Br. J. Pharmacol. (1991), 102, 621-626
(D Macmillan Press Ltd, 1991
Effects of vasoactive intestinal peptide (VIP) on contractile responses of smooth muscle in rat stomach Toshio Ohta, Shigeo Ito & Akira Ohga Department of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan 1 The effects of vasoactive intestinal peptide (VIP) on contractile responses to carbachol (CCh), KCl and caffeine of the circular smooth muscle in rat stomach were examined by the isometric tension recording method and by measurement of the intracellular Ca level, [Ca]j, with fura 2. 2 Removal of extracellular Ca or nifedipine (0.1 UM) inhibited contractions induced by KCl (40 mM) and a low concentration (1 UM) of CCh but not that induced by caffeine (3mM). After these treatments, the contraction induced by a high concentration of CCh (100pM) changed to a phasic response. 3 VIP dose-dependently inhibited the contraction induced by 1,UM CCh, but not those caused by 40mm KCl or 3 mm caffeine. 4 In Ca-free solution containing 2mM EGTA, VIP inhibited the phasic contraction induced by 100pM CCh, but not that induced by 30 mm caffeine. 5 CCh caused dose-dependent tension development concomitant with the increase in [Ca]j. VIP reduced both responses and thus did not affect the [Ca]i-force relation for CCh. In the chemically skinned muscle fibres, VIP had no effect on the pCa-tension relation. 6 It is suggested that the inhibitory effects of VIP on CCh-induced contractions are due to the inhibition of the processes of signal transduction from muscarinic receptors to voltage-dependent Ca channels and to intracellular Ca stores.
Introduction Vasoactive intestinal peptide (VIP) produces relaxation of various smooth muscles (Piper et al., 1970; Said & Mutt, 1970). VIP has been suggested as a neurotransmitter candidate in non-adrenergic, non-cholinergic (NANC) inhibitory nerves in several tissues, including rat stomach (De Beurma & Lefebvre, 1988; Ito et al., 1988; 1990; Kamata et al., 1988). In the rat stomach, we have found that there is a good correlation between the ontogenesis of the functional innervation of NANC inhibitory nerves and that of VIP immunoreactive fibres (Ito et al., 1988), and that the electrical and cyclic nucleotide responses to activation of these nerves are similar to those to VIP (Ito et al., 1990). The contractions of various smooth muscles depend on the increase in the intracellular free-Ca concentration produced by an influx of extracellular Ca through voltage-dependent or receptor-operated Ca channels and by the release of Ca from intracellular Ca stores (Bolton, 1979; Kuriyama et al., 1982). VIP has been shown to produce a decoupling of electrical and mechanical activities of smooth muscle (Morgan et al., 1978; Ito & Takeda, 1982). However, the effect of VIP on the regulatory function of Ca is not well understood in visceral smooth muscles, though it has been described in vascular smooth muscles (Itoh et al., 1985). KCl, carbachol (CCh) and caffeine have been reported to produce contractions through different sources of Ca in many tissues (Bolton, 1979). Therefore, the present study attempts to examine the effects of VIP on contractile responses to these agents, on intracellular Ca levels in intact smooth muscles and on the pCa-tension relation in skinned muscles of rat stomach.
Methods Wistar rats of either sex, weighing 200-300 g, were stunned and exsanguinated. Their stomachs were excised and cut in the longitudinal direction along the greater curvature. With the help of a stereomicroscope, the mucosal, submucosal and longitudinal muscle layers were carefully removed from the
circular muscle layer of the antrum in a dissection chamber filled with HEPES-Krebs solution. Then antral circular muscle strips (about 0.1 mm in width and 0.7-1 mm in length) were prepared with fine forceps and small knives made from razor blades.
Recording of mechanical activity Mechanical activities of intact and skinned muscle fibres were measured isometrically by attaching a circular strip to a strain gauge transducer (AE801; AME, Norway). Each end of the strip was knotted by a single silk thread and tied to two tungsten needles. One of the needles was connected to the strain gauge transducer and the other to a manipulator. Experimental solutions were poured into small wells (volume about 0.5 ml) engraved on an aluminium plate in such a way that the surface of the solution was raised by surface tension from the plate surface. The muscle strip was placed horizontally into the meniscus of the solution, so that by moving the plate horizontally it could be transferred from one well into another rapidly (Ohta et al., 1989). After a 30 min equilibration period, the strip was repeatedly exposed to isotonic KCI (150 mM) for 1 min until responses become stable, after which the experiments were started. The temperature of the plate was controlled by circulating water or exothermic transistors underneath the plate. The experiments were carried out at 32-34°C for intact muscle fibres and at 200C for skinned muscle fibres. Chemically skinned muscle fibres were obtained by exposing the fibres to saponin (50gml- 1) in relaxing solution for 30 min at 20°C. To prevent the deterioration of the contractile response of skinned muscle fibres, calmodulin (1 pM) was added throughout the experiments. Calmodulin was prepared from bovine brain according to the method of Gopalakrishna & Anderson (1982).
Measurement of intracellular Ca level The intracellular Ca level was measured with a fluorescent indicator, fura 2 (Grynkiewicz et al., 1985). Antral circular
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muscle strips were exposed to 1OflM fura 2 acetoxymethyl ester (fura 2-AM) in HEPES-Krebs solution for 2-3 h at 32-340C with constant mixing. Pluronic F127 (0.005%), a noncytotoxic detergent, was also added in order to increase the solubility of fura 2-AM. After the fura 2 loading, muscle strips were washed with fura 2-AM-free HEPES-Krebs solution for more than 30min and then used for experiments. Experiments were performed with a fluorometer using dual wavelength excitation (CAM-200, Japan Spectroscopic, Tokyo, Japan) as described by Yanagisawa et al. (1989). In brief, the muscle strip loaded with fura 2 was positioned horizontally in a plastic chamber (volume about 0.5ml) on an inverted microscope stage for simultaneous measurement of tension and the fluorescent signal. Experimental solutions were rapidly injected by a syringe from one end of the organ bath and overflowing solution was aspirated from the other end. The tension of the muscle strip was recorded with a strain gauge transducer as described above. The excitation light was obtained from a Xenon high-pressure lamp (150W) equipped with a monochrometer and the excitation light alternating between 340 and 380 nm (10 nm-band path) was obtained with a chopping wheel (100Hz). The ratio of the fluorescence due to excitation at 340 nm to that of 380 nm was calculated from successive illumination periods and is referred to as the F340/F380. The fluorescent signal was obtained by using a Nikon CF UV (Fluor) lens and the light emitted from the muscle fibres was collected by a photomultiplier through a 500 nm (20 nm-band path) filter. The present results were obtained from the preparations in which the fluorescent signals excited by F340 and F380 became mirror images. In the present experiments, the absolute intracellular Ca concentration was not determined, since intracellular fura 2 binds to some soluble proteins and the binding constant of fura 2 to Ca in cytoplasm is not known. Therefore, F340/F380 was used as an indicator of relative [Ca2 ]i (Himpens & Somlyo,
1988; Ozaki et al., 1988).
Solutions The ionic composition of the HEPES-Krebs solution was as follows (mM): NaCl 144, KCl 5.8, MgCl2 1.2, CaCl2 2.5, 2-[4(2-hydroxyethyl)-1-piperazinyl]-ethanesulphonate (HEPES) 5, glucose 11.1 (pH 7.4). High-KCI solutions were prepared by replacing NaCl with KCl isosmotically. In Ca-free solution, CaCl2 was omitted and 2 mm ethyleneglycol-bis-(#-aminoethylether)N,N'-tetraacetic acid (EGTA) was always added. The pH was adjusted with NaOH or KOH. In the majority of the experiments, the bathing solutions were not pre-gassed with 02. The contractile responses in the solutions pre-gassed with 02 were not different from those in the solutions without gassing.
All solutions used to soak the skinned muscle fibres contained 20 mm piperazine-N,N'-bis(ethylenesulphonate) (PIPES) as a pH buffer (pH 7.0 with KOH) and 3.5 mm MgATP, and had an ionic strength of 0.2 M, adjusted with K methanesulphonate (Ms). Solutions activating the skinned muscle fibres contained various concentrations of free Ca buffered by 10 mM EGTA. The free-Ca concentration was changed by adding appropriate amounts of CaMs2 to EGTA. The apparent binding constant of EGTA for Ca was calculated by the formula given by Harafuji & Ogawa (1980). The relaxing solution was composed of (mM): MgMs2 5.1, ATPNa2 4.2, KMs 126, EGTA 2 and PIPES 20.
a
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Figure 1 The effects of nifedipine (b) and extracellular Ca removal (c) on contractile responses to KCI, carbachol (CCh) and caffeine. (a) Control. CCh (IpM; *, 100pM; El), KCI (40mM; 0) and caffeine (3mM; lD) were applied for min. Nifedipine (0.1 pM) was applied more than 10 min before application of these agents. Extracellular Ca removal was performed by treatment with Ca-free solution containing 2 mM EGTA for 2 min.
Results
Effects of extracellular Ca removal and nifedipine on carbachol-, KCl- and caffeine-induced contractions In order to determine the source of Ca contributing to contractions evoked by CCh, KCI and caffeine, the effects of extracellular Ca removal and nifedipine, a specific inhibitor of dihydropyridine (DHP)-sensitive voltage-dependent Ca channels, were examined. Typical records of contractions evoked by CCh, KCI and caffeine before and after these treatments are shown in Figure 1 and their relative amplitudes are summarized in Table 1. Application of these three agents for 1 min evoked dose-dependent contractions (CCh, 0.1-100puM; KCI,
10-150mM; caffeine; 0.5-30mM) and the 50-75% effective concentration was 1 gM for CCh, 40mM for KCI and 3 mm for
caffeine. As shown in Figure la, both KC1 (40mM) and CCh (1 fM and 100pM) produced a phasic contraction followed by a tonic one. In some cases, CCh at 1 gM caused oscillatory contractions superimposed on the tonic component. In contrast, 3 mm caffeine elicited a phasic contraction only. After treatment with 0.1 uM nifedipine (Figure lb) or Ca-free solution containing 2mm EGTA (Figure ic), 40mM KCI evoked neither a phasic nor a tonic response and the 1 fM CCh-induced contraction was also greatly inhibited, while the Table 1 The effects of extracellular Ca removal and nifedipine on carbachol (CCh)-, KCI- and caffeine-induced contractions
Drugs
CCh
The chemicals used were: ATPNa2 (Boehringer), caffeine (Wako), carbachol and verapamil (Sigma), methoxyverapamil (D600, Ezai), EGTA, fura 2-AM, HEPES, PIPES and pluronic F127 (Dojin), Ms (Tokyo Kasei), nifedipine (Bayer), saponin (ICN) and VIP (Protein Inc.).
KCl Caffeine
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Ca-free
Nifedipine
4.2 + 0.8 (n = 7) 38.5 + 5.2 (n = 6) 0 (n= 7) 86.6 + 6.9 (n = 5)
3.3+ 1.0(n-6) 34.6 + 4.1 (n = 8) (n= 8) 0 91.2 + 3.8 (n = 8)
Each value indicates the % of the peak contractile response to each drug before Ca removal or treatment with nifedipine (0.1 pM). For further explanation, see the legend to Figure 1.
INHIBITORY EFFECT OF VIP ON SMOOTH MUSCLE a
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VIP (nM) Figure 2 Effects of vasoactive intestinal peptide (VIP) on contractions evoked by carbachol (CCh), KCI and caffeine. (a) Typical records of effects of VIP (lOOnM) on CCh- (1,aM; M, 100pUM; l), KCI(40mM; E) and caffeine- (3mM; ED) induced contractions. (b) Concentration-response curves for VIP on contractions evoked by CCh (1 AIm; 0, n = 8) and caffeine (3 mM; El, n = 6) and on the phasic (A, n = 8) and tonic (A, n = 8) responses of KCI- (40mM) induced contractions. The amplitude of contraction evoked in the absence of VIP was normalized as 100%. Vertical lines indicate s.e.mean.
phasic contraction evoked by 3 mm caffeine was only slightly reduced. The tonic contraction but not the phasic one induced by a high concentration of CCh (100piM) was inhibited by these treatments. Other Ca channel blockers, such as D600 and verapamil, had effects similar to that of nifedipine.
Effect of vasoactive intestinal peptide on the contractile responses to carbachol-, KCI- and caffeine The inhibitory effect of VIP on CCh-induced contraction developed gradually. When CCh (1 gM) and VIP (30 nM) were applied simultaneously, the peak amplitude of the CChinduced contraction was 97.3 + 2.4% of the control. The contractile response to CCh was reduced to 74.8 + 4.1% for 1 min pretreatment with VIP, 55.3 + 2.6% for 2 min, 40.4 + 5.0% for 4min and 41.7 + 1.9% for 8 min (n = 4). Therefore, the contractile responses to the agents were examined more than 4min after treatment with VIP. Figure 2a shows typical contractile responses to CCh, KCI and caffeine in the presence and absence of VIP (100 nM). VIP greatly inhibited the 1 M CCh-induced contraction. Although VIP markedly slowed the rising phase of the contraction induced by CCh at 100pM, it did not affect its peak amplitude of contraction. KCI and caffeine-induced contractions were hardly affected by VIP. Figure 2b shows the effects of various concentrations of VIP on the 1 am CCh-, 40 mM KCI- and 3 mm caffeine-induced contractions. VIP inhibited the contraction evoked by 1 gUM CCh in a dose-dependent manner and its 50% inhibitory dose was about 20 nm. In contrast, contractions evoked by KCI and caffeine were only slightly affected even after much longer pretreatment with VIP.
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Relative F340/F380 (%) Figure 3 Effects of vasoactive intestinal peptide (VIP) on increases in [Ca]i and contractions in response to carbachol (CCh). (a) Typical chart recording. The intracellular Ca level, [Ca]j, is indicated by F340/F380. (b) Relation between the increase in [Ca]i and tension in response to CCh in the presence (0; n = 5) and absence (0; n = 6) of VIP (100nM). Numbers below symbols indicate CCh concentrations (AM). The amplitude of tension and increase in [Ca]i in response to CCh (100#M) in the absence of VIP were normalized as 100%. Vertical and horizontal lines indicate s.e.mean.
Effects of vasoactive intestinal peptide on the intracellular Ca level increase induced by carbachol Figure 3a shows the effects of VIP (100 nM) on the rise in [Ca]i and tension development in response to various concentrations of CCh. The increase in [Ca]i in response to CCh preceded the development of tension. Although VIP did not affect the resting [Ca]i level, it reduced the increase in the [Ca]i level and contraction in response to CCh simultaneously. Figure 3b shows the relationship between the increase in the [Ca]1 level and the amplitude of contraction in response to CCh in the presence and absence of VIP (100 nM). This relation, obtained in the presence of VIP, was not different from that in the absence of VIP. This result indicates that the inhibition of CCh-induced contraction by VIP is accompanied by a decrease in intracellular Ca.
Effects of vasoactive intestinal peptide on contractions utilizing intracellular stored Ca In gastric circular smooth muscles, CCh at a high concentration and caffeine evoked monophasic contractions in the Ca-free solution as shown in Figure 1c. We examined the effects of VIP on these contractions in detail. After the Ca stored in muscle cells was completely depleted by repetitive applications of caffeine (30 mM) in Ca-free solution containing 2 mm EGTA, the muscle was exposed to 2.5 mm Ca together with isotonic KCl (150 mM) for 2 min to load the Ca in intracellular Ca stores (Ca-loading). After the tissue was washed for 4min with repeatedly changing Ca-free solutions containing
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Figure 4 Effects of vasoactive intestinal peptide (VIP) on carbachol (CCh)- and caffeine-induced contractions in Ca-free solution containing 2mM EGTA. After 2.5mm Ca was applied together with isotonic KCI (150mM) for 2min (Ca-loading), the muscle fibre was washed with Ca-free solution containing 2mM EGTA for 4min (Ca-washing). Thereafter, (a) CCh (100pM; *) or (b) caffeine (30mM; El) was applied for 30s in Ca-free solution containing 2mM EGTA. Chart speed is 4 times slower in the Ca-loading and Ca-washing steps than in the next steps. (i) Control; (ii) VIP (100nM) was applied after Ca-loading; (iii) VIP was applied before Ca-loading. (c) The % amplitude of contractions evoked by CCh and caffeine in Ca-free solutions containing 2mm EGTA. Open columns show the data obtained when VIP is introduced after Ca-loading and hatched columns before Ca-loading (n 5). Horizontal lines indicate s.e.mean. =
0 Figure 5 Effects of vasoactive intestinal peptide (VIP) on successive carbachol (CCh)- and caffeine-induced contractions in Ca-free solution containing 2mM EGTA. After Ca-loading, CCh (100pM; *) and caffeine (30mM; El) were applied successively for 30s with a min interval. (a) Control; (b) VIP (100nM) was applied after Ca-loading. For further explanations, see the legend to Figure 4.
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2 mm EGTA (Ca-washing), application of CCh (100puM) or caffeine (30 mM) for 30s produced a transient contraction in the Ca-free solution containing 2mM EGTA (Figures 4a(i) and 4b(i)). Contractions no longer occurred when KCI (40 mM) was applied instead of CCh or caffeine, indicating that external Ca was sufficiently removed during the Ca-washing period (data not shown). VIP (100 nM) was applied during the periods of Ca-washing and subsequent CCh or caffeine stimulation. VIP significantly inhibited the contraction evoked by CCh in a dose-dependent manner, but not that caused by caffeine (Figures 4a(ii), 4b(ii) and open columns in 4c). When VIP was applied from the time of Ca-loading, the inhibitory effect of VIP was less pronounced (Figure 4a(iii) and hatched columns in 4c). In order to see whether this inhibitory effect of VIP was due to the inhibition of Ca release from intracellular Ca stores or to an enhancement of Ca extrusion through the plasma membrane, the contractions induced in Ca-free solution containing 2 mm EGTA by successive applications of CCh and caffeine were examined in the presence or absence of 100 nm VIP (Figure 5). After CCh had evoked a contraction, a small contraction could be evoked by the application of caffeine.
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pCa Figure 6 Effects of vasoactive intestinal peptide (VIP) on the Cainduced contraction in skinned fibres. (a) (i) Isotonic KCI (150mM)induced contraction in intact muscles. (ii) After skinning with saponin, various concentrations of Ca were applied cumulatively. After the pCa 6.3-induced contraction reached a steady level, VIP (100nM) was applied. (b) pCa-tension relation observed in the presence (C; n = 4) or absence (0; n = 5) of VIP (100nM). The amplitude of contraction evoked by pCa 5.0 in the absence of VIP was normalized as 100%. Vertical lines indicate s.e.mean.
INHIBITORY EFFECT OF VIP ON SMOOTH MUSCLE
However, after caffeine evoked a contraction, CCh or caffeine did not (data not shown). When the contraction induced by a preceding application of CCh was inhibited by VIP, the caffeine-induced contraction was enhanced (3.7 + 0.3 times the control, n = 10)
Effects of vasoactive intestinal peptide on the contractile machinery in skinnedfibres Figure 6a shows a typical record of contraction induced by cumulative application of Ca and the effect of VIP (100pM) on the contraction evoked by pCa 6.3 in skinned muscle fibres. VIP did not modify the amplitude of contraction induced by Ca. The effect of VIP (100nM) on the cumulative pCa-tension relation is shown in Figure 6b. The minimal concentration of Ca required to produce the contraction of skinned muscle fibres was pCa 6.7 and the amplitude of contraction was increased with increasing concentrations of Ca up to pCa 5.7. VIP had no effect on the pCa-tension relation.
Discussion The present results indicate that VIP inhibits the contraction induced by CCh but not those caused by KCl and caffeine in the gastric circular smooth muscle of the rat. VIP inhibited the increase in [Ca]i together with tension development in response to CCh and thus did not affect the [Ca]i-tension relation for CCh. Therefore, it seems likely that the change in the Ca sensitivity of the contractile system is not responsible for the inhibitory action of VIP on CCh-induced contraction. Furthermore, VIP did not influence the pCa-tension relation in skinned fibres of gastric smooth muscles, as was the case of vascular smooth muscles (Itoh et al., 1985). The contraction evoked by CCh at a low concentration was inhibited by the removal of extracellular Ca or by treatment with the Ca channel blocker, nifedipine. As VIP was effective in inhibition of the contractions and the rise in [Ca]i in response to CCh, VIP probably inhibits the CCh-induced Ca influx across the cell membrane through DHP-sensitive voltage-dependent Ca channels. However, the KCl-induced contraction was resistant to VIP. Therefore, it seems unlikely that the action of VIP on CCh-induced contractions is due to a direct inhibition of voltage-dependent Ca channels. VIP could, however, interefere with the processes of signal transduction from muscarinic receptors to voltage-dependent Ca channels. The phasic contractions evoked by CCh and caffeine in Ca-free solution are known to be due to Ca release from intracellular Ca stores (Endo et al., 1980; Itoh et al., 1981). Similar phasic contractions induced by these agents were observed in gastric circular smooth muscles. In Ca-free solution contain-
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ing 2 mm EGTA, VIP inhibited the contraction induced by CCh but not that induced by caffeine. After VIP inhibited the CCh-induced contraction, the contraction induced by a subsequent application of caffeine was significantly enlarged in the presence of VIP. These results indicate that the Ca stores in this tissue are probably activated by both drugs and that the inhibitory action of VIP on the CCh-induced contraction in Ca-free solution may result from an inhibition of the CChinduced Ca release from intracellular Ca stores rather than from an increase in Ca extrusion through the cell membrane. The difference between the effects of VIP on CCh- and caffeine-induced contractions could be attributable to the fact that caffeine acts directly on intracellular Ca stores (Endo et al., 1980; Itoh et al., 1981) but CCh does not. It seems likely that VIP inhibits some steps between muscarinic receptor activation and the Ca release from intracellular Ca stores. It has been shown that muscarinic receptors couple to guanine nucleotide binding proteins (Haga et al., 1987) and stimulation of these receptors leads to the activation of phospholipase C, which causes inositol breakdown and the production of inositol 1,4,5-trisphosphate (UP3; Gardner et al., 1988; Secrest et al., 1989), a putative messenger for the mobilization of Ca from non-mitochondrial stores (Berridge & Irvine, 1984). Therefore, the contraction induced by CCh in the absence of extracellular Ca may result from IP3 formation in the rat gastric smooth muscle. More recently, we have found that VIP increases intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentrations and that the time course was comparable to that of relaxation (Ito et al., 1990). The time-dependence of the inhibitory effect of VIP on CCh-induced contraction may imply the involvement of second messenger systems, such as cyclic AMP. Furthermore, Ca sequestering action into the Ca stores has been reported to be increased by cyclic AMP (Bhalla et al., 1978; Itoh et al., 1982). The inhibitory effect of VIP on CCh-induced contractions in Ca-free solution containing 2mm EGTA was less pronounced with application of VIP before the Ca-loading period than after it, suggesting that VIP has a Ca-sequestering action on Ca storage sites in addition to the inhibition of CCh-induced Ca release. In conclusion, VIP inhibits CCh-induced contraction in the presence and absence of extracellular Ca. VIP seems not to have direct inhibitory effects on DHP-sensitive voltagedependent Ca channels, the Ca-release mechanism from intracellular Ca stores and the contractile system. The site of the inhibitory action of VIP may be the processes of signal transduction from muscarinic receptors to DHP-sensitive voltagedependent Ca channels and to intracellular Ca stores. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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Factors modifying contraction-relaxation cycle in vascular smooth muscles. Am. J. Physiol., 243, H641-H662. MORGAN, K.G., SCHMALZ, P.F. & SZURSZEWSKI, J.H. (1978). The inhibitory effects of vasoactive intestinal polypeptide on the mechanical and electrical activity of canine antral smooth muscle. J. Physiol., 282, 437-450. OHTA, T., ENDO, M., NAKANO, T., MOROHOSHI, Y., WANIKAWA, K.
& OHGA, A. (1989). Ca-induced Ca release in malignant hyperthermia-susceptible pig skeletal muscle. Am. J. Physiol., 256, C358-C367. OZAKI, H., SATOH, T., KARAKI, H. & ISHIDA, Y. (1988). Regulation of
metabolism and contraction by cytoplasmic calcium in the intestinal smooth muscle. J. Biol. Chem., 263, 14074-14079. PIPER, R.J., SAID, S.I. & VANE, J.R. (1970). Effects on smooth muscle preparations of unidentified vasoactive peptide from intestinal and lung. Nature, 225, 1144-1146. SAID, S.I. & MUT1T, V. (1970). Potent peripheral and splanchnic vasodilator peptide from normal gut. Nature, 225, 863-864. SECREST, R.A., SCHOEPP, D.D. & COHEN, M.L. (1989). Comparison of contractions to serotonin, carbamylcholine and prostaglandin F2, in rat stomach fundus. J. Pharmacol. Exp. Ther., 25 971-978. YANAGISAWA, T., KAWADA, M. & TAIRA, N. (1989). Nitroglycerin relaxes canine coronary arterial smooth muscle without reducing intracellular Ca2+ concentrations measured with fura-2. Br. J. Pharmacol., 98, 469-482.
(Received July 20, 1990 Revised October 29, 1990 Accepted November 5, 1990)
(D Macmillan Press Ltd, 1991
Effects of vasoactive intestinal peptide (VIP) on contractile responses of smooth muscle in rat stomach Toshio Ohta, Shigeo Ito & Akira Ohga Department of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan 1 The effects of vasoactive intestinal peptide (VIP) on contractile responses to carbachol (CCh), KCl and caffeine of the circular smooth muscle in rat stomach were examined by the isometric tension recording method and by measurement of the intracellular Ca level, [Ca]j, with fura 2. 2 Removal of extracellular Ca or nifedipine (0.1 UM) inhibited contractions induced by KCl (40 mM) and a low concentration (1 UM) of CCh but not that induced by caffeine (3mM). After these treatments, the contraction induced by a high concentration of CCh (100pM) changed to a phasic response. 3 VIP dose-dependently inhibited the contraction induced by 1,UM CCh, but not those caused by 40mm KCl or 3 mm caffeine. 4 In Ca-free solution containing 2mM EGTA, VIP inhibited the phasic contraction induced by 100pM CCh, but not that induced by 30 mm caffeine. 5 CCh caused dose-dependent tension development concomitant with the increase in [Ca]j. VIP reduced both responses and thus did not affect the [Ca]i-force relation for CCh. In the chemically skinned muscle fibres, VIP had no effect on the pCa-tension relation. 6 It is suggested that the inhibitory effects of VIP on CCh-induced contractions are due to the inhibition of the processes of signal transduction from muscarinic receptors to voltage-dependent Ca channels and to intracellular Ca stores.
Introduction Vasoactive intestinal peptide (VIP) produces relaxation of various smooth muscles (Piper et al., 1970; Said & Mutt, 1970). VIP has been suggested as a neurotransmitter candidate in non-adrenergic, non-cholinergic (NANC) inhibitory nerves in several tissues, including rat stomach (De Beurma & Lefebvre, 1988; Ito et al., 1988; 1990; Kamata et al., 1988). In the rat stomach, we have found that there is a good correlation between the ontogenesis of the functional innervation of NANC inhibitory nerves and that of VIP immunoreactive fibres (Ito et al., 1988), and that the electrical and cyclic nucleotide responses to activation of these nerves are similar to those to VIP (Ito et al., 1990). The contractions of various smooth muscles depend on the increase in the intracellular free-Ca concentration produced by an influx of extracellular Ca through voltage-dependent or receptor-operated Ca channels and by the release of Ca from intracellular Ca stores (Bolton, 1979; Kuriyama et al., 1982). VIP has been shown to produce a decoupling of electrical and mechanical activities of smooth muscle (Morgan et al., 1978; Ito & Takeda, 1982). However, the effect of VIP on the regulatory function of Ca is not well understood in visceral smooth muscles, though it has been described in vascular smooth muscles (Itoh et al., 1985). KCl, carbachol (CCh) and caffeine have been reported to produce contractions through different sources of Ca in many tissues (Bolton, 1979). Therefore, the present study attempts to examine the effects of VIP on contractile responses to these agents, on intracellular Ca levels in intact smooth muscles and on the pCa-tension relation in skinned muscles of rat stomach.
Methods Wistar rats of either sex, weighing 200-300 g, were stunned and exsanguinated. Their stomachs were excised and cut in the longitudinal direction along the greater curvature. With the help of a stereomicroscope, the mucosal, submucosal and longitudinal muscle layers were carefully removed from the
circular muscle layer of the antrum in a dissection chamber filled with HEPES-Krebs solution. Then antral circular muscle strips (about 0.1 mm in width and 0.7-1 mm in length) were prepared with fine forceps and small knives made from razor blades.
Recording of mechanical activity Mechanical activities of intact and skinned muscle fibres were measured isometrically by attaching a circular strip to a strain gauge transducer (AE801; AME, Norway). Each end of the strip was knotted by a single silk thread and tied to two tungsten needles. One of the needles was connected to the strain gauge transducer and the other to a manipulator. Experimental solutions were poured into small wells (volume about 0.5 ml) engraved on an aluminium plate in such a way that the surface of the solution was raised by surface tension from the plate surface. The muscle strip was placed horizontally into the meniscus of the solution, so that by moving the plate horizontally it could be transferred from one well into another rapidly (Ohta et al., 1989). After a 30 min equilibration period, the strip was repeatedly exposed to isotonic KCI (150 mM) for 1 min until responses become stable, after which the experiments were started. The temperature of the plate was controlled by circulating water or exothermic transistors underneath the plate. The experiments were carried out at 32-34°C for intact muscle fibres and at 200C for skinned muscle fibres. Chemically skinned muscle fibres were obtained by exposing the fibres to saponin (50gml- 1) in relaxing solution for 30 min at 20°C. To prevent the deterioration of the contractile response of skinned muscle fibres, calmodulin (1 pM) was added throughout the experiments. Calmodulin was prepared from bovine brain according to the method of Gopalakrishna & Anderson (1982).
Measurement of intracellular Ca level The intracellular Ca level was measured with a fluorescent indicator, fura 2 (Grynkiewicz et al., 1985). Antral circular
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muscle strips were exposed to 1OflM fura 2 acetoxymethyl ester (fura 2-AM) in HEPES-Krebs solution for 2-3 h at 32-340C with constant mixing. Pluronic F127 (0.005%), a noncytotoxic detergent, was also added in order to increase the solubility of fura 2-AM. After the fura 2 loading, muscle strips were washed with fura 2-AM-free HEPES-Krebs solution for more than 30min and then used for experiments. Experiments were performed with a fluorometer using dual wavelength excitation (CAM-200, Japan Spectroscopic, Tokyo, Japan) as described by Yanagisawa et al. (1989). In brief, the muscle strip loaded with fura 2 was positioned horizontally in a plastic chamber (volume about 0.5ml) on an inverted microscope stage for simultaneous measurement of tension and the fluorescent signal. Experimental solutions were rapidly injected by a syringe from one end of the organ bath and overflowing solution was aspirated from the other end. The tension of the muscle strip was recorded with a strain gauge transducer as described above. The excitation light was obtained from a Xenon high-pressure lamp (150W) equipped with a monochrometer and the excitation light alternating between 340 and 380 nm (10 nm-band path) was obtained with a chopping wheel (100Hz). The ratio of the fluorescence due to excitation at 340 nm to that of 380 nm was calculated from successive illumination periods and is referred to as the F340/F380. The fluorescent signal was obtained by using a Nikon CF UV (Fluor) lens and the light emitted from the muscle fibres was collected by a photomultiplier through a 500 nm (20 nm-band path) filter. The present results were obtained from the preparations in which the fluorescent signals excited by F340 and F380 became mirror images. In the present experiments, the absolute intracellular Ca concentration was not determined, since intracellular fura 2 binds to some soluble proteins and the binding constant of fura 2 to Ca in cytoplasm is not known. Therefore, F340/F380 was used as an indicator of relative [Ca2 ]i (Himpens & Somlyo,
1988; Ozaki et al., 1988).
Solutions The ionic composition of the HEPES-Krebs solution was as follows (mM): NaCl 144, KCl 5.8, MgCl2 1.2, CaCl2 2.5, 2-[4(2-hydroxyethyl)-1-piperazinyl]-ethanesulphonate (HEPES) 5, glucose 11.1 (pH 7.4). High-KCI solutions were prepared by replacing NaCl with KCl isosmotically. In Ca-free solution, CaCl2 was omitted and 2 mm ethyleneglycol-bis-(#-aminoethylether)N,N'-tetraacetic acid (EGTA) was always added. The pH was adjusted with NaOH or KOH. In the majority of the experiments, the bathing solutions were not pre-gassed with 02. The contractile responses in the solutions pre-gassed with 02 were not different from those in the solutions without gassing.
All solutions used to soak the skinned muscle fibres contained 20 mm piperazine-N,N'-bis(ethylenesulphonate) (PIPES) as a pH buffer (pH 7.0 with KOH) and 3.5 mm MgATP, and had an ionic strength of 0.2 M, adjusted with K methanesulphonate (Ms). Solutions activating the skinned muscle fibres contained various concentrations of free Ca buffered by 10 mM EGTA. The free-Ca concentration was changed by adding appropriate amounts of CaMs2 to EGTA. The apparent binding constant of EGTA for Ca was calculated by the formula given by Harafuji & Ogawa (1980). The relaxing solution was composed of (mM): MgMs2 5.1, ATPNa2 4.2, KMs 126, EGTA 2 and PIPES 20.
a
Nifedipine 0.1 ,wM
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Figure 1 The effects of nifedipine (b) and extracellular Ca removal (c) on contractile responses to KCI, carbachol (CCh) and caffeine. (a) Control. CCh (IpM; *, 100pM; El), KCI (40mM; 0) and caffeine (3mM; lD) were applied for min. Nifedipine (0.1 pM) was applied more than 10 min before application of these agents. Extracellular Ca removal was performed by treatment with Ca-free solution containing 2 mM EGTA for 2 min.
Results
Effects of extracellular Ca removal and nifedipine on carbachol-, KCl- and caffeine-induced contractions In order to determine the source of Ca contributing to contractions evoked by CCh, KCI and caffeine, the effects of extracellular Ca removal and nifedipine, a specific inhibitor of dihydropyridine (DHP)-sensitive voltage-dependent Ca channels, were examined. Typical records of contractions evoked by CCh, KCI and caffeine before and after these treatments are shown in Figure 1 and their relative amplitudes are summarized in Table 1. Application of these three agents for 1 min evoked dose-dependent contractions (CCh, 0.1-100puM; KCI,
10-150mM; caffeine; 0.5-30mM) and the 50-75% effective concentration was 1 gM for CCh, 40mM for KCI and 3 mm for
caffeine. As shown in Figure la, both KC1 (40mM) and CCh (1 fM and 100pM) produced a phasic contraction followed by a tonic one. In some cases, CCh at 1 gM caused oscillatory contractions superimposed on the tonic component. In contrast, 3 mm caffeine elicited a phasic contraction only. After treatment with 0.1 uM nifedipine (Figure lb) or Ca-free solution containing 2mm EGTA (Figure ic), 40mM KCI evoked neither a phasic nor a tonic response and the 1 fM CCh-induced contraction was also greatly inhibited, while the Table 1 The effects of extracellular Ca removal and nifedipine on carbachol (CCh)-, KCI- and caffeine-induced contractions
Drugs
CCh
The chemicals used were: ATPNa2 (Boehringer), caffeine (Wako), carbachol and verapamil (Sigma), methoxyverapamil (D600, Ezai), EGTA, fura 2-AM, HEPES, PIPES and pluronic F127 (Dojin), Ms (Tokyo Kasei), nifedipine (Bayer), saponin (ICN) and VIP (Protein Inc.).
KCl Caffeine
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40mM 3mM
Ca-free
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4.2 + 0.8 (n = 7) 38.5 + 5.2 (n = 6) 0 (n= 7) 86.6 + 6.9 (n = 5)
3.3+ 1.0(n-6) 34.6 + 4.1 (n = 8) (n= 8) 0 91.2 + 3.8 (n = 8)
Each value indicates the % of the peak contractile response to each drug before Ca removal or treatment with nifedipine (0.1 pM). For further explanation, see the legend to Figure 1.
INHIBITORY EFFECT OF VIP ON SMOOTH MUSCLE a
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VIP (nM) Figure 2 Effects of vasoactive intestinal peptide (VIP) on contractions evoked by carbachol (CCh), KCI and caffeine. (a) Typical records of effects of VIP (lOOnM) on CCh- (1,aM; M, 100pUM; l), KCI(40mM; E) and caffeine- (3mM; ED) induced contractions. (b) Concentration-response curves for VIP on contractions evoked by CCh (1 AIm; 0, n = 8) and caffeine (3 mM; El, n = 6) and on the phasic (A, n = 8) and tonic (A, n = 8) responses of KCI- (40mM) induced contractions. The amplitude of contraction evoked in the absence of VIP was normalized as 100%. Vertical lines indicate s.e.mean.
phasic contraction evoked by 3 mm caffeine was only slightly reduced. The tonic contraction but not the phasic one induced by a high concentration of CCh (100piM) was inhibited by these treatments. Other Ca channel blockers, such as D600 and verapamil, had effects similar to that of nifedipine.
Effect of vasoactive intestinal peptide on the contractile responses to carbachol-, KCI- and caffeine The inhibitory effect of VIP on CCh-induced contraction developed gradually. When CCh (1 gM) and VIP (30 nM) were applied simultaneously, the peak amplitude of the CChinduced contraction was 97.3 + 2.4% of the control. The contractile response to CCh was reduced to 74.8 + 4.1% for 1 min pretreatment with VIP, 55.3 + 2.6% for 2 min, 40.4 + 5.0% for 4min and 41.7 + 1.9% for 8 min (n = 4). Therefore, the contractile responses to the agents were examined more than 4min after treatment with VIP. Figure 2a shows typical contractile responses to CCh, KCI and caffeine in the presence and absence of VIP (100 nM). VIP greatly inhibited the 1 M CCh-induced contraction. Although VIP markedly slowed the rising phase of the contraction induced by CCh at 100pM, it did not affect its peak amplitude of contraction. KCI and caffeine-induced contractions were hardly affected by VIP. Figure 2b shows the effects of various concentrations of VIP on the 1 am CCh-, 40 mM KCI- and 3 mm caffeine-induced contractions. VIP inhibited the contraction evoked by 1 gUM CCh in a dose-dependent manner and its 50% inhibitory dose was about 20 nm. In contrast, contractions evoked by KCI and caffeine were only slightly affected even after much longer pretreatment with VIP.
0
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Relative F340/F380 (%) Figure 3 Effects of vasoactive intestinal peptide (VIP) on increases in [Ca]i and contractions in response to carbachol (CCh). (a) Typical chart recording. The intracellular Ca level, [Ca]j, is indicated by F340/F380. (b) Relation between the increase in [Ca]i and tension in response to CCh in the presence (0; n = 5) and absence (0; n = 6) of VIP (100nM). Numbers below symbols indicate CCh concentrations (AM). The amplitude of tension and increase in [Ca]i in response to CCh (100#M) in the absence of VIP were normalized as 100%. Vertical and horizontal lines indicate s.e.mean.
Effects of vasoactive intestinal peptide on the intracellular Ca level increase induced by carbachol Figure 3a shows the effects of VIP (100 nM) on the rise in [Ca]i and tension development in response to various concentrations of CCh. The increase in [Ca]i in response to CCh preceded the development of tension. Although VIP did not affect the resting [Ca]i level, it reduced the increase in the [Ca]i level and contraction in response to CCh simultaneously. Figure 3b shows the relationship between the increase in the [Ca]1 level and the amplitude of contraction in response to CCh in the presence and absence of VIP (100 nM). This relation, obtained in the presence of VIP, was not different from that in the absence of VIP. This result indicates that the inhibition of CCh-induced contraction by VIP is accompanied by a decrease in intracellular Ca.
Effects of vasoactive intestinal peptide on contractions utilizing intracellular stored Ca In gastric circular smooth muscles, CCh at a high concentration and caffeine evoked monophasic contractions in the Ca-free solution as shown in Figure 1c. We examined the effects of VIP on these contractions in detail. After the Ca stored in muscle cells was completely depleted by repetitive applications of caffeine (30 mM) in Ca-free solution containing 2 mm EGTA, the muscle was exposed to 2.5 mm Ca together with isotonic KCl (150 mM) for 2 min to load the Ca in intracellular Ca stores (Ca-loading). After the tissue was washed for 4min with repeatedly changing Ca-free solutions containing
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Figure 4 Effects of vasoactive intestinal peptide (VIP) on carbachol (CCh)- and caffeine-induced contractions in Ca-free solution containing 2mM EGTA. After 2.5mm Ca was applied together with isotonic KCI (150mM) for 2min (Ca-loading), the muscle fibre was washed with Ca-free solution containing 2mM EGTA for 4min (Ca-washing). Thereafter, (a) CCh (100pM; *) or (b) caffeine (30mM; El) was applied for 30s in Ca-free solution containing 2mM EGTA. Chart speed is 4 times slower in the Ca-loading and Ca-washing steps than in the next steps. (i) Control; (ii) VIP (100nM) was applied after Ca-loading; (iii) VIP was applied before Ca-loading. (c) The % amplitude of contractions evoked by CCh and caffeine in Ca-free solutions containing 2mm EGTA. Open columns show the data obtained when VIP is introduced after Ca-loading and hatched columns before Ca-loading (n 5). Horizontal lines indicate s.e.mean. =
0 Figure 5 Effects of vasoactive intestinal peptide (VIP) on successive carbachol (CCh)- and caffeine-induced contractions in Ca-free solution containing 2mM EGTA. After Ca-loading, CCh (100pM; *) and caffeine (30mM; El) were applied successively for 30s with a min interval. (a) Control; (b) VIP (100nM) was applied after Ca-loading. For further explanations, see the legend to Figure 4.
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2 mm EGTA (Ca-washing), application of CCh (100puM) or caffeine (30 mM) for 30s produced a transient contraction in the Ca-free solution containing 2mM EGTA (Figures 4a(i) and 4b(i)). Contractions no longer occurred when KCI (40 mM) was applied instead of CCh or caffeine, indicating that external Ca was sufficiently removed during the Ca-washing period (data not shown). VIP (100 nM) was applied during the periods of Ca-washing and subsequent CCh or caffeine stimulation. VIP significantly inhibited the contraction evoked by CCh in a dose-dependent manner, but not that caused by caffeine (Figures 4a(ii), 4b(ii) and open columns in 4c). When VIP was applied from the time of Ca-loading, the inhibitory effect of VIP was less pronounced (Figure 4a(iii) and hatched columns in 4c). In order to see whether this inhibitory effect of VIP was due to the inhibition of Ca release from intracellular Ca stores or to an enhancement of Ca extrusion through the plasma membrane, the contractions induced in Ca-free solution containing 2 mm EGTA by successive applications of CCh and caffeine were examined in the presence or absence of 100 nm VIP (Figure 5). After CCh had evoked a contraction, a small contraction could be evoked by the application of caffeine.
100
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pCa Figure 6 Effects of vasoactive intestinal peptide (VIP) on the Cainduced contraction in skinned fibres. (a) (i) Isotonic KCI (150mM)induced contraction in intact muscles. (ii) After skinning with saponin, various concentrations of Ca were applied cumulatively. After the pCa 6.3-induced contraction reached a steady level, VIP (100nM) was applied. (b) pCa-tension relation observed in the presence (C; n = 4) or absence (0; n = 5) of VIP (100nM). The amplitude of contraction evoked by pCa 5.0 in the absence of VIP was normalized as 100%. Vertical lines indicate s.e.mean.
INHIBITORY EFFECT OF VIP ON SMOOTH MUSCLE
However, after caffeine evoked a contraction, CCh or caffeine did not (data not shown). When the contraction induced by a preceding application of CCh was inhibited by VIP, the caffeine-induced contraction was enhanced (3.7 + 0.3 times the control, n = 10)
Effects of vasoactive intestinal peptide on the contractile machinery in skinnedfibres Figure 6a shows a typical record of contraction induced by cumulative application of Ca and the effect of VIP (100pM) on the contraction evoked by pCa 6.3 in skinned muscle fibres. VIP did not modify the amplitude of contraction induced by Ca. The effect of VIP (100nM) on the cumulative pCa-tension relation is shown in Figure 6b. The minimal concentration of Ca required to produce the contraction of skinned muscle fibres was pCa 6.7 and the amplitude of contraction was increased with increasing concentrations of Ca up to pCa 5.7. VIP had no effect on the pCa-tension relation.
Discussion The present results indicate that VIP inhibits the contraction induced by CCh but not those caused by KCl and caffeine in the gastric circular smooth muscle of the rat. VIP inhibited the increase in [Ca]i together with tension development in response to CCh and thus did not affect the [Ca]i-tension relation for CCh. Therefore, it seems likely that the change in the Ca sensitivity of the contractile system is not responsible for the inhibitory action of VIP on CCh-induced contraction. Furthermore, VIP did not influence the pCa-tension relation in skinned fibres of gastric smooth muscles, as was the case of vascular smooth muscles (Itoh et al., 1985). The contraction evoked by CCh at a low concentration was inhibited by the removal of extracellular Ca or by treatment with the Ca channel blocker, nifedipine. As VIP was effective in inhibition of the contractions and the rise in [Ca]i in response to CCh, VIP probably inhibits the CCh-induced Ca influx across the cell membrane through DHP-sensitive voltage-dependent Ca channels. However, the KCl-induced contraction was resistant to VIP. Therefore, it seems unlikely that the action of VIP on CCh-induced contractions is due to a direct inhibition of voltage-dependent Ca channels. VIP could, however, interefere with the processes of signal transduction from muscarinic receptors to voltage-dependent Ca channels. The phasic contractions evoked by CCh and caffeine in Ca-free solution are known to be due to Ca release from intracellular Ca stores (Endo et al., 1980; Itoh et al., 1981). Similar phasic contractions induced by these agents were observed in gastric circular smooth muscles. In Ca-free solution contain-
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ing 2 mm EGTA, VIP inhibited the contraction induced by CCh but not that induced by caffeine. After VIP inhibited the CCh-induced contraction, the contraction induced by a subsequent application of caffeine was significantly enlarged in the presence of VIP. These results indicate that the Ca stores in this tissue are probably activated by both drugs and that the inhibitory action of VIP on the CCh-induced contraction in Ca-free solution may result from an inhibition of the CChinduced Ca release from intracellular Ca stores rather than from an increase in Ca extrusion through the cell membrane. The difference between the effects of VIP on CCh- and caffeine-induced contractions could be attributable to the fact that caffeine acts directly on intracellular Ca stores (Endo et al., 1980; Itoh et al., 1981) but CCh does not. It seems likely that VIP inhibits some steps between muscarinic receptor activation and the Ca release from intracellular Ca stores. It has been shown that muscarinic receptors couple to guanine nucleotide binding proteins (Haga et al., 1987) and stimulation of these receptors leads to the activation of phospholipase C, which causes inositol breakdown and the production of inositol 1,4,5-trisphosphate (UP3; Gardner et al., 1988; Secrest et al., 1989), a putative messenger for the mobilization of Ca from non-mitochondrial stores (Berridge & Irvine, 1984). Therefore, the contraction induced by CCh in the absence of extracellular Ca may result from IP3 formation in the rat gastric smooth muscle. More recently, we have found that VIP increases intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentrations and that the time course was comparable to that of relaxation (Ito et al., 1990). The time-dependence of the inhibitory effect of VIP on CCh-induced contraction may imply the involvement of second messenger systems, such as cyclic AMP. Furthermore, Ca sequestering action into the Ca stores has been reported to be increased by cyclic AMP (Bhalla et al., 1978; Itoh et al., 1982). The inhibitory effect of VIP on CCh-induced contractions in Ca-free solution containing 2mm EGTA was less pronounced with application of VIP before the Ca-loading period than after it, suggesting that VIP has a Ca-sequestering action on Ca storage sites in addition to the inhibition of CCh-induced Ca release. In conclusion, VIP inhibits CCh-induced contraction in the presence and absence of extracellular Ca. VIP seems not to have direct inhibitory effects on DHP-sensitive voltagedependent Ca channels, the Ca-release mechanism from intracellular Ca stores and the contractile system. The site of the inhibitory action of VIP may be the processes of signal transduction from muscarinic receptors to DHP-sensitive voltagedependent Ca channels and to intracellular Ca stores. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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ICHIYAMA, A. (1987). Molecular properties of muscarinic receptors. Trends Pharmacol. Sci., 8 (Suppl. Subtypes Muscarinic Receptor III), 12-18. HARAFUJI, H. & OGAWA, Y. (1980). Re-examination of the apparent binding constant of ethylene glycol bis(fi-aminoethyl ether)-N,N, N',N'- tetraacetic acid with calcium around neutral pH. J. Biochem., 87, 1305-1312. HIMPENS, B. & SOMLYO, A.P. (1988). Free-calcium and force tran-
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(Received July 20, 1990 Revised October 29, 1990 Accepted November 5, 1990)
