EndothelinB receptor on guinea pig small intestinal smooth muscle cells MASAHIRO YOSHINAGA, YOSHIHARU CHIJIIWA, TADASHI MISAWA, NAOHIKO HARADA, AND HAJIME NAWATA Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Yoshinaga, Masahiro, Yoshiharu Chijiiwa, Tadashi Misawa, Naohiko Harada, and Hajime Nawata. EndothelinB receptor on guinea pig small intestinal smooth muscle cells. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G308-G311, 1992.-We investigated the binding characteristics of the endothelin (ET) receptor and the mechanism by which ET induces contraction of longitudinal smooth muscle cells of the guinea pig small intestine by using vasoactive intestinal contractor (VIC), a mouse variant of ET-Z. A functional receptor for VIC was found to exist on longitudinal smooth muscle cells. These cells showed a similar binding of and contractile response to ET-l, ET-Z, and ET-3. Inhibitors of both intracellular and extracellular Ca2’ movement attenuated the VIC-induced contraction of longitudinal smooth muscle cells. These results suggest that smooth muscle cells of the guinea pig small intestine express the ETB receptor that primarily mediates the contractile effect on smooth muscle cells. In addition, ET-induced contraction depends on intracellular as well as extracellular Ca2+. endothelin-l-3;
endothelin
receptor
subtype; binding
assay
(ET) is a peptide consisting of 21 amino acid residues that was isolated from vascular endothelium and has been reported to exert a potent vasoconstrictive effect (24). Cloning studies of genomic DNA have shown that there are three distinct members of the ET family (7). All three isopeptides (ET-l, ET-2, and ET-3) cause contraction of smooth muscle from the vas deferens, bronchus, urinary bladder, and ileum, as well as vascular smooth muscle (10). A novel peptide, vasoactive intestinal contractor (VIC), which differs from ET-l in only three amino acid residues, has been cloned and sequenced from the mouse genome (17) and is now considered a mouse variant of ET-2. Northern blot analysis has indicated that the gene for VIC is expressed only in the intestine and not in other tissues or endothelial cells (17). Ishida et al. (8) have reported that VIC causes a potent, long-lasting contraction of guinea pig ileal longitudinal muscle strips. This contraction has a slow onset and is inhibited by nicardipine. We also have reported the direct contractile effect of VIC on isolated longitudinal smooth muscle cells from the guinea pig small intestine (25). These results strongly suggest the existence of the functional receptor for VIC on longitudinal smooth muscle cells of guinea pig small intestine. The present study was designed to investigate by using VIC, a variant of ET-2, 1) the binding characteristics of ET receptor on isolated longitudinal smooth muscle cells obtained from guinea pig small intestine and 2) the role of intracellular and extracellular Ca2+ in ET-induced smooth muscle cell contraction.
ENDOTHELIN
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0193~1857/92
$2.00
Copyright
MATERIALS
AND
METHODS
Materials. VIC, ET-l, ET-Z, and ET-3 were obtained from the Peptide Institute (Osaka, Japan); collagenase (CLS type II) from Worthington Biochemicals (Freehold, NJ); trypsin inhibitor (type I-S), 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB -8)) bovine serum albumin (BSA), and bacitracin were from Sigma Chemical (St. Louis, MO); N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) from Wako Pure Chemical (Osaka, Japan); acrolein from Tokyo Kasei (Tokyo); and 1251-VIC (sp act 74 TBq/mmol) from Amersham International (Amersham, UK). Preparation of dispersed smooth muscle cells. Smooth muscle cells were isolated from the longitudinal muscle layer of the guinea pig small intestine by the method of Bitar and Makhlouf (2). In brief, male Hartley guinea pigs (300-600 g) raised on a standard diet at the Animal Center of Kyushu University (Fukuoka, Japan) were fasted overnight and killed by the intracardiac injection of pentobarbital sodium (100 mg/kg). The small intestine was excised from each animal and placed in oxygenated ice-cold HEPES medium [(in mM) 120 NaCl, 2.6 KH2P04, 4 KCl, 2 CaC12, 0.6 MgC12, 25 HEPES, 14 glucose, and 0.01% trypsin inhibitor, pH 7.41. The longitudinal muscle layer was peeled off by tangential stroking with wet gauze along the mesenteric border, cut into small pieces, and incubated for two successive 45-min periods at 31°C in 15 ml of HEPES medium containing 150 U/ml of collagenase. After incubation, the partly digested strips were washed with 50 ml of enzyme-free HEPES medium and reincubated in 15 ml of fresh HEPES medium for 20-30 min at 31°C to allow the cells to disperse spontaneously. Cells were then harvested by filtration through a 500~pm polyester mesh. 1251-VIC binding to dispersed cells. HEPES medium containing 2% BSA and 0.3 mg/ml bacitracin was used for the binding studies. Aliquots (0.5 ml) of the dispersed cell suspension (10” cells/mm”) were preincubated at room temperature for 10 min, with or without 0.1 ml of unlabeled VIC, and then incubated with 0.1 ml of 1251-VIC (final concentration 25 PM) at room temperature for a given period. All binding studies were performed in duplicate. At the end of incubation, bound and free 1251-VIC was separated by centrifugation at 900 g for 2 min at 4°C. The radioactivity bound to cells was measured using a gamma scintillation counter. Specific binding was defined as the total binding minus the nonspecific binding determined in the presence of an excess (IO-” M) of unlabeled VIC. The timecourse changes of 1251-VIC binding at room temperature were determined in a separate series of experiments in which binding proceeded for 5, 10, 20, 40, 60, or 90 min. The ability of ET family peptides (ET-l, ET-Z, VIC, and ET-3) to displace 1251VIC was examined using unlabeled peptides in the concentration range of IO-l2 M to lOA M. Scatchard plot were analyzed by linear regression to determine dissociation constant (&) and the maximum binding capacity (B,,,). Contractile response of dispersed cells. Dispersed cells were stimulated by test agents, and the contractile response was measured by the method of Bitar and Makhlouf (2). Aliquots
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Physiological
Society
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ETB
RECEPTOR
ON
INTESTINAL
(0.5 ml) of cells were added to 0.2 ml of a solution containing each test agent for a given time. The kinetics of the contractile response to VIC were studied by incubating cells for various periods in the presence of 10e7 M VIC. The reaction was interrupted by the addition of acrolein at a final concentration of 1%. The lengths of 50 cells in microscopic fields were measured by image-splitting micrometry, and the percent decrease in mean cell length was determined by comparison with the control. To examine the role of intracellular and extracellular Ca2+ in the VIC-induced contraction of longitudinal smooth muscle cells, we added TMB-8 [an inhibitor of intracellular Ca2’ release (ll)] or verapamil (a Ca2’ channel blocker) to aliquots of cells simultaneously with 10e7 M VIC. The percentage contraction was determined in comparison with the VIC-induced contraction. Student’s t test or Welch’s method after inspection of variances was used to determine the significance of suppressive effect of TMB-8 and verapamil on VIC-induced contraction. Probability values ~0.05 were considered to be significant.
SMOOTH
MUSCLE
G309
CELLS
ent manner and with the same affinity (Fig. 2). The 50% inhibitory concentration (IC50) value was 0.7 nM. Scatchard analysis of these data showed that Kd and B,,, were 0.9 nM and 37.3 fmol/5 x 10” cells, respectively. Contractile response of dispersed smooth muscle cells. The maximal contractile response of longitudinal smooth muscle cells was attained after 45 s, with a 16.9 t 2.3% (mean t SE) (Fig. 3) decrease in cell length from control. Subsequent experiments were performed with an incubation time of 45 s. VIC induced contraction of smooth
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1251-VIC binding to dispersed cells. The specific binding of 1251-VICto dispersed longitudinal smooth muscle cells of the guinea pig small intestine reached an equilibrium after 60 min (Fig. 1). Total VIC binding was 8.3 t 0.7% of the added radioactivity, and specific binding was 72 t 4.1% (mean t SE, n = 4) of the total binding. To determine the specificity of VIC binding sites, a variety of peptides were tested to inhibit 1251-VIC with smooth muscle cells. Inclusion of up to lOA M vasoactive intestinal peptide, glucagon, calcitonin gene-related peptide, neuropeptide Y, substance P, somatostatin, motilin, and cholecystokinin-8 did not affect 12’I-VIC binding in longitudinal smooth muscle cells of guinea pig small intestine. Specific binding of 1251-VICwas inhibited by unlabeled VIC, ET-l, ET-Z, and ET-3 in a concentration-depend-
01-1 0
30 TIME
60
; 40-I G
0 cl
I
x 0
-12
-11
-10
-9
Log
[ PEPTIDE
-8
-7
WC
ET-1 ET-2 ET-3
-6
]
Fig. 2. Inhibition of binding of 12”I-VIC to isolated longitudinal smooth muscle cells by unlabeled VIC, endothelin (ET)-1, ET-2, and ET-3. Specific binding of 12”1-VIC is expressed as percentage of radioactivity bound to cells in absence of unlabeled peptide. Values are means t SE of 4 separate experiments.
I 90
(MINUTES)
Fig. 1. Time course of specific binding of l”“I-vasoactive intestinal contractor (VIC) to isolated longitudinal smooth muscle cells of guinea pig small intestine. Muscle cells were incubated with 25 pM “‘1-VIC in presence or absence of 1 PM unlabeled VIC at room temperature. Data are expressed as percentage of radioactivity specifically bound to cells. Values are means t SE of 4 separate experiments.
0
30
TIME
60
90
120
(SECONDS)
Fig. 3. VIC (10e7 M)-induced contractile response of isolated longitudinal smooth muscle cells from guinea pig small intestine. Values are means t SE of 5-7 experiments. Vertical axis shows percent decrease in mean cell length compared with control.
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G310
ETB
RECEPTOR
ON
INTESTINAL
muscle cells in a dose-dependent manner, with and 50% effective dose (ED& value of 5 nM. ET-l, ET-Z, and ET-3 also evoked contraction of smooth muscle cells with the same potency as VIC (Fig. 4). TMB-8 and verapamil significantly inhibited the contractile response of longitudinal smooth cells to VIC in a dose-dependent manner (Fig. 5), with IC& values of 0.1 and 0.9 PM, respectively. DISCUSSION
To our knowledge, this is the first study demonstrating the existence of an ETB receptor on longitudinal smooth
WC ET-1 ET-2 ET-3
1
1
1
1
’
’
1
-11
-10
-9
-8
-7
-6
Log
[ PEPTIDE
]
Fig. 4. Contractile response of isolated longitudinal smooth muscle cells of guinea pig small intestine to various concentrations of VIC, ET-l, ET-2, and ET-3. Each value shows mean t SE (n = 5 experiments).
---o--
o-1 0
Ca2+
TMB-8 VERAPAMIL
1
'
'
'
'
1o-g
1o-8
1o-7
lo+
ANTAGONIST
'
1o-5
(mol /L)
Fig. 5. Inhibitory effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) and verapamil on contraction of isolated longitudinal smooth muscle cells produced by VIC (10s7 M). Each value shows mean k SE (n = 4-6 experiments). *p < 0.05. **P c 0.01. ***ip < 0.001.
SMOOTH
MUSCLE
CELLS
muscle cells of the guinea pig small intestine. We showed that the affinity of 1251-VIC binding to smooth muscle cells (I&o 0.7 nM, & 0.9 nM) is similar to the pharmacological effect of VIC in producing smooth muscle cell contraction (ED50 5 nM). This result indicates that a functional receptor for VIC is expressed on the smooth muscle cells. VIC is now considered a mouse variant of ET-Z. Our study showed VIC and ET-Z had a similar binding affinity and contractile potency in these cells. These results therefore indicate a functional receptor for VIC can be regarded as an ET receptor on smooth muscle cells of guinea pig small intestine. ET receptors are classified into subtypes. Sakurai et al. (18) proposed two ET receptor subtypes: the ETA receptor (ET-l/ET-Z selective subtype) and the ETB receptor (non-isopeptide-selective subtype). Recently, Arai et al. (1) reported the cloning of cDNA encoding a bovine ET receptor that shows high affinity for ET-l. Sakurai et al. (18) also reported the cloning of cDNA encoding a rat nonselective ET (ETB) receptor. The ETA receptor is expressed on vascular and nonvascular tissues, such as chick cardiac membranes (23), rat vascular smooth muscle cells (6), and rat uterus (5). In a pharmacological study, ET-l and ET-2 caused stronger contraction of vascular and uterine smooth muscle strips than did ET-3 (5,7). On the other hand, the ETB receptor is expressed on many rat tissues but not on vascular smooth muscle cells (18). In isolated perfused rat mesentery, Warner et al. (22) reported that ET-l and ET-3, at low doses, were equipotent as vasodilators acting through the release of endothelium-derived relaxing factor. On the contrary, ET-l, at high doses, was -10 times more potent as a vasoconstrictor than ET-3 (22). It was also suggested that the ET-l/ET-2-selective receptor (ETA receptor) primarily mediated vasoconstriction, and the non-isopeptide-selective receptor (ETB receptor) led to the formation of endothelium-derived relaxing factor and vasodilation (12). In our study, the functional ET receptor on longitudinal smooth muscle cells of guinea pig small intestine showed similar affinities for ET-l and ET-3. Moreover, ET-3 induced smooth muscle cell contraction as effectively as did ET-l. These results strongly suggest that the functional ET receptor on longitudinal smooth muscle cells of the guinea pig small intestine is classified into the ETB receptor and that the ETB receptor primarily mediates small intestinal smooth muscle contraction. Thus there is a difference between ETinduced intestinal contraction and vasoconstriction. ET-l and ET-3 have been isolated from the porcine brain (19), and Takayanagi et al. (20) have shown that ET-3 was almost equipotent with ET-l in binding to the porcine cerebellum. Nambi et al. (16) performed crosslinking experiments with 1251-ET-l and 1251-ET-3, using membranes from various parts of the rat brain, and found that two specific bands were labeled (mol wt 45,000 and 37,000). These labeled bands were diminished in the presence of excess unlabeled ET-l or ET-3 (16). Crawford et al. (3) have reported that ET-3 as well as ET-l stimulated phosphoinositide hydrolysis in the rat cerebellum and cerebral cortex. From these findings and our results, the ET-receptor subtype expressed by longitu-
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ETB
RECEPTOR
ON
INTESTINAL
dinal smooth muscle cells of the guinea pig small intestine is suggested to be similar to that found in the central nervous system. ET-3 is abundant in the intestine, pituitary gland, and brain of the rat (13), suggesting the possibility that it may act as a brain-gut peptide. It has been variously reported that the mechanism of the ET-induced contraction of vascular smooth muscle cells is dependent on Ca2+ release from intracellular storage sites (14, 15), Ca2+ influx through Ca2+ channels (4, 21, 24), or protein kinase C activation (9). In the present study, we used two Ca2+ antagonists with different mechanisms to examine the role of intracellular and extracellular Ca2+ in VIC-induced longitudinal smooth muscle cell contraction. Contraction of the cells was inhibited by both TMB-8 and verapamil in a dose-dependent manner. So the VIC-induced contraction of longitudinal smooth muscle cells appears to depend on both intracellular and extracellular Ca2+. In conclusion, our new findings were as follows: 1) the functional ET receptor that shows a similar affinity for ET-l, ET-2, VIC, and ET-3 exists on longitudinal smooth muscle cells of the guinea pig small intestine; 2) ET-l, ET-2, VIC, and ET-3 cause contraction of longitudinal cells with similar potency; and 3) both TMB-8 and verapamil inhibit the VIC-induced contraction of longitudinal smooth muscle cells. These results strongly suggest that longitudinal sm 00th muscle cells of the small intestine express the ETB receptor that primarily mediates contraction of the small intestinal smooth muscle cell. In addition, not only the release of Ca2+ from intracellular stores but also the influx of Ca2+ appears to be required for the maximal smooth muscle cell contractile response to ET. Address Received
reprint 23 January
requests
to M. Yoshinaga.
1991; accepted
in final
form
9 September
1991.
REFERENCES 1. Arai, H., S. Hori, I. Aramori, H. Ohkubo, and S. Nakanishi. Cloning and expression of a cDNA encoding an endothelin receptor. Nature Lond. 348: 730-732, 1990. 2. Bitar, K. N., and G. M. Makhlouf. Receptors on smooth muscle cells: characterization by contraction and specific antagonists. Am. J. Physiol. 242 (Gustrointest. Liver Physiol. 5): G4OO-G407, 1982. 3. Crawford, M. L. A., C. R. Hiley, and J. M. Young. Characteristics of endothelin-1 and endothelin-3 stimulation of phosphoinositide breakdown differ between regions of guinea-pig and rat brain. Naunyn-Schmiedeberg’s Arch. Pharmacol. 341: 268-271, 1990. 4. Goto, K., Y. Kasuya, N. Matsuki, Y. Takuwa, H. Kurihara, T. Ishikawa, S. Kimura, M. Yanagisawa, and T. Masaki. Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle. Proc. NatZ. Acad. Sci. USA 86: 3915-3918,1989. 5. Hagiwara, H., M. Kozuka, T. Ito, S. Eguchi, and S. Hirose. Properties of rat uterus endothelin receptor sites. Biomed. Res. 11: 93-98,199O. 6. Hirata, Y., H. Yoshimi, Y. Takagi, K. Kanno, S. Eguchi, and F. Marumo. Interaction of endothelin isopeptides with vascular endothelin-1 receptor. Biomed. Res. 11: 195198, 1990. 7. Inoue, A., M. Yanagisawa, S. Kimura, Y. Kasuya, T. Miyauchi, K. Goto, and T. Masaki. The human endothelin family: three structurally and pharmacologically distinct isopeptodes pre-
SMOOTH dieted
MUSCLE by three
G311
CELLS separate
genes.
Proc.
Natl.
Acad.
Sci.
USA
86:
2863-2867,1989. 8. Ishida,
N., K. Tsujioka, M. Tomoi, K. Saida, and Y. Mitsui. Differential activities of two distinct endothelin family peptides on ileum and coronary artery. FEBS Lett. 247: 337-340, 1989. 9. Komada, M., H. Kanaide, S. Abe, K. Hirano, H. Kai, and M. Nakamura. Endothelin-induced Ca-independent contraction of the porcine coronary artery. Biochem. Biophys. Res. Commun. 160:
1302-1308,1989. 10. Maggi, C. A., S. Giuliani, R. Patacchini, P. Revero, A. Giachetti, and A. Meli. The activity of peptides of the endothelin family in various mammalian smooth muscle preparations. Eur. J. Pharmacol. 174: 23-31, 1989. 11. Malagodi, M. H., and C. Y. Chiou. Pharmacological evaluation of a new Ca2+ antagonist, 8-( N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8): studies in smooth muscles. Eur. J. Pharmacol. 27: 25-33, 1974. 12. Martin, E. R., B. M. Brenner, and B. J. Ballermann. Heterogeneity of cell surface endothelin receptors. J. Biol. Chem. 265: 14044-14049,199o. 13. Matsumoto, H., N. Suzuki, H. Onda, and M. Fujino. Abundance of endothelin-3 in rat intestine, pituitary gland and brain. Biochem. Biophys. Res. Commun. 164: 74-80, 1989. 14. Miasiro, N., H. Yamamoto, H. Kanaide, and M. Nakamura. Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture? Biochem. Biophys. Res. Commun. 156: 312-317,1988. 15. Muldoon, L. L., K. D. Rodland, M. L. Forsythe, and B. E. Magun. Stimulation of phosphatidylinositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells. J. BioZ. Chem. 264: 8529-8536, 1989. 16. Nambi, P., M. Pullen, and G. Feuerstein. Identification of endothelin receptors in various regions of rat brain. Neuropeptides 16: 195-199,199O. 17. Saida, K., Y. Mitsui, and N. Ishida. A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. J. Biol. Chem. 264: 14613-14616, 1989. 18. Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, K. Goto, and T. Masaki. Cloning of cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature Lond. 348: 732-735,199O. 19. Shinmi, O., S. Kimura, T. Sawarura, Y. Sugita, T. Yoshizawa, Y. Uchiyama, M. Yanagisawa, K. Goto, T. Masaki, and I. Kanazawa. Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain. Biochem. Biophys. Res. Commun. 164: 587-593,1989. 20. Takayanagi, R., K. Ohnaka, T. Takasaki, M. Ohashi, and H. Nawata. Multiple subtypes of endothelin receptors in porcine tissues: characterization by ligand binding, affinity labeling and regional distribution. Regul. Pept. 32: 23-37, 1991. 21. Takuwa, Y., Y. Kasuya, N. Takuwa, M. Kudo, M. Yanagisawa, T. Masaki, and K. Yamashita. Endothelin receptor is coupled to phospholipase C via a pertussis toxin-insensitive guanine nucleotide-binding regulatory protein in vascular smooth muscle cells. J. Clin. Invest. 85: 653-658, 1990. 22. Warner, T. D., G. D. Nucci, and J. R. Vane. Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat. Eur. J. Pharmacol. 159: 325-326,1989. 23. Watanabe, H., H. Miyazaki, M. Kondoh, Y. Masuda, S. Kimura, M. Yanagisawa, T. Masaki, and K. Murakami. Two distinct types of endothelin receptors are present on chick cardiac membranes. Biochem. Biophys. Res. Commun. 161: 12521259,1989. 24. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazawa, K. Goto, and T. Masaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988. 25. Yoshinaga, M., Y. Chijiiwa, T. Misawa, N. Harada, and H. Nawata. Contractile effect of the vasoactive intestinal contractor on isolated smooth muscle cells of guinea pig small intestine. Med. Sci. Res. 18: 509-510, 1990.
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endothelin
receptor
subtype; binding
assay
(ET) is a peptide consisting of 21 amino acid residues that was isolated from vascular endothelium and has been reported to exert a potent vasoconstrictive effect (24). Cloning studies of genomic DNA have shown that there are three distinct members of the ET family (7). All three isopeptides (ET-l, ET-2, and ET-3) cause contraction of smooth muscle from the vas deferens, bronchus, urinary bladder, and ileum, as well as vascular smooth muscle (10). A novel peptide, vasoactive intestinal contractor (VIC), which differs from ET-l in only three amino acid residues, has been cloned and sequenced from the mouse genome (17) and is now considered a mouse variant of ET-2. Northern blot analysis has indicated that the gene for VIC is expressed only in the intestine and not in other tissues or endothelial cells (17). Ishida et al. (8) have reported that VIC causes a potent, long-lasting contraction of guinea pig ileal longitudinal muscle strips. This contraction has a slow onset and is inhibited by nicardipine. We also have reported the direct contractile effect of VIC on isolated longitudinal smooth muscle cells from the guinea pig small intestine (25). These results strongly suggest the existence of the functional receptor for VIC on longitudinal smooth muscle cells of guinea pig small intestine. The present study was designed to investigate by using VIC, a variant of ET-2, 1) the binding characteristics of ET receptor on isolated longitudinal smooth muscle cells obtained from guinea pig small intestine and 2) the role of intracellular and extracellular Ca2+ in ET-induced smooth muscle cell contraction.
ENDOTHELIN
G308
0193~1857/92
$2.00
Copyright
MATERIALS
AND
METHODS
Materials. VIC, ET-l, ET-Z, and ET-3 were obtained from the Peptide Institute (Osaka, Japan); collagenase (CLS type II) from Worthington Biochemicals (Freehold, NJ); trypsin inhibitor (type I-S), 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB -8)) bovine serum albumin (BSA), and bacitracin were from Sigma Chemical (St. Louis, MO); N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) from Wako Pure Chemical (Osaka, Japan); acrolein from Tokyo Kasei (Tokyo); and 1251-VIC (sp act 74 TBq/mmol) from Amersham International (Amersham, UK). Preparation of dispersed smooth muscle cells. Smooth muscle cells were isolated from the longitudinal muscle layer of the guinea pig small intestine by the method of Bitar and Makhlouf (2). In brief, male Hartley guinea pigs (300-600 g) raised on a standard diet at the Animal Center of Kyushu University (Fukuoka, Japan) were fasted overnight and killed by the intracardiac injection of pentobarbital sodium (100 mg/kg). The small intestine was excised from each animal and placed in oxygenated ice-cold HEPES medium [(in mM) 120 NaCl, 2.6 KH2P04, 4 KCl, 2 CaC12, 0.6 MgC12, 25 HEPES, 14 glucose, and 0.01% trypsin inhibitor, pH 7.41. The longitudinal muscle layer was peeled off by tangential stroking with wet gauze along the mesenteric border, cut into small pieces, and incubated for two successive 45-min periods at 31°C in 15 ml of HEPES medium containing 150 U/ml of collagenase. After incubation, the partly digested strips were washed with 50 ml of enzyme-free HEPES medium and reincubated in 15 ml of fresh HEPES medium for 20-30 min at 31°C to allow the cells to disperse spontaneously. Cells were then harvested by filtration through a 500~pm polyester mesh. 1251-VIC binding to dispersed cells. HEPES medium containing 2% BSA and 0.3 mg/ml bacitracin was used for the binding studies. Aliquots (0.5 ml) of the dispersed cell suspension (10” cells/mm”) were preincubated at room temperature for 10 min, with or without 0.1 ml of unlabeled VIC, and then incubated with 0.1 ml of 1251-VIC (final concentration 25 PM) at room temperature for a given period. All binding studies were performed in duplicate. At the end of incubation, bound and free 1251-VIC was separated by centrifugation at 900 g for 2 min at 4°C. The radioactivity bound to cells was measured using a gamma scintillation counter. Specific binding was defined as the total binding minus the nonspecific binding determined in the presence of an excess (IO-” M) of unlabeled VIC. The timecourse changes of 1251-VIC binding at room temperature were determined in a separate series of experiments in which binding proceeded for 5, 10, 20, 40, 60, or 90 min. The ability of ET family peptides (ET-l, ET-Z, VIC, and ET-3) to displace 1251VIC was examined using unlabeled peptides in the concentration range of IO-l2 M to lOA M. Scatchard plot were analyzed by linear regression to determine dissociation constant (&) and the maximum binding capacity (B,,,). Contractile response of dispersed cells. Dispersed cells were stimulated by test agents, and the contractile response was measured by the method of Bitar and Makhlouf (2). Aliquots
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 14, 2019.
ETB
RECEPTOR
ON
INTESTINAL
(0.5 ml) of cells were added to 0.2 ml of a solution containing each test agent for a given time. The kinetics of the contractile response to VIC were studied by incubating cells for various periods in the presence of 10e7 M VIC. The reaction was interrupted by the addition of acrolein at a final concentration of 1%. The lengths of 50 cells in microscopic fields were measured by image-splitting micrometry, and the percent decrease in mean cell length was determined by comparison with the control. To examine the role of intracellular and extracellular Ca2+ in the VIC-induced contraction of longitudinal smooth muscle cells, we added TMB-8 [an inhibitor of intracellular Ca2’ release (ll)] or verapamil (a Ca2’ channel blocker) to aliquots of cells simultaneously with 10e7 M VIC. The percentage contraction was determined in comparison with the VIC-induced contraction. Student’s t test or Welch’s method after inspection of variances was used to determine the significance of suppressive effect of TMB-8 and verapamil on VIC-induced contraction. Probability values ~0.05 were considered to be significant.
SMOOTH
MUSCLE
G309
CELLS
ent manner and with the same affinity (Fig. 2). The 50% inhibitory concentration (IC50) value was 0.7 nM. Scatchard analysis of these data showed that Kd and B,,, were 0.9 nM and 37.3 fmol/5 x 10” cells, respectively. Contractile response of dispersed smooth muscle cells. The maximal contractile response of longitudinal smooth muscle cells was attained after 45 s, with a 16.9 t 2.3% (mean t SE) (Fig. 3) decrease in cell length from control. Subsequent experiments were performed with an incubation time of 45 s. VIC induced contraction of smooth
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RESULTS
iii
1251-VIC binding to dispersed cells. The specific binding of 1251-VICto dispersed longitudinal smooth muscle cells of the guinea pig small intestine reached an equilibrium after 60 min (Fig. 1). Total VIC binding was 8.3 t 0.7% of the added radioactivity, and specific binding was 72 t 4.1% (mean t SE, n = 4) of the total binding. To determine the specificity of VIC binding sites, a variety of peptides were tested to inhibit 1251-VIC with smooth muscle cells. Inclusion of up to lOA M vasoactive intestinal peptide, glucagon, calcitonin gene-related peptide, neuropeptide Y, substance P, somatostatin, motilin, and cholecystokinin-8 did not affect 12’I-VIC binding in longitudinal smooth muscle cells of guinea pig small intestine. Specific binding of 1251-VICwas inhibited by unlabeled VIC, ET-l, ET-Z, and ET-3 in a concentration-depend-
01-1 0
30 TIME
60
; 40-I G
0 cl
I
x 0
-12
-11
-10
-9
Log
[ PEPTIDE
-8
-7
WC
ET-1 ET-2 ET-3
-6
]
Fig. 2. Inhibition of binding of 12”I-VIC to isolated longitudinal smooth muscle cells by unlabeled VIC, endothelin (ET)-1, ET-2, and ET-3. Specific binding of 12”1-VIC is expressed as percentage of radioactivity bound to cells in absence of unlabeled peptide. Values are means t SE of 4 separate experiments.
I 90
(MINUTES)
Fig. 1. Time course of specific binding of l”“I-vasoactive intestinal contractor (VIC) to isolated longitudinal smooth muscle cells of guinea pig small intestine. Muscle cells were incubated with 25 pM “‘1-VIC in presence or absence of 1 PM unlabeled VIC at room temperature. Data are expressed as percentage of radioactivity specifically bound to cells. Values are means t SE of 4 separate experiments.
0
30
TIME
60
90
120
(SECONDS)
Fig. 3. VIC (10e7 M)-induced contractile response of isolated longitudinal smooth muscle cells from guinea pig small intestine. Values are means t SE of 5-7 experiments. Vertical axis shows percent decrease in mean cell length compared with control.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 14, 2019.
G310
ETB
RECEPTOR
ON
INTESTINAL
muscle cells in a dose-dependent manner, with and 50% effective dose (ED& value of 5 nM. ET-l, ET-Z, and ET-3 also evoked contraction of smooth muscle cells with the same potency as VIC (Fig. 4). TMB-8 and verapamil significantly inhibited the contractile response of longitudinal smooth cells to VIC in a dose-dependent manner (Fig. 5), with IC& values of 0.1 and 0.9 PM, respectively. DISCUSSION
To our knowledge, this is the first study demonstrating the existence of an ETB receptor on longitudinal smooth
WC ET-1 ET-2 ET-3
1
1
1
1
’
’
1
-11
-10
-9
-8
-7
-6
Log
[ PEPTIDE
]
Fig. 4. Contractile response of isolated longitudinal smooth muscle cells of guinea pig small intestine to various concentrations of VIC, ET-l, ET-2, and ET-3. Each value shows mean t SE (n = 5 experiments).
---o--
o-1 0
Ca2+
TMB-8 VERAPAMIL
1
'
'
'
'
1o-g
1o-8
1o-7
lo+
ANTAGONIST
'
1o-5
(mol /L)
Fig. 5. Inhibitory effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) and verapamil on contraction of isolated longitudinal smooth muscle cells produced by VIC (10s7 M). Each value shows mean k SE (n = 4-6 experiments). *p < 0.05. **P c 0.01. ***ip < 0.001.
SMOOTH
MUSCLE
CELLS
muscle cells of the guinea pig small intestine. We showed that the affinity of 1251-VIC binding to smooth muscle cells (I&o 0.7 nM, & 0.9 nM) is similar to the pharmacological effect of VIC in producing smooth muscle cell contraction (ED50 5 nM). This result indicates that a functional receptor for VIC is expressed on the smooth muscle cells. VIC is now considered a mouse variant of ET-Z. Our study showed VIC and ET-Z had a similar binding affinity and contractile potency in these cells. These results therefore indicate a functional receptor for VIC can be regarded as an ET receptor on smooth muscle cells of guinea pig small intestine. ET receptors are classified into subtypes. Sakurai et al. (18) proposed two ET receptor subtypes: the ETA receptor (ET-l/ET-Z selective subtype) and the ETB receptor (non-isopeptide-selective subtype). Recently, Arai et al. (1) reported the cloning of cDNA encoding a bovine ET receptor that shows high affinity for ET-l. Sakurai et al. (18) also reported the cloning of cDNA encoding a rat nonselective ET (ETB) receptor. The ETA receptor is expressed on vascular and nonvascular tissues, such as chick cardiac membranes (23), rat vascular smooth muscle cells (6), and rat uterus (5). In a pharmacological study, ET-l and ET-2 caused stronger contraction of vascular and uterine smooth muscle strips than did ET-3 (5,7). On the other hand, the ETB receptor is expressed on many rat tissues but not on vascular smooth muscle cells (18). In isolated perfused rat mesentery, Warner et al. (22) reported that ET-l and ET-3, at low doses, were equipotent as vasodilators acting through the release of endothelium-derived relaxing factor. On the contrary, ET-l, at high doses, was -10 times more potent as a vasoconstrictor than ET-3 (22). It was also suggested that the ET-l/ET-2-selective receptor (ETA receptor) primarily mediated vasoconstriction, and the non-isopeptide-selective receptor (ETB receptor) led to the formation of endothelium-derived relaxing factor and vasodilation (12). In our study, the functional ET receptor on longitudinal smooth muscle cells of guinea pig small intestine showed similar affinities for ET-l and ET-3. Moreover, ET-3 induced smooth muscle cell contraction as effectively as did ET-l. These results strongly suggest that the functional ET receptor on longitudinal smooth muscle cells of the guinea pig small intestine is classified into the ETB receptor and that the ETB receptor primarily mediates small intestinal smooth muscle contraction. Thus there is a difference between ETinduced intestinal contraction and vasoconstriction. ET-l and ET-3 have been isolated from the porcine brain (19), and Takayanagi et al. (20) have shown that ET-3 was almost equipotent with ET-l in binding to the porcine cerebellum. Nambi et al. (16) performed crosslinking experiments with 1251-ET-l and 1251-ET-3, using membranes from various parts of the rat brain, and found that two specific bands were labeled (mol wt 45,000 and 37,000). These labeled bands were diminished in the presence of excess unlabeled ET-l or ET-3 (16). Crawford et al. (3) have reported that ET-3 as well as ET-l stimulated phosphoinositide hydrolysis in the rat cerebellum and cerebral cortex. From these findings and our results, the ET-receptor subtype expressed by longitu-
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (150.216.068.200) on January 14, 2019.
ETB
RECEPTOR
ON
INTESTINAL
dinal smooth muscle cells of the guinea pig small intestine is suggested to be similar to that found in the central nervous system. ET-3 is abundant in the intestine, pituitary gland, and brain of the rat (13), suggesting the possibility that it may act as a brain-gut peptide. It has been variously reported that the mechanism of the ET-induced contraction of vascular smooth muscle cells is dependent on Ca2+ release from intracellular storage sites (14, 15), Ca2+ influx through Ca2+ channels (4, 21, 24), or protein kinase C activation (9). In the present study, we used two Ca2+ antagonists with different mechanisms to examine the role of intracellular and extracellular Ca2+ in VIC-induced longitudinal smooth muscle cell contraction. Contraction of the cells was inhibited by both TMB-8 and verapamil in a dose-dependent manner. So the VIC-induced contraction of longitudinal smooth muscle cells appears to depend on both intracellular and extracellular Ca2+. In conclusion, our new findings were as follows: 1) the functional ET receptor that shows a similar affinity for ET-l, ET-2, VIC, and ET-3 exists on longitudinal smooth muscle cells of the guinea pig small intestine; 2) ET-l, ET-2, VIC, and ET-3 cause contraction of longitudinal cells with similar potency; and 3) both TMB-8 and verapamil inhibit the VIC-induced contraction of longitudinal smooth muscle cells. These results strongly suggest that longitudinal sm 00th muscle cells of the small intestine express the ETB receptor that primarily mediates contraction of the small intestinal smooth muscle cell. In addition, not only the release of Ca2+ from intracellular stores but also the influx of Ca2+ appears to be required for the maximal smooth muscle cell contractile response to ET. Address Received
reprint 23 January
requests
to M. Yoshinaga.
1991; accepted
in final
form
9 September
1991.
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