Functional and Molecular Characterization of Beta Adrenoceptors in the Internal Anal Sphincter

44 Pages·2003·0.78 MB·English

JPET Fast Forward. Published on February 11, 2003 as DOI: 10.1124/jpet.102.048462
JPET Fast Forward. Published on February 11, 2003 as DOI:10.1124/jpet.102.048462
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Functional and Molecular
Characterization of Beta Adrenoceptors
in the Internal Anal Sphincter
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Sandeep Rathi, Shiva Kazerounian, Kuldip Banwait, jp
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Stephanie Schulz, Scott A Waldman, and Satish sp
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Rattan urn
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Department of Medicine, Division of Gastroenterology & Hepatology, (S.R., K.B., E
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S.R.); and Clinical Pharmacology (S.K., S.S., S.A.W.), Jefferson Medical College o
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of Thomas Jefferson University, Philadelphia, PA a
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Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: β-adrenoceptors in IAS smooth muscle

1Corresponding Author: Dr. Satish Rattan, Jefferson Medical College, Thomas Jefferson
University, 1025 Walnut Street, Room # 901 College; Philadelphia, PA 19107
Tel # [phone]; Fax # [phone]; Email: [email protected]

Number of text pages: 24 D
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Number of tables: 3 loa
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Figures: 8 fro
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References: 47 t.a
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Number of words in abstract: 242 u
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Number of words in introduction: 566 als
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Number of words in discussion: 1520 rg
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ABBREVIATIONS: -$5(cid:15)(cid:3) –adrenergic receptor; IAS, internal anal sphincter; CRC, J
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concentration response curve; EC , concentration causing maximal relaxation; EC , rn
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concentration causing 50% of maximal relaxation; ICYP, iodocyanopindolol; ZD 7114 o
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hydrochloride,(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2- n
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methoxyethyl)phenoxyacetamide); SR 59230A hydrochloride, (1-(2-ethylphenoxy)-3-[[(1S)- ry
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1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride); ICI 118,551 , 2
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2
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hydrochloride, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-
butanol; CGP 20712A methanesulfonate salt, (±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-
(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide
methanesulfonate salt)

Recommended section: Gastrointestinal, Hepatic, Pulmonary, & Renal
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ABSTRACT

7KH(cid:3)SXUSRVH(cid:3)RI(cid:3)WKH(cid:3)SUHVHQW(cid:3)VWXG\(cid:3)ZDV(cid:3)WR(cid:3)FKDUDFWHUL]H(cid:3)GLIIHUHQW(cid:3) -DGUHQRFHSWRUV(cid:3)(cid:11) -ARs) and
determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter
(cid:11),$6(cid:12)(cid:17)(cid:3) (cid:3) 7KH(cid:3) -AR subtypes in the opossum IAS were investigated by functional in vitro,
radioligand binding, western blot, and reverse transcription polymerase chain reaction (RT-PCR)
VWXGLHV(cid:17)(cid:3)(cid:3)=’(cid:3)(cid:26)(cid:20)(cid:20)(cid:23)(cid:15)(cid:3)D(cid:3)VHOHFWLYH(cid:3) -AR agonist, caused a potent and concentration-dependent
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UHOD[DWLRQ(cid:3)RI(cid:3)WKH(cid:3),$6(cid:3)VPRRWK(cid:3)PXVFOH(cid:3)WKDW(cid:3)ZDV(cid:3)DQWDJRQL]HG(cid:3)E\(cid:3)WKH(cid:3) -AR antagonist SR 59230A.
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&RQYHUVHO\(cid:15)(cid:3)WKH(cid:3),$6(cid:3)VPRRWK(cid:3)PXVFOH(cid:3)UHOD[DWLRQ(cid:3)FDXVHG(cid:3)E\(cid:3) (cid:3)DQG(cid:3) -AR agonists (xamoterol and n
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procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 fro
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and ICI 118551. Saturation binding of [125I]iodocyanopindolol ([125,@&<3(cid:12)(cid:3)WR(cid:3) -AR subtypes jpe
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revealed the presence of a high affinity site (Kd1 = 96.4 ± 8.7 pM; Bmax1 = 12.5 ± 0.6 fmol/mg tjo
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protein) and a low affinity site (Kd2 = 1.96 ± 1.7 nM; Bmax2 = 58.7 ± 4.3 fmol/mg protein). ls.o
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&RPSHWLWLRQ(cid:3) ELQGLQJ(cid:3) ZLWK(cid:3) VHOHFWLYH(cid:3) -AR antagonists revealed that the high affinity site t A
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FRUUHVSRQG(cid:3)WR(cid:3) 1(cid:18) 2-$5(cid:3)DQG(cid:3)WKH(cid:3)ORZ(cid:3)DIILQLW\(cid:3)VLWH(cid:3)WR(cid:3) 3-AR. Receptor binding data suggest the Jo
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predominant presence of β -AR over β /β -$5(cid:17)(cid:3)(cid:3):HVWHUQ(cid:3)EORW(cid:3)VWXGLHV(cid:3)LGHQWLILHG(cid:3) (cid:15)(cid:3) (cid:15)(cid:3)DQG(cid:3) -AR ls
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VXEW\SHV(cid:17)(cid:3)(cid:3)7KH(cid:3)SUHVHQFH(cid:3)RI(cid:3) (cid:15)(cid:3) (cid:15)(cid:3)DQG(cid:3) -AR was further demonstrated by mRNA analysis using u
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RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of , 2
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(cid:15)(cid:3) (cid:15)(cid:3)DQG(cid:3) -ARs in IAS smooth muscle. These studies may have important implications in
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anorectal and other gastrointestinal motility disorders.
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It is well known that post-junctional β-adrenoceptors (β-ARs) mediate the inhibitory effects of
sympathetic nerve stimulation in different smooth muscles including those of the gastrointestinal
tract (Gauthier et al., 2000; Manara et al., 1995b). The intestinal β-AR was originally described
as a β -and β -AR (Lands et al., 1967). Further studies with gastrointestinal preparations from
1 2
several species established the relaxant effect of classical β-AR (β and β ) agonists (Bennett,
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1965; Hedges and Turner, 1969; De Ponti et al., 1996a). Subsequently, studies investigating β-
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ARs in gastrointestinal smooth muscle from several species demonstrated relaxation responses lo
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that were resistant to propranolol and displayed lower affinity to other conventional β-AR fro
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antagonists (Arch and Kaumann, 1993; Strosberg, 1997; Manara et al., 2000; Goldberg and t.a
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Frishman, 1995). This finding along with the emergence of a new class of β-AR agonists tjou
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described first in adipocytes (Feve et al., 1991) suggested the presence of an “atypical” class of .org
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β-ARs. In 1989, Emorine and colleagues (Emorine et al., 1989) cloned and sequenced the β - S
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AR and found that it shared the pharmacological characteristics of the “atypical” β-AR. ou
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The β3-AR has been found in a variety of mammalian tissues (Berkowitz et al., 1995) including ary 2
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white and brown adipocytes (Muzzin et al., 1991), trachea (Webber and Stock, 1992), heart 0
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(Kaumann and Molenaar, 1996; Gauthier et al., 2000), gastrointestinal tract (Bardou et al., 1998;
De Ponti et al., 1995), and the urinary tract (Tomiyama et al., 1998). In the GI tract, recent
VWXGLHV(cid:3)KDYH(cid:3)IRFXVHG(cid:3)RQ(cid:3)WKH(cid:3)DELOLW\(cid:3)RI(cid:3) -AR specific agonists to cause relaxation in a number of
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different smooth muscle tissues including rat ileum, jejunum, colon, guinea-pig ileum, and
duodenum (Manara et al., 1995b). One of the problems in delineating the pharmacology of β-
ARs in the gastrointestinal tract has been the lack of subtype-selective agonists and antagonists,
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HVSHFLDOO\(cid:3)WKRVH(cid:3)IRU(cid:3) -AR. Recent in vivo studies have demonstrated the selective, potent, and
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SURORQJHG(cid:3)UHOD[DQW(cid:3)HIIHFW(cid:3)RI(cid:3)&/(cid:3)(cid:22)(cid:20)(cid:25)(cid:15)(cid:21)(cid:23)(cid:22)(cid:15)(cid:3)D(cid:3)VHOHFWLYH(cid:3) -AR agonist, on the sphincteric smooth
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muscles of the opossum LES (DiMarino et al., 2002), without the significant systemic
FDUGLRYDVFXODU(cid:3)VLGH(cid:3)HIIHFWV(cid:3)WKDW(cid:3)DUH(cid:3)DVVRFLDWHG(cid:3)ZLWK(cid:3) -(cid:3)DQG(cid:3) -AR agonists.
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In the past few years, β -agonists have emerged as potential therapeutic agents for several
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gastrointestinal motility disorders including irritable bowel syndrome (Scarpignato and Pelosini,
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1999). Anorectal dysfunctions such as Hirschsprung’s disease, constipation, anal fissures, and n
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hemorrhoids may also be associated with either hypertensive IAS or failure of sphincteric fro
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relaxation in response to the rectoanal inhibitory reflex (Azpiroz and Whitehead, 2002). jpe
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Characterization of neurohumoral receptors that mediate selective, potent, and prolonged tjo
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relaxation of IAS and other GI smooth muscles without untoward systemic effects will be of ls.o
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considerable interest in the treatment of anorectal and other GI motility disorders. t A
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Present investigation was carried out to characterize β-AR in the gastrointestinal tonic smooth ls
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muscle of the IAS by comprehensive studies using a combination of classical pharmacology, u
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receptor binding, and molecular biology approaches. , 2
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The aim of the present study is to determine the presence of and to characterize the β-AR
subtypes involved in mediating relaxation of the IAS smooth muscle. We used selective agonists
and antagonists, to determine the receptor binding profiles of each β-AR subtype. The presence
of membrane bound β-AR and mRNA encoding for the three β-AR subtypes was determined
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through western blot studies and reverse transcription polymerase chain reaction (RT-PCR)
analysis, respectively.
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Materials and Methods

Preparation of Smooth Muscle Strips. Adult male opossums (Didelphis virginiana), weighing
2.5 to 3.5 kg, were anesthetized with sodium pentobarbital (50 mg/kg; i.p.). Laparotomy was
performed and a part of the rectum along with the anal canal was removed using sharp
dissection. The IAS was identified by manometry as high-pressure zone and marked by means
of sutures in situ. The animals were sacrificed by exsanguination, the anorectal region was then
dissected out and transferred immediately to oxygenated (95% O + 5% CO ) Krebs’ D
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physiological solution of the following composition: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM loa
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CaCl , 1.16 mM MgSO , 1.01 mM NaH PO , 25 mM NaHCO , and 11.10 mM glucose. A fro
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longitudinal incision along the length of isolated anorectal region was made and the tissue was t.a
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pinned flat in a Sylgard (Dow Corning Corp., Midland, MI) coated petri dish. Once the lumen u
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was fully exposed, the mucosa and submucosa were removed carefully by sharp dissection. The rg
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tissue was then turned on the serosal side and all extraneous tissue including the outer S
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longitudinal muscle was removed. Circular smooth muscle strips of the IAS (approximately ou
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1x10 mm) were prepared and tied on either end using 3-0 silk suture in preparation for o
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measurement of isometric tension. a
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The experimental protocol of the study was approved by the Institutional Animal Care and Use
Committee of Thomas Jefferson University in accordance with the recommendations of the
American Association for the Accreditation of Laboratory Animal Care.

Measurement of Isometric Tension. The smooth muscle strips were transferred to 2 ml
muscle baths (Radnoti Glass Technology, Inc., Monrovia, CA) containing oxygenated Krebs’
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solution at 35oC. One end of the muscle strip was anchored at the bottom of the muscle bath
while the other end was connected to a force transducer (model FT03; Grass Instruments,
Quincy, MA). Isometric tension was recorded by the PowerLab/8SP data acquisition system
using Chart 4.1.2 (ADInstruements, Grand Junction, CO). Each smooth muscle strip was
initially stretched to a tension of 0.7 g. The muscle strips were then given at least an hour to
equilibrate during which they were washed with Krebs’ solution every 15 min. Only smooth
muscle strips that developed spontaneous tone and responded to electrical field stimulation (EFS)
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were used in this study. The changes in tension from various drugs were expressed as the n
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percent maximal relaxation achieved by 50 mM EGTA, at the end of each experiment. Each d fro
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smooth muscle served as its own control. jp
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Drug Responses. To determine the concentration-response curves (CRC) with β -, β -, and β - ls
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AR agonists on the basal tone of the IAS smooth muscles, xamoterol, procaterol, and ZD 7114, t A
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respectively were added to the muscle bath in cumulative concentrations (Rattan and Moummi, J
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1989). Successive concentrations of the agonists were not added till the response of the previous ls
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concentration stabilized. Ten minutes between additions of different concentrations were nu
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allowed when no effect was observed. In preliminary studies, when a single concentration was 2, 2
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used, we noted that this was an appropriate time needed to gauge the maximal effect of a given
concentration of the agonist. No difference in the results occurred with longer exposures. In
order to determine the effects of β -, β -, and β -AR antagonists, CGP 20712A, ICI 118551, and
1 2 3
SR 59230A, respectively (in concentrations ranging from 1x10-8 to 1x10-6 M) were added 30 min
before obtaining the CRC of the test agonist.
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Beta-Adrenoceptor (β-AR) Analysis by Western Blot. Western blot analysis of β -, β -, and
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β - in the IAS and rectum of the opossum was performed according to the protocol of Santa Cruz
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Biotechnology (Santa Cruz, CA). Circular smooth muscles tissues of the IAS and rectum were
cut into small pieces (2 x 2 mm cubes) and rapidly homogenized in 3 ml of boiling lysis buffer
(1% SDS, 1.0 mM sodium orthovanadate, 10 mM Tris pH 7.4), then put into the microwave for
10 seconds. The homogenates were centrifuged (16,000g, 4oC) for 15 min. The pellet obtained
was dissolved in Krebs’ buffer (composition already described above) containing 1 mM EDTA,
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1mM DTT, and 1 mM PMSF (combined pH of 7.6). The protein contents were determined by nlo
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the method described by Lowry et al (Lowry et al., 1951) using BSA as the standard. fro
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All of the samples were mixed with 2X sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% e
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glycerol, 0.006% bromophenol blue, 2% -PHUFDSWRHWKDQRO(cid:12)(cid:3)DQG(cid:3)ERLOHG(cid:3)IRU(cid:3)(cid:23)(cid:3)PLQXWHV(cid:17)(cid:3)(cid:3)(cid:21)(cid:19)(cid:3) O(cid:3)(cid:11)(cid:23)(cid:19)(cid:3) tjo
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J(cid:3) WRWDO(cid:3) SURWHLQ(cid:12)(cid:3) RI(cid:3) HDFK(cid:3) VDPSOH(cid:3) ZDV(cid:3) DSSOLHG(cid:3) WR(cid:3) FRPPHUFLDOO\(cid:3) DYDLODEOH(cid:3) (cid:26)(cid:17)(cid:24)(cid:8)(cid:3) 6’6(cid:3) ls.o
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polyacrylamide gel PAGEr Gold Gel (BioWhitaker Molecular Applications, Rockland, ME) t A
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applied to 7.5% sodium dodecyl sulfate polyacrylamide gel (SDS Page) apparatus by the method J
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of Laemmli (Laemmli, 1970) using 150 V for 1 hr. The separated proteins were ls
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electrophoretically transferred to nitrocellulose membrane (NCM) at 4oC for 90 min at 100 V. nu
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To block nonspecific antibody binding, the NCM were immersed overnight at 4oC in Super 2, 2
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Block Tris-buffered saline Tween (TBS) blocking buffer (Pierce Biotechnology, Rockford, IL).
The NCM was divided into three smaller sections labeled as β , β , and β . The NCM were then
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incubated with the respective diluted isoform specific primary (1o) antibodies corresponding to
the specific -AR subtype. The NCM were incubated with rabbit , β , and goat β polyclonal
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antibodies, respectively (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a dilution of 1:500.
All membranes were incubated with 1o antibody for 1 hr at room temperature. The membranes
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JPET/2002/48462 10
were then washed with TBS-T three times. Afterwards, the membranes corresponding to and
1
were incubated separately in 1:1000 diluted horseradish peroxidase-conjugated donkey anti-
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rabbit IgG (Amersham Biosciences, Piscataway, NJ) in 2o antibody buffer for 1 hr at room
temperature. The remaining membrane was incubated in 1: 5,000 diluted horseradish peroxidase
conjugated bovine anti-goat IgG (Santa Cruz Biotechnology) in 2o antibody buffer. The bands
were identified by chemiluminescence using the ECL detection system and Hyperfilm MP
(Amersham Biosciences). Densitometric analysis of the bands was performed using Image Pro
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Plus 4.0 software (Media Cybernetics, Silver Spring, MD). n
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Membrane Preparation For Receptor Binding Studies. The circular smooth muscle of the jp
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IAS was dissected free by the aforementioned procedure and placed immediately in ice-cold tjo
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Krebs’ buffer (composition already described above) containing 1 mM EDTA, 1mM DTT, and 1 ls
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mM PMSF (combined pH of 7.6). The IAS was minced with scissors and homogenized in 5 t A
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volumes of ice-cold TED buffer by the use of Tekmar Tissuemizer (Tekmar & Co., Cincinnati, J
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OH) for 15 s. The homogenates were centrifuged at 100,000 g for 1 hr at 4°C. The supernatant ls
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was filtered through a 500 µ Nitex mesh. The pellets were resuspended in cold Krebs’ buffer and an
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stored at -80°C until used. Protein content was determined by the method of Lowry et al (Lowry 2, 2
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et al., 1951).

Radioligand Binding Studies. The radioligand (-)-3-[125I]Iodocyanopindolol ([125I]CYP);
Amersham Pharmacia Biotech UK limited, Buckinghamshire, UK) was used for identifying β-
AR. For equilibrium determination, membranes at a protein concentration of 40 µg per tube
were incubated with [125I]CYP (specific activity: 2,000 Ci/mmol) for 0, 15, 30, 45, 60, 90, 120,