the same vessels (107.3 ± 25.3 µm; p<0.05). The calyculin-A constrictions were irreversible in the subsequent 15 min washout period. These maximum maintained contractions to calyculin-A occurred even in the absence of extracellular Ca2+ in two experiments (Fig. 4A). Furthermore, when compared to the magnitude of global Ca2+ changes induced by high K+ stimulation, calyculin-A treatment did not significantly (0.15 ± 0.10-fold of high K+) alter basal Ca2+ (Fig. 4B). However, calyculin-A treatment resulted in significantly fewer cells undergoing oscillations in Ca2+ at rest or following stimulation with one of two G-protein-coupled receptor agonists (phenylephrine or U46619 [GenBank] ) compared to before treatment (Fig. 4C). Consistent with this, the elevation in global Ca2+ in response to maximal dose of phenylephrine following calyculin-A treatment was 47+26% (n=3) of that to phenylephrine before calyculin-A.


Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Calyculin-A effects on pressurised artery diameter global [Ca2+]i and [Ca2+]i oscillations. (A) Calyculin-A (2 x 10–6 mol/l) resulted in constrictions, both in the presence and absence of Ca2+, that were greater than those to 10–5 mol/l phenylephrine obtained in the same tissues in the presence of Ca2+. (B) Although the mean change in diameter following calyculin-A application was greater than that due to addition of high K+ solution, the change in global [Ca2+]i was not elevated above basal. (C) Following calyculin-A treatment, the percentage of cells exhibiting Ca2+ oscillations in response to phenylephrine (25 ± 8.0% of 83 cells from 6 vessels) or U46619 (63 ± 5.8% of 172 cells from 11 vessels) was markedly reduced (open bars) compared to before treatment (black bars, 88 ± 5.0% of 80 cells from 6 vessels for PE and 20 ± 7.5% of 86 cells from 5 vessels for U46619). At rest, the number of cells exhibiting Ca2+ oscillations was also reduced from 19% to 3%.

 
All the fixed calyculin-A-treated arteries examined electron microscopically (n=7) exhibited different features from untreated arteries (Fig. 5). Regions of the hyper-constricted arteries had the appearance of prominent notches. These were enriched in caveolae but the SR was no longer in such close juxtaposition with the invaginations. Rather, the peripheral SR was arranged in concentric swirls that were not apparent in untreated tissues. On some occasions the caveolae appeared to have become invaginated/fused with these altered SR structures (e.g. Fig. 5). Such gross rearrangements were not observed in any of the calyculin-A untreated vessels that had been fixed whilst constricted with physiological stimuli.


Figure 5
View larger version (78K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of calyculin-A on the ultrastructure of isolated pressurised arterial smooth muscle. A, B and C demonstrate that calyculin-A treatment resulted in the appearance of prominent membrane notches. These contained caveolae (asterisks) and swirls of SR (denoted by white triangles) that were no longer in close apposition to caveolae. Some indication of invagination of caveolae is evident. White arrows show the directions of the myofilaments. Tissues were treated with calyculin-A in the presence (A–C) or absence (D) of extracellular Ca2+. White arrows indicate myofilaments. Scale bars=800 nm (A) and 200 nm (B–D).

 
Electron microscopic examination of the smooth muscle cells following treatment with the cholesterol binding drug mβcd (n=10) indicated flattened plasma membranes with few caveolae even though, in many cases, peripheral SR coupling close to the plasmalemma could still be observed in the absence of caveolae (Fig. 6A–B, n=6). In mβcd-treated arteries supplemented with water soluble cholesterol this was not the case–here, caveolae and peripheral SR close associations could still be observed (Fig. 6C). Mβcd treatment also resulted in a blunting of the arterial responses to agonist stimulation (Figs. 7 and 8Go). This was partly reversed by supplementation with water soluble cholesterol (mean diameter changes to phenylephrine were 158 ± 16 µm control, 50 ± 19 µm after mβcd and 86 ± 18 µm following cholesterol supplementation (p<0.05)). As in calyculin-A-treated arteries, mβcd treatment also resulted in a reduction of the number of cells exhibiting Ca2+ oscillations to stimulation with either phenylephrine or U46619 [GenBank] (Fig. 6D–E).


Figure 6
View larger version (136K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects of mβcd treatment and cholesterol replenishment on smooth muscle plasma membrane structure. (A, B) Following mβcd treatment caveolae are absent even though peripheral SR localisation close to the plasmalemma remains visible. (C) Shows smooth muscle ultrastructure of an artery which received cholesterol supplementation following mβcd treatment. Here the peripheral SR is in close association with caveolae. Scale bars=200 nm.

 

Figure 7
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 The influence of mβcd treatment on agonist-sensitivity. (A) Representative tracing of phenylephrine concentration–diameter responses before and after mβcd treatment. B and C show that dose-responsiveness to phenylephrine or U44619 is blunted in pressurised arteries after mβcd treatment.

 

Figure 8
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Mβcd effects on pressurised artery diameter and on smooth muscle [Ca2+]i oscillations. (A) Diameter reductions to maximal doses of phenylephrine and U44619 were less after mβcd treatment (160 ± 14 µm before and 72 ± 37 µm after for phenylephrine; 126 ± 2.4 µm before and 15 ± 13 µm after for U46619). (B) The blunted responsiveness following mβcd treatment is accompanied by a reduction in the percentage of cells exhibiting Ca2+ oscillations in response to 3 x 10–7 mol/l phenylephrine (79 ± 6.2%, n=3 cells from 6 vessels before (black box) and 22 ± 12%, n=88 cells from 6 vessels after) or U46619 (63 ± 5.8%, n=172 cells from 11 vessels before (black box) and 16 ± 10%, n=45 cells from 5 vessels after).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we have performed a structural and functional analysis of the relationship between caveolae and the peripheral SR in smooth muscles of resistance arteries. Electron microscopy revealed that the peripheral SR often enshrouds individual caveolae resulting in a close association of the two membrane structures. The possible implications of this arrangement for the regulation of vessel tone were evidenced by the actions of calyculin-A and mβcd.

Calyculin-A, an inhibitor of serine/threonine phosphatases, maximally constricted vessels both in the presence and absence of extracellular Ca2+ consistent with a mechanism of action attributable to inhibition of myosin phosphatase activity. This produced a rearrangement of the peripheral SR–caveolae distribution with prominent notches of membrane containing condensed caveolae and peripheral SR. The SR now appeared as tight swirls and no longer as intimately associated with rows of caveolae. These notched regions of the cell were not immediately overlying the myofilament lattice as in untreated cells. Caveolae in a number of cell types have been reported to be internalised by phosphatase inhibition [28–30]. The fact that agonist-mediated Ca2+ oscillations were reduced in treated arteries is in agreement with the suggestion of Lee et al., in venous smooth muscle [27], that the caveolae–peripheral SR arrangements disrupted by calyculin-A are normally important for the generation of Ca2+ oscillations.

Mβcd treatment resulted in a disruption of caveolae and a reduction in agonist-mediated constrictions. Thus, in common with calyculin-A-treated tissues, the caveolar–peripheral SR close association was disrupted although the SR remained in close apposition to flattened areas of the plasmalemma. Nonetheless, agonist-mediated Ca2+ oscillations were again reduced providing further indirect evidence supporting an important role for caveolae–SR couplings in the origin of Ca2+ oscillations. However, one must be cautious in the interpretation as changes in receptor distribution or Ca2+ entry may occur with mβcd treatment [8,9]. Nonetheless, in addition to pressurised vessels reported here, calyculin-A or mβcd treatment has resulted in alteration of smooth muscle Ca2+ oscillations [8,12,27].

A burgeoning body of evidence suggests that resistance arteries responded to G-protein-coupled contractile stimuli with waves of Ca2+ propagating along the length of individual smooth muscle cells [14,18,26,33]. As indicated in the data presented here, the Ca2+ oscillations are not unidirectional; on occasions when there was more than one discreet point of origin of Ca2+ waves in the same cell, the waves were observed to progress in opposite directions. In isolated smooth muscle cells, and intact smooth muscle tissues, such Ca2+ oscillations are disrupted by modulators of SR Ca2+ homeostasis, including inhibitors of calcium-induced Ca2+ release or IP3-sensitive Ca2+ release [[21–23,26] suggesting a dependence on regenerative SR Ca2+ release [19,34]. This suggests that the progression of a Ca2+ wave will be dependent upon the intracellular arrangement of the SR.

The separation of the peripheral SR and the caveolar plasma membrane of only ~10–20 nm may give rise to so-called focal discharge sites underlying Ca2+ spark activity in mesenteric and other smooth muscles [20,35–37]. Agonist-mediated Ca2+ waves appear to originate at focal discharge/Ca2+ spark sites, although controversy exists as to whether summation or inhibition of sparks contributes to cellular Ca2+ waves [20,26]. In several regions of a cell, peripheral SR strands were found to connect with more centrally located SR that, in turn, followed a course along the longitudinal axis of the cell in proximity to the nuclear membrane and mitochondria. The appearance of several focal discharge sites per cell [37] could arise from multiple caveolae–peripheral SR couplings, and together with a peripheral–central SR continuum, may explain the observations of Ca2+ waves (i) propagating away from that site along the longitudinal axis of the cell and (ii) not necessarily being unidirectional. The latter would offer the advantage of Ca2+ wave generation occurring in response to extraneous excitatory stimuli that might originate at quite distant (feasibly>100 µm) plasmalemmal sites. Future experiments with better spatial and temporal resolution will be required to directly confirm this possibility.

Caveolae–peripheral SR couplings, and portions of central SR along the longitudinal axis of the cell, are closely positioned adjacent to the tightly packed myofilaments. A major implication of these arrangements is that any elevations in Ca2+ as a result of propagated waves from the SR will likely activate the myofilaments that are in close proximity. Notably, myosin light chain kinase and a proportion of calmodulin–proteins essential for Ca2+-dependent contractile activation–are tightly bound to the myofilaments [38,39].

In summary, a close structural link between caveolae and peripheral SR exists in pressurised rat mesenteric arteries similar to that reported for guinea pig aorta, vas deferens [32,39,40], bladder [27] and human arteries [41]. Disruption of the caveolae–SR associations with the underlying myofilament lattice can be invoked by calyculin-A or mβcd treatment with a consequent impairment of Ca2+ oscillations and function. These data, together with an increasing body of evidence suggesting localisation of proteins involved in Ca2+ homeostasis to caveolar–SR domains [3,5–11], support a role for caveolae in the generation of Ca2+ oscillations and development of vascular tone. Future studies investigating (i) the immunogold localisation for IP3 and/or ryanodine receptor [5,29,37] close to caveolae and (ii) Ca2+ and tone changes in vessels of caveolin knockout mice will be instructive in this matter.


   Acknowledgements
 
Supported by the British Heart Foundation and Tommy's, the Baby Charity.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Taggart M. Smooth muscle excitation–contraction coupling: a role for caveolae and caveolins? NIPS (2001) 16:61–65.[Abstract/Free Full Text]
  2. Bergdahl A., Sward K. Caveolae-associated signalling in smooth muscle. Can J Physiol Pharmacol (2004) 82:289–299. [& Sward].[CrossRef][Web of Science][Medline]
  3. Moore E.D., Voigt T., Kobayashi Y.M., Isenberg G., Fay F.S., Gallitelli M.F., et al. Organization of Ca2+ release units in excitable smooth muscle of guinea-pig urinary bladder. Biophys J (2004) 87:1836–1847.[CrossRef][Web of Science][Medline]
  4. Je H.D., Gallant C., Leavis P.C., Morgan K.G. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol (2003) 286:H91–H98.[Web of Science]
  5. Fujimoto T., Nakade S., Miyawaki A., Mikoshiba K., Ogawa K. Localisation of inositol 1,4,5-triphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol (1992) 119:1507–1513.[Abstract/Free Full Text]
  6. Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol (1993) 120:1147–1157.[Abstract/Free Full Text]
  7. Brainard A.M., Miller A.J., Martens J.R., England S.K. Maxi-K channels localize to caveolae in human myometrium: a role for an actin–channel–caveolin complex in the regulation of myometrial smooth muscle K+ current. Am J Physiol (2005) 289:C49–C57.[CrossRef][Web of Science]
  8. Dreja K., Voldstedlund M., Vinten J., Tranum-Jensen J., Hellstrand P., Sward K. Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction. Arterioscler Thromb Vasc Biol (2002) 22:1267–1272.[Abstract/Free Full Text]
  9. Berghal A., Gomez M.F., Dreja K., Xu S., Adner M., Beech D.J., et al. Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TrpC1. Circ Res (2003) 93:839–847.[Abstract/Free Full Text]
  10. Cho W.J., Daniel E.E. Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1. Am J Physiol (2005) 288:571–585.
  11. Darby P.J., Daniel E.E. Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+ handling. Am J Physiol (2000) 279:L1226–L1235.[Web of Science]
  12. Lohn M., Furstenau M., Sagach V., Elger M., Schulze W., Luft F.C., et al. Ignition of Ca sparks in arterial and cardiac muscle through caveolae. Circ Res (2000) 87:1034–1039.[Abstract/Free Full Text]
  13. Iino M., Kasai H., Yamazawa T. Visualisation of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ. EMBO J (1994) 13:5026–5031.[Web of Science][Medline]
  14. Miriel V.A., Mauban J.R.H., Blaustein M.P., Wier G.W. Local and cellular Ca2+ transients in smooth muscle of pressurized rat resistance arteries during myogenic and agonist stimulation. J Physiol (1999) 518:815–824.[Abstract/Free Full Text]
  15. Jaggar J.H., Nelson M.T. Differential regulation of Ca2+ sparks and Ca2+ waves by UTP in rat cerebral artery smooth muscle cells. Am J Physiol (2000) C1528–C1539.
  16. Lee C.H., Poburko D., Sahota P., Sandhu J., Ruehlmann D.O., Van Breeman C. The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol (2001) 534:641–650.[Abstract/Free Full Text]
  17. Peng H., Matchkov V., Ivarsen A., Aalkjaer C., Nilsson H. Hypothesis for the initiation of vasomotion. Circ Res (2001) 88:810–815.[Abstract/Free Full Text]
  18. Shaw L., O'Neill S.C., Jones C.J.P., Austin C., Taggart M.J. Comparison of U46619-, endothelin-1- or phenylephrine-induced changes in cellular Ca2+ profiles and Ca2+-sensitisation of constriction of pressurised rat resistance arteries. Br J Pharmacol (2004) 141:678–688.[CrossRef][Web of Science][Medline]
  19. Lee C.H., Poburko D., Kuo K., Seow Y., Van Breeman C. Ca2+ oscilations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol (2002) H1571–H1583.
  20. Bolton T.B., rdienko V., Povstyan V.O., Harhun M.I., Pucovsky V. Smooth muscle cells and interstitial cells of blood vessels. Cell Calcium (2004) 35:643–657.[CrossRef][Web of Science][Medline]
  21. White C., McGeown J.G. Carbachol triggers RyR-dependent Ca2+ release via activation of IP3 receptors in isolated rat gastric myocytes. J Physiol (2002) 542:725–733.[Abstract/Free Full Text]
  22. Gordienko D.V., Bolton T.B. Crosstalk between ryanodine receptors and IP3 receptors as a factor shaping spontaneous Ca2+ release events in rabbit portal vein myocytes. J Physiol (2002) 542:743–762.[Abstract/Free Full Text]
  23. McCarron J.G., MacMillan D., Bradley K.N., Chalmers S., Muir T.C. Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localised photolysis of caged inositol 1,4,5-triphosphate. J Biol Chem (2004) 279:8417–8427.[Abstract/Free Full Text]
  24. Zhang W.J., Balke C.W., Wier W.G. Graded {alpha}1-adrenoceptor activation of arteries involves recruitment of smooth muscle cells to produce ‘all or none’ Ca2+ signals. Cell Calcium (2001) 29:327–334.[CrossRef][Web of Science][Medline]
  25. Jaggar H. Intravascular pressure regulates local and global Ca2+ signalling in cerebral artery smooth muscle cells. Am J Physiol (2001) 281:C439–C448.[Web of Science]
  26. Lamont C., Weir G. Different roles of ryanodine receptors and inositol (1,4,5)-triphosphate receptors in adrenergically stimulated contractions of small arteries. Am J Physiol (2004) 287:H617–H625.[Web of Science]
  27. Lee C., Kuo K., Dai J., Leo J.M., Seow C.Y., Van Breeman C. Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle. Cell Calcium (2005) 37:9–16.[CrossRef][Web of Science][Medline]
  28. Parton R.G., Joggesrt B., Simons K. Regulated internalization of caveolae. J Cell Biol (1994) 127:1199–1215.[Abstract/Free Full Text]
  29. Thomsen P., Roepstorff K., Satlhult M., van Deurs B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytotic trafficking. Mol Cell Biol (2002) 13:238–250.[CrossRef]
  30. Kiss, AL, Botos, E, Turi, A, Mullner, N. Okadaic acid treatment causes tyrosine phosphorylation of caveolin-2 and induces internalisation of caveolae in rat peritoneal macrophages.
  31. Forbes M.S., Plantholt B.A., Sperelakis N. Cytochemical staining procedures selective for sarcotubular systems of muscle: modifications and applications. J Ultrastruct Res (1977) 60:306–327.[CrossRef][Web of Science][Medline]
  32. Nixon G.F., Mignery G.A., Somlyo A.V. Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle. J Muscle Res Cell Motil (1994) 15:682–700.[CrossRef][Web of Science][Medline]
  33. Mauban J.R.H., Lamont C., Balke W., Wier G.W. Adrenergic stimulation of rat resistance arteries affects Ca2+ sparks, Ca2+ waves, and Ca2+ oscillations. Am J Physiol (2001) 280:H2399–H2405.[Web of Science]
  34. Berridge M. The endoplasmic reticulum: a multifunctional signalling organelle. Cell Calcium (2002) 32:235–249.[CrossRef][Web of Science][Medline]
  35. Pucovsky V., Gordienko D.V., Bolton T.B. Effect of nitric oxide donors and noradrenaline on Ca2+ release sites and global intracellular Ca2+ in myocytes from guinea-pig small mesenteric arteries. J Physiol (2002) 539:25–39.[Abstract/Free Full Text]
  36. Cheranov S.Y., Jagger J.H. Mitochondrial modulation of Ca2+ sparks and transient Kca currents in smooth muscle cells of rat cerebral arteries. J Physiol (2004) 556:755–771.[Abstract/Free Full Text]
  37. Zhuge, R, Fogarty, KE, Baker, SP, McCarron, JG, Tuft, RA, Lifshitz, LM, Walsh, JV. Ca2+ spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large conductance K+ channels, and coupling ratio between them. Am J Physiol 287: C1577–C1588.
  38. Lin P., Luby-Phelps K., Stull J.T. Binding of myosin light chain kinase to actin myosin filaments. J Biol Chem (1997) 272:7412–7420.[Abstract/Free Full Text]
  39. Wilson D.P., Sutherland C., Walsh M.P. Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+. J Biol Chem (2002) 277:2186–2192.[Abstract/Free Full Text]
  40. Lesh R.E., Nixon G.F., Flesicher S., Airey J.A., Somlyo A.P., Somlyo A.V. Localisation of ryanodine receptors in smooth muscle. Circ Res (1998) 82:175–185.[Abstract/Free Full Text]
  41. Sweeney, M, Jones, CJ, Greenwood, SL, Baker, PN, Taggart, MJ. Ultrastructural features of smooth muscle and endothelial cells of isolated isobaric human placental and maternal arteries. Placenta, Epub ahead of print. doi:10.1016/j.placenta.2005.05.010.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. J. Clarke, S. Forman, J. Pritchett, V. Ohanian, and J. Ohanian
Phospholipase C-{delta}1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H826 - H834.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. J. Clarke, V. Ohanian, and J. Ohanian
Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2248 - H2256.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Adebiyi, G. Zhao, S. Y. Cheranov, A. Ahmed, and J. H. Jaggar
Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries
Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1584 - H1592.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Zacharia, J. Zhang, and W. G. Wier
Ca2+ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction
Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1523 - H1532.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
I. Fleming
Segregation and integration: Roles played by caveolae and caveolins in the cardiovascular system
Cardiovasc Res, March 1, 2006; 69(4): 784 - 787.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Shaw, L.
Right arrow Articles by Taggart, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shaw, L.
Right arrow Articles by Taggart, M. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?