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Received 12 March 1998; accepted after revision 30 September 1998.
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ABSTRACT |
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INTRODUCTION |
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Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and shown to stimulate adenylate cyclase (Miyata et al. 1989). Later, PACAP was also shown to be widely distributed in the gastrointestinal tract (Sundler et al. 1991). Katsoulis & Schmidt (1996) and Schwörer et al. (1992) reported that PACAP relaxed the smooth muscle preparations of guinea-pig ileum and taenia coli, rat fundus, ileum and colon, pig ileum, and human colon. We have previously suggested that PACAP mediates non-adrenergic, non-cholinergic (NANC) inhibitory responses of longitudinal muscle of rat distal colon via the activation of apamin-sensitive K+ channels (Kishi et al. 1996). PACAP has also been shown to be released by nerve stimulation and to be responsible for the apamin-sensitive component of electrical field stimulation (EFS)-induced relaxation and inhibitory junction potentials (IJPs) of guinea-pig taenia coli (Jin et al. 1994a; McConalogue et al. 1995b). These findings strongly suggest that PACAP is a member of a group of potential neurotransmitters responsible for NANC relaxation, which activates apamin-sensitive K+ channels in some regions of the gastrointestinal tract. However, the mechanism by which PACAP activates apamin-sensitive K+ channels is still unknown.
Increases in cyclic AMP concentrations result in the stimulation of large conductance Ca2+-activated K+ channels in rat aorta (Sadoshima et al. 1988) and rabbit trachea (Kume et al. 1989). Cyclic AMP-dependent phosphorylation of large conductance Ca2+-activated K+ channels also increases the channel activity in smooth muscle cells from canine proximal colon (Carl et al. 1991) and rat aorta (Sadoshima et al. 1988). Phosphorylation of large conductance Ca2+-activated K+ channels has been suggested to increase their sensitivity to Ca2+ and result in their activation at lower concentrations of Ca2+ (Archer et al. 1994; Esguerra et al. 1994; Perez & Toro, 1994). However, in guinea-pig taenia coli, PACAP induces relaxation without a concomitant increase in cyclic AMP levels and this relaxation is inhibited by apamin, an inhibitor of small conductance Ca2+-activated K+ channels (Jin et al. 1994a). This finding indicates that a cyclic AMP-mediated mechanism is not involved in the opening of apamin-sensitive K+ channels by PACAP. Although the amino acid sequence of the apamin-sensitive K+ channel has been identified, the intracellular mechanism of activation of apamin-sensitive K+ channels is not yet known. We therefore investigated the intracellular mechanism of opening of apamin-sensitive K+ channels by PACAP in rat distal colon.
Neurotransmitters, hormones and growth factors have been shown to regulate the activity of various types of K+ channels through tyrosine kinase-dependent pathways (Lev et al. 1995; Prevarskaya et al. 1995). In vascular smooth muscle cells isolated from the rat tail artery, neuropeptide Y inhibits Ca2+-activated K+ channels and this inhibition is reversed by inhibitors of tyrosine kinase (Xiong & Cheung, 1995). In Xenopus oocytes and cultured mammalian cells that express muscarinic acetylcholine receptors and delayed rectifier K+ channels, the activation of muscarinic receptors suppresses K+ channel activity through direct phosphorylation by tyrosine kinase (Huang et al. 1993). In addition, some reports indicate that certain types of K+ channels are phosphorylated directly by tyrosine kinase and that tyrosine phosphorylation modulates the activity of the channels (Holmes et al. 1996a,b; Bowlby et al. 1997). Recently, PACAP has been suggested to activate tyrosine kinase, since the cell growth and mitogenic effects induced by the peptide were inhibited by inhibitors of tyrosine kinase (Morisset et al. 1995; Schafer et al. 1996). Thus, in this study we have investigated the mechanism by which PACAP induces activation of apamin-sensitive K+ channels in the longitudinal muscle of rat distal colon. We have also studied the relationship between changes in membrane potential and intracellular calcium ion concentration ([Ca2+]i) with relaxation of the muscle.
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METHODS |
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Male Wistar-ST rats (250-350 g) obtained from Nippon SLC (Shizuoka, Japan) were lightly anaesthetized with ether, stunned by a blow on the head and exsanguinated via the carotid artery. The colon was removed and placed in Tyrode solution consisting of (mM): 137 NaCl, 2·7 KCl, 1·8 CaCl2, 1·1 MgCl2, 0·42 NaH2PO4, 11·9 NaHCO3 and 5·6 glucose. The contents of the excised segment were flushed out with Tyrode solution. The portion of descending colon attached by the mesentery to the small intestine was defined as the distal colon.
Recording of isometric responses of longitudinal muscle of distal colon to PACAP
Small strips (0·2-0·3 mm × 1·0-2·0 mm) of longitudinal muscle were prepared from the distal colon, and tied with monofilament silk to the fine tips of two tungsten needles, one of which was connected to a force transducer (AM801, SensoNor, Horten, Norway). Then they were mounted to the wall on a bubble plate (Horiuti, 1988) containing Tyrode solution to allow moderately rapid solution exchange. Carbachol (CCh) induced a contraction consisting of a transient phasic and a subsequent tonic component. The relaxant effect of PACAP on the tonic contraction was examined after the tonic contraction reached a constant amplitude. The muscle strips were treated with cyclic AMP-dependent protein kinase inhibitor or tyrosine kinase inhibitors for at least 15 min before PACAP was applied. The effects of inhibitors were expressed as a percentage of the peptide-induced relaxation in the absence of inhibitors (control).
Recording of isotonic responses of longitudinal muscle of distal colon to EFS
Entire segments of the distal colon (2·5-3·0 cm in length) were suspended in an organ bath filled with Tyrode solution aerated with 5 % CO2 in O2 and maintained at 37°C. The anal end of each segment was attached to a transducer and the oral end was mounted on an anodal electrode placed at the bottom of the bath. Atropine (1 µM) and guanethidine (5 µM) were present throughout the experiment to block cholinergic and adrenergic responses, respectively. Responses of the distal colon to EFS with trains of 100 pulses of 0·5 ms width and supramaximal voltage (50 V) at a frequency of 10 Hz were recorded isotonically with a 10 min interval between tests. The longitudinal muscle was subjected to a resting load of 1·6 g. After an equilibration period of 30 min, the muscle exhibited moderate spontaneous contractile activity and the resting tone gradually increased during successive trains of EFS given at 10 min intervals. With progressive increases in tone, the segments began to exhibit clear relaxations on EFS. Drugs were added to the organ bath in volumes of less than 1·0 % of the bathing solution for 10 min before EFS or an application of PACAP. The volume of vehicle (distilled water) for the drugs did not affect the spontaneous contractile activity or the muscle tone. When examining the effects of K+ channel blockers or tyrosine kinase inhibitors on the EFS-induced relaxation, at least three trials were run after addition of the inhibitors. The extent of the EFS-induced relaxation was expressed as the area under the line of resting tone that was drawn through the centre of resting spontaneous contractile activity immediately before EFS. Relaxation after treatment with drugs was expressed as the percentage of values before treatment.
Recording of IJPs induced by EFS in longitudinal muscle of distal colon
Segments of the distal colon were mounted in a 1·5 ml organ bath maintained at 30°C and perfused continuously with Tyrode solution at a rate of 3 ml min-1. This temperature allowed stable recording of the membrane potentials, since the spontaneous and evoked mechanical responses were reduced. Atropine (1 µM) and guanethidine (5 µM) were present in the bathing solution throughout the experiment. Membrane potentials were recorded with conventional glass microelectrodes filled with 3 M KCl with resistances of 50-80 M
. IJPs were elicited by EFS to the intramural nerves within the segment with square-wave pulses of 0·5 ms duration at an appropriate intensity (10-30 V) (Takewaki & Ohashi, 1977). The electrode was impaled into the longitudinal muscle cells of the superficial layer from the serosal side. Stimulus pulses were delivered with a pair of Ag-AgCl wire electrodes, one on the serosal surface 1-2 mm from the impaled glass microelectrode and the other in the solution. The distance between the two electrodes was about 20 mm.
Measurement of intracellular Ca2+ levels of longitudinal muscle cells of distal colon
Small strips (2-3 mm × 2·0-3·0 mm) of longitudinal muscle were incubated in Tyrode solution containing 5 µM fura-2 acetoxymethyl ester (fura-2 AM) and 0·02 % (w/v) cremophor EL for 5-6 h at 25°C under 5 % CO2 in O2. Then the fura-2-loaded muscle was washed and mounted on a strain gauge transducer (U gauge, UL-2GR, Minebea, Tokyo, Japan) to record contractile responses isometrically. Changes in Ca2+ levels were measured simultaneously in a Nihonbunko CAF-100 spectrofluorometer (Nihonbunko, Tokyo), using 340 nm/380 nm excitation with emission at 510 nm. A load of 0·5 g was applied as a resting tension. Contractions and increases in Ca2+ levels of the muscle strips were induced by 100 mM K+ or 1 µM CCh. The relaxations and changes in Ca2+ levels induced by PACAP in the tonic phase were examined after the contraction reached a constant level. K+ channel blocker or tyrosine kinase inhibitor was applied for at least 15 min before the effects of PACAP were examined.
Measurement of cyclic AMP content of longitudinal muscle cells of distal colon
Small longitudinal muscle strips (10-30 mg wet wt) prepared from the distal colon were equilibrated with Tyrode solution aerated with 95 % O2, 5 % CO2 for 20 min at 37°C. The preparations were then incubated with various concentrations of PACAP for 2 min, and quickly frozen by putting them between two metal plates frozen on dry ice. The frozen preparations were homogenized in 2 ml of 6·0 % (w/v) trichloroacetic acid solution. After removal of the trichloroacetic acid with ether, the cyclic AMP content of the preparations was measured by radioimmunoassay with a cyclic AMP assay kit (Amersham Japan, Tokyo).
Drugs and chemicals
Apamin, daidzein, tyrphostin 25 and cremophor EL were purchased from Sigma. 8-Bromoadenosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-bromo-cAMPS) and Sp isomer (Sp-8-bromo-cAMPS) were from BIOLOG Life Science Institute (Bremen, Germany). Lavendustin A and tyrphostin A1 were from Calbiochem. Fura-2 AM was from Dojin (Osaka, Japan). Pituitary adenylate cyclase activating peptide (PACAP), vasoactive intestinal peptide (VIP) and charybdotoxin were from the Peptide Institute (Osaka, Japan). Genistein and tetrodotoxin were from Wako Pure Chemicals (Osaka, Japan). All other chemicals were of analytical grade. Tyrosine kinase inhibitors were dissolved in dimethyl sulfoxide (DMSO) at stock solutions. The final DMSO concentration was 0·1 %, which did not have any effect on the preparations. Other drugs were added as solutions in redistilled water in volumes of less than 1·0 % of the bathing solution. A similar volume of redistilled water alone had no effect on the muscle.
Statistical analysis
Data are expressed as means ±
Effects of tyrosine kinase inhibitors and cyclic AMP-dependent protein kinase inhibitor on PACAP-induced relaxation
PACAP induced concentration-dependent, gradual relaxation of longitudinal muscle pre-contracted with 1 µM CCh, exhibiting an almost maximum effect at 100 nM (Fig. 1). We first examined whether the cyclic AMP-dependent kinase (protein kinase A (PKA)) system is involved in the PACAP-mediated relaxation, as PACAP has been suggested as mediating relaxation via this system. Our results showed that PACAP (10-100 nM) did not significantly increase the cyclic AMP content of the longitudinal muscle (control (resting content): 165·0 ± 1·65 fmol (100 mg tissue wt)-1, n = 3; PACAP at 100 nM: 181·9 ± 21·5 fmol (100 mg tissue wt)-1, n = 3). Moreover, 30 µM Rp-8-bromo-cAMPS, an inhibitor of PKA, had no effect on relaxation induced by 100 nM PACAP (115·2 ± 13·8 % of control values, n = 5). This concentration of Rp-8-bromo-cAMPS maximally inhibited cyclic AMP-mediated relaxation induced by vasoactive intestinal peptide (VIP) (Makhlouf & Grider, 1993) to 55·5 ± 9·3 % of control values (n = 6) and inhibited the relaxation induced by Sp-8-bromo-cAMPS (100 µM), a non-hydrolysable activator of PKA, to 32·2 ± 10·6 % of control values (n = 3). On the other hand, genistein, tyrphostin 25 and lavendustin A, which are inhibitors of tyrosine kinase, significantly inhibited PACAP-induced relaxation in a concentration-dependent manner (Fig. 2), suggesting the involvement of tyrosine kinase in PACAP-mediated relaxation of the muscle. The IC50 values of tyrphostin 25 and lavendustin A were about 14 µM. The effect of genistein was moderate; 10 µM inhibited PACAP-induced relaxation to about 60 % of the control value (Fig. 2). Tyrophostin 25 at 10 µM did not inhibit relaxation induced by 10 µM isoprenaline (98·0 ± 14·2 % of control value, n = 4) or 1 µM VIP (102·6 ± 13·2 % of control value, n = 5). Daidzein and tyrphostin A1, inactive analogues of genistein and tyrphostin 25, respectively, did not inhibit the PACAP-induced relaxation at concentrations of 10 µM (Fig. 2).
Distal colon segments were first contracted with 1 µM carbachol. When contraction reached a constant level, various concentrations of PACAP were applied. Relaxations are expressed as percentages of the amplitude of pre-contraction with 1 µM CCh. Values are means ±
Relaxation was induced by 100 nM PACAP in the absence or presence of 1-30 µM tyrphostin 25 (
Effects of tyrosine kinase inhibitors and K+ channel blockers on EFS-induced relaxation
EFS induced a rapid transient relaxation (rapid relaxation), a subsequent rebound contraction and a delayed sustained relaxation (delayed relaxation) of the longitudinal muscle of the distal colon segments. These responses were abolished by tetrodotoxin (n = 6). Three tyrosine kinase inhibitors significantly inhibited the spontaneous contractile activity and partially inhibited EFS-induced relaxation, but did not affect the resting tone of the preparations (Fig. 3). As shown in Table 1, genistein at 10 µM, tyrphostin 25 at 10 µM and lavendustin A at 30 µM inhibited EFS-induced relaxation to 75 %, 50 % and 36 % of control values, respectively, while daidzein and tyrphostin A1 had no effect. We have previously proposed that PACAP and VIP mediate NANC inhibitory responses of this preparation via K+ channels sensitive to apamin (a blocker of small conductance Ca2+-activated K+ channels) in the case of PACAP, and charybdotoxin (a blocker of large conductance Ca2+-activated K+ channels) in the case of VIP (Kishi et al. 1996). We therefore examined the relationship between the tyrosine kinase inhibitor-sensitive component of EFS-induced relaxation, and charybdotoxin- or apamin-sensitive relaxation. A combination of 10 µM tyrphostin 25 and 100 nM charybdotoxin, which showed maximal inhibitory effects on preparations (Kishi et al. 1996), inhibited EFS-induced rapid relaxation further to about 25 % of the control value (Fig. 4), whereas a combination of tyrphostin 25 and apamin did not induce inhibition further to that obtained with each drug alone (Fig. 5). These results suggest that the tyrphostin 25-sensitive component of relaxation is independent of the charybdotoxin-sensitive component, but is closely associated with the apamin-sensitive component. In other words, PACAP may open apamin-sensitive K+ channels via activation of tyrosine kinase.
Relaxation was induced by EFS (10 Hz, 30 V, 0·5 ms for 10 s) in the absence (left side) or presence (right side) of tyrosine kinase inhibitors; 10 µM genistein (A), 10 µM tyrphostin 25 (B) or 30 µM lavendustin A (C) was applied for 10 min before EFS. Bars indicate the 10 s duration of EFS. After recording normal spontaneous movement, the chart speed was increased immediately before the stimulation (shown by the dashed lines) to make the relaxant response clearer, and then returned to the slow speed.
Relaxation was induced by EFS (10 Hz, 30 V, 0·5 ms for 10 s) in the absence (A) or presence of 100 nM charybdotoxin (ChTX) without (B) or with 10 µM tyrphostin 25 (C). The muscle preparations were treated with inhibitors for 10 min before EFS. Bars indicate the 10 s duration of EFS. After recording normal spontaneous movement, the chart was run faster from immediately before the stimulation to make the relaxant response clearer (dashed lines). D, summary of the effects of charybdotoxin and tyrphostin 25. Relaxations are expressed as percentages of those before addition of the antagonist (control). Values are means ±
Relaxation was induced by EFS (10 Hz, 30 V, 0·5 ms for 10 s) in the absence (A) or presence of 1 µM apamin without (B) or with 10 µM tyrphostin 25 (C). The muscle preparations were treated with inhibitors for 10 min before EFS. Bars indicate the 10 s duration of EFS. After recording normal spontaneous movement, the chart recorder was run faster from immediately before the stimulation to make the relaxant response clearer (dashed lines). D, summary of effects of apamin and tyrphostin 25. Relaxations are expressed as the percentages of those obtained before addition of antagonist (control). Values are means ±
Table 1. Effects of tyrosine kinase inhibitors on EFS-induced relaxation
Effects of tyrosine kinase inhibitors on changes in membrane potentials induced by EFS and PACAP
The resting membrane potential of longitudinal muscle cells of the rat distal colon was -61·0 ± 1·2 mV (n = 60). In the presence of atropine (1 µM) and guanethidine (5 µM), EFS with a single pulse or two pulses at 10 Hz induced IJPs. The IJPs induced by two pulses at 10 Hz often consisted of two phases, rapid and subsequent slow hyperpolarization. But the latter was hardly ever recorded on application of a single pulse. The amplitude of the slow IJPs varied from one cell to another. At the concentrations used, genistein and tyrphostin 25 did not have any significant effect on the resting membrane potentials; in the presence of genistein at 10 µM or tyrphostin 25 at 5 µM these were -61·8 ± 1·8 mV (n = 10) and -60·6 ± 2·2 mV (n = 10), respectively. Genistein inhibited the IJPs (mean amplitude: 17·7 ± 1·4 mV, n = 10) induced by a single pulse in a concentration-dependent manner (Fig. 6A and D). Tyrphostin 25 also significantly inhibited the IJPs (mean amplitude: 18·2 ± 1·1 mV, n = 10) induced by a single pulse (Fig. 6B and D). The IC50 values of genistein and tyrphostin 25 were 5 and 3·5 µM, respectively. Tyrphostin 25 at 10 µM completely inhibited the rapid phase (Fig. 6C), but did not affect the slow phase of the IJPs induced by two pulses (n = 7). Genistein at 10 µM had a similar effect (n = 2; data not shown). Daidzein did not inhibit the IJPs (control: 18·5 ± 1·2 mV, n = 6; 10 µM daidzein: 17·5 ± 1·5 mV, n = 3). Tyrphostin A1 also had no effect on the IJPs (control: 16·8 ± 1·8 mV, n = 8; 10 µM tyrphostin A1: 16·0 ± 2·0 mV, n = 3).
IJPs were induced by a single pulse (A and B) or two pulses at 10 Hz (C) in the absence (left side) or presence (right side) of 10 µM genistein (A) or 25 µM tyrphostin 25 (B and C). The smooth muscle cells were treated with inhibitors for 10 min before EFS. Note that EFS with two pulses in the presence of tyrphostin 25 (C) induced delayed hyperpolarization without a rapid phase. D, concentration-response curves of inhibition of IJPs by tyrosine kinase inhibitors. IJPs were induced by a single pulse in the absence or presence of various concentrations of tyrphostin 25 (
Bath application of PACAP induced hyperpolarization of longitudinal muscle cells (3·6 ± 1·1 mV, n = 5 at 1 µM or 7·8 ± 0·8 mV, n = 10 at 3 µM; Fig. 7). Genistein at 10 µM inhibited the hyperpolarization induced by 1 µM PACAP completely (n = 4) and by 3 µM PACAP significantly (up to 1·8 ± 0·6 mV, n = 6) (Fig. 7). Tyrphostin 25 at 10 µM also inhibited the PACAP (3 µM)-induced hyperpolarization completely (0 mV, n = 6) and at 3 µM, significantly (4·0 ± 0·7 mV, n = 4).
Exogenously added PACAP (3 µM) induced slow hyperpolarization. Treatment with 10 µM genistein for 15 min resulted in almost complete inhibition of PACAP-induced hyperpolarization. Horizontal lines indicate the presence of PACAP.
Effects of tyrphostin 25 and apamin on changes in intracellular Ca2+ concentration ([Ca2+]i) induced by PACAP
To determine the relationship between the inhibitory effects of tyrosine kinase inhibitors on PACAP-induced relaxation and changes in [Ca2+]i levels, we measured changes in muscle tension and [Ca2+]i simultaneously. Small strips of the distal colonic longitudinal muscle which had been loaded with fura-2 AM were pre-contracted with 1 µM CCh. These CCh-treated strips exhibited rapid contraction and subsequent spontaneous activity. The increase in [Ca2+]i also showed a transient phasic component followed by a tonic sustained component, the amplitude of which was smaller than that of the phasic one. When the tonic phases of contraction and the increase in [Ca2+]i induced by CCh (1 µM) reached constant levels, PACAP (100 nM) added to the organ bath induced decreases in tension and [Ca2+]i simultaneously. Pretreatment with tyrphostin 25 (10 µM) or apamin (1 µM) had no significant effects on these CCh-induced responses. However, the effects of PACAP on [Ca2+]i and tension were significantly inhibited by both tyrphostin 25 (Fig. 8) and apamin (Fig. 9) treatment (n = 4 and n = 3, respectively).
A, muscle strips preloaded with fura-2 AM were first contracted with 1 µM CCh and 100 mM K+. When the CCh-induced responses reached a constant level, the effects of 100 nM PACAP were examined in the absence (left side) or presence (right side) of 10 µM tyrphostin 25. The top panel shows PACAP-induced relaxation and the bottom panel shows the changes in [Ca2+]i as indicated by the fluorescence ratio, F340/F380. Bars above and below the panels show the times of application of the indicated chemicals. B, summary of the effects of tyrphostin 25 on PACAP-induced relaxation (
Effects of PACAP were examined in the absence or presence of 1 µM apamin (A) and are summarized in B (n = 3).
We have previously proposed that VIP and PACAP are involved in the NANC inhibitory response of longitudinal muscle of rat distal colon by activation of charybdotoxin- and apamin-sensitive K+ channels, respectively (Kishi et al. 1996). In this study, we investigated the intracellular mechanism of relaxation mediated by PACAP. There are some reports that PACAP activates adenylate cyclase and increases the intracellular cyclic AMP content in rabbit gastric muscle cells (Murthy & Makhlouf, 1994; Murthy et al. 1997) and guinea-pig taenia coli (McConalogue et al. 1995a). However, in our preparations, PACAP-induced relaxation was not affected by an inhibitor of protein kinase A. Nor did PACAP at a concentration that induced significant relaxation increase the cyclic AMP content of the longitudinal muscle. These findings suggest that PACAP induces relaxation of longitudinal muscle through a mechanism(s) independent of the cyclic AMP-PKA system. Jin et al. (1994b) also showed that PACAP induced relaxation without concomitant changes in intracellular cyclic AMP content of guinea-pig taenia coli. Recently, PACAP has been shown to stimulate proliferation of cells from a rat pancreatic acinar tumour line, AR4-2J, by activating tyrosine kinase (Morisset et al. 1995). Ogata et al. (1997) showed that genistein inhibits ATP-sensitive K+ channels in rabbit vein smooth muscle. We have also reported that EFS induces PACAP-mediated, apamin-sensitive relaxation of longitudinal muscle of rat distal colon (Kishi et al. 1996). Therefore, in this study we have investigated the effects of tyrosine kinase inhibitors on PACAP-induced relaxation. From the following findings, we propose that PACAP mediates apamin-sensitive relaxation by activating tyrosine kinase. Firstly, various tyrosine kinase inhibitors inhibited exogenous PACAP-induced relaxation. Secondly, these tyrosine kinase inhibitors also inhibited EFS-induced relaxation to about 50 % of control values, but had no appreciable effect in the presence of apamin. We have previously suggested that VIP also mediates about half the component of relaxation by activating charybdotoxin-sensitive K+ channels in the rat distal colon (Kishi et al. 1996). However, VIP-induced relaxation was not inhibited by tyrphostin 25, and charybdotoxin further inhibited the relaxation which persisted after the tyrphostin 25 treatment in the present study. These results suggest that inhibitors of tyrosine kinase selectively inhibited the PACAP-mediated component of relaxation in the distal colon. Thirdly, these tyrosine kinase inhibitors inhibited exogenous PACAP-induced hyperpolarization. In addition, they also inhibited apamin-sensitive IJPs. We have reported previously that stimulation by two pulses at 10 Hz induced IJPs consisting of both rapid and delayed phases and that the rapid component was inhibited by a PACAP antagonist and apamin (Kishi et al. 1996). Tyrosine kinase inhibitors inhibited only the rapid phase of IJPs in the present study. These findings suggest that PACAP induces an inhibitory response by opening apamin-sensitive K+ channels via activation of tyrosine kinase.
In the present study, inhibitors of tyrosine kinase inhibited PACAP-induced relaxation and EFS-induced IJPs over a similar concentration range: IC50 values were between 3·5 and 14 µM in every case. PACAP-induced hyperpolarization was also inhibited by similar concentrations of genistein and tyrphostin 25. Genistein and tyrphostin have been reported to inhibit the epidermal growth factor receptor (EGFR) kinase with IC50 values of about 3 µM (Akiyama et al. 1987; Gazit et al. 1989). The IC50 value of the effects of lavendustin A on PACAP-induced relaxation was 14 µM. Although lavendustin A inhibits EGFR kinase with an IC50 value of about 0·01 µM in a cell-free system, micromolar concentrations were needed in intact preparations (Di Salvo et al. 1997). Tyrphostin 25 did not inhibit isoprenaline- and VIP-induced relaxation and inactive analogues of the inhibitors had no effect on PACAP-mediated responses in the present study. These tyrosine kinase inhibitors have also been reported as inhibiting tyrosine kinase more selectively than other protein kinases (Onoda et al. 1989; Levitzki & Gazit, 1995). These results therefore suggest that the effects of tyrosine kinase inhibitors shown in this study are attributable mainly to their inhibitory action on tyrosine kinase.
Exogenously applied PACAP induced slow, gradual relaxation of longitudinal muscle of the distal colon in our previous study (Kishi et al. 1996) and slow hyperpolarization in the present study. In contrast, EFS induced fast IJPs, which have been suggested as being mediated by PACAP. This discrepancy in the speed of action seems to result from the difference in accessibility to PACAP receptors between endogenous and exogenous PACAP molecules; the former were released by EFS from the nerve terminals and the latter were added to the bathing solution. However, another explanation cannot necessarily be excluded.
Opening of K+ channels may hyperpolarize membrane potentials. Missiaen et al. (1990) suggested that hyperpolarization of cell membranes results in inactivation of voltage-dependent Ca2+ channels, which in turn decreases Ca2+ influx into the cells. Thus, there seems to be a close association between changes in membrane potential and the [Ca2+]i level. In this study, PACAP hyperpolarized the membrane potential of smooth muscle cells and the response was significantly inhibited by apamin or tyrosine kinase inhibitors. PACAP also lowered the [Ca2+]i level and simultaneously induced the relaxation of preparations pre-contracted with carbachol. Inhibitors of tyrosine kinase and apamin inhibited both responses. These results suggest that the decrease in [Ca2+]i levels is due to the opening of apamin-sensitive K+ channels activated by tyrosine kinase. Our findings indicate the following sequence of events in the relaxation of longitudinal muscle of the rat distal colon: (1) activation of PACAP receptors; (2) activation of tyrosine kinase; (3) opening of apamin-sensitive K+ channels; (4) induction of membrane hyperpolarization; (5) decrease in [Ca2+]i; and finally (6) relaxation of the muscle.
However, the sequence has not yet been completely clarified by the present study, with the following issues requiring further investigation. Firstly, there are two different ways in which tyrosine kinase regulates apamin-sensitive K+ channels. One is by phosphorylating K+ channels directly - although tyrosine kinase has been reported to phosphorylate some types of K+ channels directly (Holmes et al. 1996a,b; Bowlby et al. 1997), there is no report that it phosphorylates an apamin-sensitive K+ channel. The other way is the same way in which other kinases, such as serine/threonine kinase and phospholipase C (activated through phosphorylation by tyrosine kinase) modulate K+ channel activity. The exact nature of the regulatory mechanism of apamin-sensitive K+ channels by tyrosine kinase therefore requires further investigation. Secondly, Ca2+ activates apamin-sensitive K+ channels, and an increase in [Ca2+]i above a certain level results in smooth muscle contraction. It seems likely, therefore, that the concentration of Ca2+ needed for activation of K+ channels is lower than that required for muscle contraction. According to Keef et al. (1994), VIP transiently increases [Ca2+]i, the increase in [Ca2+]i activates K+ channels, and this activation induces relaxation of isolated smooth muscle cells. In the present study, however, PACAP induced only a decrease, not an increase in [Ca2+]i. Nelson et al. (1995) analysed the localization of [Ca2+]i in isolated smooth muscle cells by confocal microscopy and found a localized increase just under the surface membrane, resulting from release of Ca2+ stored in the sarcoplasmic reticulum which activated Ca2+-activated K+ channels. Perhaps we did not detect localized [Ca2+]i increases because we measured the Ca2+ concentration at the tissue level, our method being more macroscopic than that of Nelson et al. (1995). Another possibility is that direct or indirect phosphorylation of the K+ channels by tyrosine kinase could increase their sensitivity to Ca2+ and result in their activation at a lower concentration of Ca2+.
In the guinea-pig and human colon, ATP has been suggested as mediating apamin-sensitive IJPs and relaxation (Zagorodnyuk & Shuba, 1986; Zagorodnyuk & Maggi, 1994). However, ATP at concentrations up to 100 µM did not induce any appreciable relaxation in the rat distal colon (data not shown). Serio et al. (1996) have also suggested that ATP is not the mediator responsible for the apamin-sensitive component of IJPs in the rat caecum. Thus, it seems likely that ATP is not associated with the apamin-sensitive NANC inhibitory response in the rat colon.
This study indicates for the first time the essential role of tyrosine kinase in PACAP-mediated relaxation of longitudinal muscle from rat distal colon.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
Corresponding author
T. Takeuchi: Department of Veterinary Pharmacology, College of Agriculture, Osaka Prefecture University, Sakai 599-8531, Japan.
Email: takeuchi{at}jyui.vet.osakafu-u.ac.jp
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RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References
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Figure 1. Relaxation of longitudinal muscle of rat distal colon in response to PACAP
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Figure 2. Effects of tyrosine kinase inhibitors on PACAP-induced relaxation of longitudinal muscle of rat distal colon
), 1-10 µM genistein (
) and 10-30 µM lavendustin A (
), and 10 µM tyrphostin A1 (
) and daidzein (
). Relaxations are expressed as percentages of those before addition of the antagonist (control). Values are means ±
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Figure 3. Effects of tyrosine kinase inhibitors on EFS-induced relaxation of longitudinal muscle of rat distal colon
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Figure 4. Effects of charybdotoxin and tyrphostin 25 on EFS-induced relaxation of longitudinal muscle of rat distal colon
Significantly different from the values with ChTX or tyrphostin 25 alone, P < 0·05.
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Figure 5. Effects of apamin and tyrphostin 25 on EFS-induced relaxation of longitudinal muscle in rat distal colon
Tyrosine kinase inhibitor Concentration (µM) EFS-induced relaxation (% of control values) Genistein 1 86·9 ± 6·6 (4) 10 75·3 ± 4·1 * (5) Daidzein 10 89·3 ± 5·5 (3) Tyrphostin 25 1 81·0 ± 3·6 * (3) 10 49·7 ± 5·2 * (4) Tyrphostin A1 10 90·3 ± 3·7 (4) Lavendustin A 30 36·0 ± 8·3 * (5)
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Figure 6. Effects of genistein and tyrphostin 25 on EFS-induced IJPs in longitudinal smooth muscle cells of rat distal colon
) or genistein (). Inhibitions are expressed as the percentage of the number of IJPs obtained before addition of antagonist. Values are means ±
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Figure 7. Effects of genistein on exogenous PACAP-induced hyperpolarization in longitudinal smooth muscle cells of rat distal colon
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Figure 8. Effects of tyrphostin 25 on PACAP-induced relaxation and decrease in [Ca2+]i in longitudinal muscle of rat distal colon
) and decrease in [Ca2+]i (
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Figure 9. Effects of apamin on PACAP-induced relaxation and decrease in [Ca2+]i in longitudinal muscle of rat distal colon
, PACAP-induced relaxation;
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References
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J. Pharmacol. Exp. Ther.,
December 1, 2002;
303(3):
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[Abstract]
[Full Text]
[PDF]
M. J. Davis, X. Wu, T. R. Nurkiewicz, J. Kawasaki, P. Gui, M. A. Hill, and E. Wilson
Regulation of ion channels by protein tyrosine phosphorylation
Am J Physiol Heart Circ Physiol,
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