endothelin-a receptor in rat skeletal muscle microvasculature

BRIEF COMMUNICATION

Endothelin-A Receptor in Rat SkeletalMuscle Microvasculature

Debra Mitchell,* Aurelia Bihari,†,‡ Martin Sandig,§ and Karel Tyml*,†,‡†A.C. Burton Vascular Biology Laboratory, LHRI, *Department of Pharmacology and Toxicology,‡Department of Medical Biophysics, and §Department of Anatomy and Cell Biology,The University of Western Ontario, London, Ontario N6A 5C1, Canada

Although the effect of endothelin-1 (ET-1) on vasculartone has been studied extensively at the arterial/arterio-lar level, little is known about the direct effect of ET-1 atthe level of the capillary. Using intravital microscopy, wedetermined capillary red blood cell velocity and arterio-lar diameter responses to ET-1, ETA-receptor blocker BQ-123, and ETB-receptor blocker BQ-788 applied locally oncapillaries in rat extensor digitorum longus (EDL) mus-cle. Using immunohistochemistry, we examined capillar-ies in this muscle and microvascular endothelial cellsisolated from this muscle for immunoreactivity with ETA-receptor antibody. ET-1 (10�9 to 10�5 M in micropipette)caused quick reductions (i.e., within several seconds),whereas BQ-123 (10�8 to 10�4 M) and BQ-788 (10�6 and10�4 M) caused quick increases, in both velocity and di-ameter. Capillaries and endothelial cells showed ETA-receptor immunoreactivity. We conclude that the micro-vasculature of the rat EDL muscle is sensitive to ET-1 andits receptor blockers and that the ETA receptor may bepresent in the capillary wall of this muscle, including theendothelium. © 2002 Elsevier Science (USA)

Key Words: capillary; endothelial cells; skeletal muscle;endothelin.

0026-2862/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

INTRODUCTION

It is well established that endothelin-1 (ET-1) plays arole in the maintenance of vascular tone (Rubanyi andPolokoff, 1994). The prevailing view is that activationof smooth muscle ETA and ETB receptors mediates along-lasting vasoconstriction, while activation of ETreceptors on endothelial cells (EC) can result in atemporary vasodilation (e.g., via release of PGI2

and/or NO). A recent study in cat skeletal muscleshowed that ET-1 can also increase microvascular per-meability (Porter et al., 2000). However, since little isknown about ET receptors on the microvascular en-dothelium in skeletal muscle (Rubanyi and Polokoff,1994), the role of a direct effect of ET-1 on the endo-thelium in the reported permeability increase has notbeen addressed (Porter et al., 2000). Our preliminaryintravital work in skeletal muscle capillaries raised thepossibility that these capillaries may, in fact, have ETreceptors (Mitchell and Tyml, 1997). In view of the

Received February 20, 2002

Microvascular Research 64, 179–185 (2002)doi:10.1006/mvre.2002.2427

potential impact of ET-1 on permeability in the capil-lary bed, the aim of the present study was to completeour intravital work and to examine whether the mi-

179

crovascular ETA receptor in skeletal muscle can bedetected by immunohistochemistry.

MATERIALS AND METHODS

Experiments were approved by the Council on An-imal Care at the University of Western Ontario. MaleWistar rats (200–350 g) were anesthetized with pento-barbital (65 mg/kg ip). The right extensor digitorumlongus (EDL) muscle was prepared for local stimula-tion of capillaries as detailed by us (Mitchell et al.,1997). Briefly, after surgical exposure, the muscle wascovered by a plastic coverslip that had a 3-mm hole inthe center. The hole was filled with degassed paraffinoil, permitting access to surface capillaries with glassmicropipettes. The epi-illuminated muscle was visu-alized on a closed-circuit video system at low magni-fication (10�/0.22 NA objective and 6.3� eyepiece;780 � 1050-�m field on the monitor) or at high mag-nification (32�/0.40 NA objective and 10� eyepiece;154 � 207-�m field). Micropipettes were backfilledwith ET-1, ETB-receptor (ETB-R) blocker BQ-788 (bothfrom Alexis Biochemicals), or ETA-receptor (ETA-R)blocker BQ-123 (Sigma Chemical Co.) dissolved indistilled water. Capillaries were stimulated 300 �mdownstream of the feeding arteriole by lowering adroplet of drug (�65 pL; ejected by pressurized air)onto the capillary (Dietrich, 1989). In this article, thegiven concentrations are those in the pipette and arehigher (100–1000�) than those in the vicinity of thecapillary following ejection (Mitchell et al., 1997).

Microvascular responses to agents were evaluatedfrom video recordings at low magnification (i.e., redblood cell velocity measurement, VRBC) and at highmagnification (diameter measurements, D). Continu-ous VRBC in the stimulated capillary was measured bythe flying spot technique (Tyml and Ellis, 1982). Wedetermined the control VRBC (VRBCcon) as the averageVRBC 1–2 min before drug application. After the stim-ulation, the microcirculatory view was recorded untilthe velocity returned to VRBCcon or to a level �15%VRBCcon. The duration of a response was defined as thetime from the onset of the response until the time

when VRBC reached either VRBCcon or a stable levelwithin �15% VRBCcon. VRBCtest was defined as the veloc-ity at its greatest change from VRBCcon. Data were ex-pressed as a percentage change from VRBCcon: �VRBC

(%) � 100% � (VRBCtest � VRBCcon)/VRBCcon. Luminal di-ameter of the feeding arteriole was measured from acalibrated video screen off-line (�1-�m resolution) for2 min prior to agent application (Dcon) and at themaximum change of the diameter within the first 3min poststimulation (Dtest). Data were expressed as apercentage change from Dcon: �D(%) � 100% � (Dtest �Dcon)/Dcon.

For immunohistochemical detection of ETA-R, themiddle portion of the EDL muscle was embedded inTissue-Tek (OCT compound, Somagen Diagnostics) andsnap-frozen in liquid nitrogen precooled isopentane.Cross-sections of the muscle (10 �m thick) were placedon a glass slide, washed with phosphate-buffered saline(PBS), fixed with 2% paraformaldehyde (5 min), rinsed,blocked with 1.0% bovine serum albumin (BSA; Sigma),and then exposed to ETA-R primary antibody (ETA-R Ab;Alexis Biochemicals; 1:50 dilution in PBS plus 1.0% BSA)for 48 h in a humidified chamber at 5°C. (In preliminaryexperiments, the specificity of the antibody was checkedby observing a single band in the range of 48–78 kDa onWestern blots of EDL muscle homogenates). The sec-tions were then washed with PBS and exposed for 1 h atroom temperature to FITC-conjugated rabbit anti-sheepIgG (1:100 dilution; Vector Laboratories) in combinationwith TRITC-labeled Griffonia simplicifolia I lectin [1:10dilution, Sigma, to identify capillaries (Nora et al., 1998)].The sections were washed with PBS, covered first withVectashield (Vector Laboratories) and then by a glasscoverslip, and viewed with an epifluorescence micro-scope equipped with filters to separate the FITC andTRITC labeling. In negative control experiments, theETA-R Ab was replaced by PBS with 1.0% BSA. Ourexamination of muscle cross-sections focused mainly oncapillaries. The rationale for this focus was the possibilitythat, in our intravital experiments, VRBC/diameter re-sponses to capillary stimulations with ET-1 could in-volve activation of ET receptors within the capillary walland a subsequent activation of a capillary–arteriolarcommunication pathway (we have hypothesized thatthis pathway includes an agonist-induced endothelial

180 Brief Communication

© 2002 Elsevier Science (USA)All rights reserved.

cell hyper/depolarization and its electrotonic spreadalong capillary endothelium to the feeding arteriole;Song and Tyml, 1993; Mitchell et al., 1997).

Using the same antibody, we also aimed to localizethe ETA receptor on microvascular EC harvested fromthe rat EDL muscle (harvesting, phenotype identifica-tion, and culturing conditions detailed by us; Wilsonet al., 1996). Cells grown on a glass coverslip werefixed either with absolute acetone (30 s) followed by2% paraformaldehyde (2 min) or with acetone alonefor 30 s, rinsed, blocked with 1.0% BSA for 1 h, rinsed,and then exposed to the ETA-R Ab (1:100) for 1 h atroom temperature. The cells were washed with PBS,exposed to the FITC-conjugated IgG (1:100) for 1 h atroom temperature, washed again, mounted withVectashield on glass slides, and viewed with epifluo-rescence microscopy.

Data are expressed as means � SEM. Student’s t testwas used to determine whether a poststimulation re-sponse was different from prestimulation levels.ANOVA was used to determine whether a responsewas dose-dependent. If the critical F ratio was ob-tained ( p � 0.05), then the Student t test with theBonferroni correction was used to determine wherethe differences existed.

RESULTS

A droplet of ET-1 applied on capillary caused aquick drop in VRBC (i.e., within several seconds) thatreached its VRBCtest value in less than 1 min. The am-plitude of VRBCtest and the duration of VRBC reductiondepended on the ET-1 concentration (Fig. 1A). For apipette concentration of 10�5 M we determined thatthe VRBC reduction (i.e., �100%) was accompanied bya 23 � 2% arteriolar constriction (Dcon � 8.7 � 0.8 �m,n � 12 arterioles in N � 7 rats), but by no change incapillary diameter (Dcon � 4.9 � 0.2 �m). To ascertainthat VRBC and arteriolar diameter responses were me-diated via ET-1 receptors (rather than by a nonspecificeffect of ET-1), we applied, in the same droplet, amixture of 10�5 M ET-1 and blocker (10�4 M BQ-123 or10�4 BQ-788). ET-1 � BQ-123 resulted in significantly

smaller �VRBC, duration, and �D values (i.e., �50 �10%, 6.8 � 1.2 min, and �5 � 2%, respectively; n �10–13, N � 4–6) compared to those elicited by ET-1alone (10�5 M). Similarly, ET-1 � BQ-788 also resultedin smaller �VRBC and duration values (i.e., �52 � 11%and 6.9 � 1.2 min, respectively; n � 9, N � 4).

A droplet of BQ-123 caused a quick increase in VRBC

that reached its VRBCtest value within 1–2 min. For con-

FIG. 1. Effect of local capillary stimulation with ET-1 and withETA-receptor blocker BQ-123 on capillary red blood cell velocity(VRBC). (A) Changes in VRBC relative to the control prestimulationVRBC (i.e., �VRBC) and durations of VRBC responses after local appli-cation of ET-1 on the capillary (10�9 to 10�5 M in the pipette). Theoverall control VRBC was 255 � 17 �m/s. n (in parentheses) is thenumber of stimulated capillaries in a total of N � 3–6 rats. (B) Effectof local BQ-123 (10�8 to 10�4 M) on capillary VRBC and duration ofVRBC response. The overall control VRBC was 350 � 32 �m/s. n is thenumber of stimulated capillaries from a total of N � 5–7 rats. Allresponses were significantly different from the baseline (0% or 0min).

181Brief Communication


centrations tested (Fig. 1B), the greatest �VRBC andresponse duration occurred at 10�4 M. A droplet ofBQ-788 also caused a quick VRBC increase (for 10�6 and10�4 M, �VRBC were 31 � 8 and 36 � 5%, while re-sponse durations were 5.3 � 0.8 and 5.7 � 1.4 min,respectively; n � 5–6, N � 5–7). For a pipette concen-tration of 10�4 M, BQ-123 and BQ-788 increased feedarteriole diameter by 34 � 9 and 22 � 11%, respec-tively (n � 6–8, N � 5–7).

Using double-labeled EDL muscle cross-sections,capillary profiles identified by lectin staining (Figs. 2Aand 2C) showed ETA-R immunoreactivity (Figs. 2Band 2D). [Note: over the 48-h period of the protocol,some capillary profiles lifted off the glass slide. Usingthe standard hematoxylin and eosin (H&E) stainingon sections taken immediately after the fixation, wetypically saw three to four capillaries around a musclefiber. When sections were examined with H&E at the

48-h time point, only one or two capillaries around afiber remained.] Overall, based on four sections fromfour muscles in four rats, more than 90% of lectin-stained capillaries showed ETA-R immunoreactivity.In negative controls (ETA-R Ab omitted), only thelectin label appeared (data not shown). As expected,arteriolar profiles also demonstrated ETA-R immuno-reactivity within the thickness of the arteriolar wall(data not shown). Figure 2 shows that capillary profilestructures labeled with lectin did not always colocal-ize with structures labeled with the ETA-R Ab. Sincethe lectin could stain the basement membrane of thecapillary (Nora et al., 1998) for both EC and pericytes(Hansen-Smith et al., 1996), the apparent lack of colo-calization suggested that one or several constituents ofthe capillary wall (e.g., EC, pericyte, mesenchymalcell, mast cell, macrophage) could have stained posi-tively with the ETA-R Ab. Figure 3 shows that EC here

FIG. 2. Immunohistochemical identification of ETA receptors on capillaries in cross-sections of the rat EDL muscle. (A and C) Identificationof capillaries in two separate sections (from two rats) using TRITC-labeled Griffonia simplicifolia I lectin. (B and D) The same sections as shownin A and C, respectively, showing capillary profiles with positive ETA-receptor Ab (ETA-R) staining using FITC-labeled IgG. The lectin appearsto label the perimeter of capillary profiles (e.g., basement membrane of endothelial cells and pericytes; Hansen-Smith et al., 1996), while theoften punctate ETA-R signal is contained within this profile (e.g., including the plasma membrane and cytoplasm of endothelial cells andpericytes). Negative controls (i.e., ETA-R Ab omitted) showed only capillaries stained with lectin (data not shown). Bar, 10 �m.



could express the ETA receptor, as cultured microvas-cular EC obtained from the rat EDL muscle stainedpositively with the ETA-R Ab. Consistent with ourprevious study (Wilson et al., 1996), EC tested posi-tively for von Willebrand factor VIII and G. simplicifo-lia I lectin, two markers of endothelial phenotype (datanot shown).

DISCUSSION

The present in vivo experiments demonstrated thatlocal capillary stimulation with ET-1, BQ-123, and BQ-788 affected both capillary blood flow and the up-stream arteriolar diameter in rat EDL muscle. Thisindicated that the microvasculature of this muscle issensitive to ET-1 and its receptor blockers. Our veloc-ity and diameter measurements obtained at post-stimulus times longer than 1 min most likely reflecteda direct diffusional effect of agents on the arteriolarsmooth muscle (Mayhan and Rubinstein, 1995; Touyz

et al., 1995). However, the immediate effect of agents(i.e., within several seconds) may be too fast to beexplained by diffusion (Mitchell et al., 1997) and couldinvolve a tissue communication pathway (Rivers andFrame, 1999), including interendothelial communica-tion along the capillary wall (Song and Tyml, 1993;Beach et al., 1998). Since the cells of this wall in vivo canrespond to pharmacological stimuli (e.g., exhibiting amembrane potential change; McGahren et al., 1998), itis possible that, among other effects, the present ap-plication of ET-1 and blockers also elicited responsesat the capillary wall. Consistent with this possibility isthe report that ET-1 constricted capillaries in the iso-lated spiral ligament from the ear (Sadanaga et al.,1997).

Although the present in vivo data did not permitmechanistic determination of the observed responses,they provided a rationale for immunohistochemicalexamination of ET-1 receptors at the capillary wall.Figures 2 and 3 indicate that the ETA receptor may bepresent both in capillaries in skeletal muscle in vivoand in cultured microvascular endothelial cells de-

FIG. 3. Presence of ETA receptors on microvascular endothelial cells harvested from the rat EDL muscle. (A) An example of endothelial cellsgrown on a coverslip in vitro (passage 4) labeled with anti-ETA-R antibody showing positive cell surface staining. (B) Negative controlexperiment (ETA-R Ab omitted) showing no such staining. Pictures are representative of other areas on the coverslips. Experiments yieldingsimilar results were obtained in triplicate. Bar, 10 �m.



rived from this muscle. Consistent with this finding,the ETA receptor has been reported in brain capillaryendothelial cells in vitro (Rubanyi and Polokoff, 1994).Using the same immunohistochemical procedure as inthe present study, we have also found ETA-R immu-noreactivity in cultured microvascular endothelialcells of human lung origin (unpublished observation).

In the present study, applications of the ETA-selec-tive blocker BQ-123 (Eguchi et al., 1992) and the ETB-selective blocker BQ-788 (Ishikawa et al., 1994) on theEDL muscle capillary increased blood flow. This sug-gests that (i) ET-1 was present within/near the capil-lary to provide a basal activation of microvascularETA/ETB receptors and that (ii) this activation wasinhibited by receptor blockers. The source of the back-ground ET-1 level could be ET-1 circulating in theblood or ET-1 synthesized within the microvascula-ture (Rubanyi and Polokoff, 1994). Since the circulat-ing level of ET-1 can be extremely low (e.g., �2 fM;Hsegawa et al., 1996) and since systemically appliedET-1 receptor blockers have no vascular effects (Ru-banyi and Polokoff, 1994), it is unlikely that there wasa circulating ET-1 level sufficient to provide this basalactivation. Rather, it is possible that ET-1 was synthe-sized within the microvasculature. In this regard, ECfrom brain (Bacic et al., 1992) and retinal capillaries(Takahashi et al., 1989) have been reported to produceET-1.

In conclusion, our intravital work showed that themicrovasculature of rat EDL muscle is sensitive toET-1 and its receptor blockers. In agreement withthese results, our immunohistochemical data indicatethat the ETA receptor may be present in the capillarywall of this muscle, including the endothelium. Thus,further studies of a direct role of ET-1 on microvascu-lar permeability may be warranted.

ACKNOWLEDGMENTS

We thank Mrs. J. Sholdice, Mr. N. Gocan, D. Anderson, M.Pollman, and D. O’Hene, and Drs. S. Chakrabarti, F. M. Hansen-Smith, and J. H. Lombard for technical help. The work was sup-ported by grants from the Heart and Stroke Foundation of Ontarioand the Canadian Institutes of Health Research awarded to K. Tyml.

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endothelin-a receptor in rat skeletal muscle microvasculature

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