FULL PAPER Pharmacology

Effects of Various Selective Phosphodiesterase Inhibitors on Carbachol-Induced Contraction and Cyclic Nucleotide Contents in the Guinea Pig Gall Bladder

Takeharu KANEDA1), Akitoshi WATANABE1), Kazumasa SHIMIZU1), Norimoto URAKAWA1) and Shinjiro NAKAJYO1)

1)Division of Veterinary Pharmacology, Nippon Veterinary and Animal Science University, 7–1 Kyonan-cho 1-chome, Musashino, Tokyo 180–8602, Japan

(Received 25 November 2004/Accepted 17 March 2005)

ABSTRACT. The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)ade- nine (EHNA, type 2), milrinone (type 3), Ro20–1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concen- tration-dependent manner. The rank order of potency for the gall bladder was Ro20–1724 > vinpocetine > EHNA >milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 µM), vinpocetine, milrinone, and Ro20– 1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20–1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta. KEY WORDS: cAMP, cGMP, Gall bladder, PDE inhibitor. J. Vet. Med. Sci. 67(7): 659–665, 2005

Cyclic adenosine monophosphate (cAMP) and cyclic Recently, we showed that the rank order of inhibitory guanosine monophosphate (cGMP) are important second potency on carbachol-induced contraction in the smooth messengers, and have been associated with smooth muscle muscle of guinea pig taenia coli was type 5 > type 1 > type relaxation [5]. These cyclic nucleotides are synthesized by 2 > type 4 > type 3, and that type 1 and type 4-induced relax- adenylyl cyclase or guanylyl cyclase, and are degraded by ation was correlated with cAMP, although type 2 and type phosphodiesterase (PDE). Currently, PDEs are classified 5-induced relaxation was correlated with cGMP [11]. These into 11 families [3, 6, 23], and selective PDE inhibitors have reports probably indicate that the differences in inhibitory been found [4]. It has been reported that the relaxation effects of these selective PDE inhibitors on muscle contrac- induced by these selective PDE (type 1-type 5) inhibitors is tility are due to the distribution of the PDE families in the involved in the increases in cAMP and/or cGMP contents in smooth muscles of the gastrointestinal tract. vascular [14], tracheal [16], urinary [15, 22], and intestinal In the present study, we have used selective PDE (type 1– smooth muscle [10, 11]. Since PDE1 and 2 hydrolyze 5) inhibitors to examine the effects of contractility of the cAMP and/or cGMP [3], it is thought that type 1 and type 2 smooth muscles in the guinea pig gall bladder, part of the inhibitor-induced relaxations may be correlated with gastrointestinal tract, and to compare with those of the tra- increases in cAMP and/or cGMP in smooth muscles. It has chea, teania coli, and aorta. In addition, we also investigated been reported that vinpocetine (type 1 inhibitor) induced the relationship between relaxation and cyclic nucleotide relaxation in the rabbit aorta through the increase in cGMP contents in carbachol-induced contractions of the gall blad- content [1, 7], and in the rat urinary bladder [15] and guinea der by type 1–5 inhibitors. pig taenia coli [10] through an increase in the cAMP con- tent. There have been few reports indicating that relaxation MATERIALS AND METHODS induced by EHNA (type 2 inhibitor) is associated with the increases in the cyclic nucleotide contents of smooth muscle Muscle preparations and tension measurement: Male [9, 11, 13]. guinea pigs (Hartley strain, 300–400 g; Funabashi Farm, Additionally, the relationship of relaxation induced by Funabashi ) were bled after being stunned, and then the tra- these selective inhibitors and cyclic nucleotides is less clear chea, thoracic aorta, taenia coli and gall bladder were in the smooth muscle of the gastrointestinal tract. Barnette quickly removed. The aorta was cut into spiral strips and the et al. [2] assessed the activity of PDE families and the relax- other muscles were made in longitudinal strips. The endo- ation induced by the PDE3, 4, and 5 inhibitors in canine thelium and epithelium were removed by gentle rubbing the colonic smooth muscle. They showed that PDE3 and 4 inner surface of the aorta and trachea with absorbent cotton. inhibitors decreased the hydrolysis of cAMP, that the PDE5 The muscle strips were incubated with physiological salt inhibitor decreased the hydrolysis of cGMP, and that the solution (PSS) containing (in mM) 136.8 NaCl, 5.4 KCl, 2.5 type 4 inhibitor was more potent than the other inhibitors. CaCl2, 1.0 MgCl2, 11.9 NaHCO3, and 5.5 glucose. PSS was 660 T. KANEDA ET AL.

aerated with 95% O2 and 5% CO2 to adjust pH to 7.2 at inhibition of EHNA and Ro20–1724 in the gall bladder were 37°C. This study was conducted in accordance with the more potent than those in other muscles. In the gall bladder, Guideline for the Care and Use of Laboratory Animals of IC50 and the maximum inhibition of milrinone were less Nippon Veterinary and Animal Science University. potent than those in the trachea and aorta, and IC50 and the Muscle tension was recorded isometrically. One end of maximum inhibition of zaprinast were much less potent than each strip was bound to a glass holder and the other end was those in the taenia coli. connected by a silk thread to a strain-gauge transducer (TB- The rank orders for the relaxant effect were Ro20–1724 > 611T; Nihon Kohden, Tokyo) in an organ bath containing vinpocetine > EHNA >milrinone > zaprinast for the gall PSS with a resting tension of 1 g. The muscle strips were bladder, Ro20–1724 > milrinone > vinpocetine > zaprinast equilibrated for 30 min to obtain a stable contractility > EHNA for the trachea, zaprinast > vinpocetine > EHNA > induced by hyperosmotically added 65 mM KCl. When the Ro20–1724 > milrinone for the taenia coli, and milrinone > contractile response induced by 0.3 µM carbachol (CCh) or vinpocetine > Ro20–1724 > zaprinast > EHNA for the aorta. 10 µM phenylephrine (PE) reached a steady level about 15– Effects of various PDE inhibitors on cGMP and cAMP 20 min after addition to a trachea, aorta, teania coli, and gall contents in the gall bladder: Vinpocetine, milrinone, and bladder, vinpocetine (type 1), EHNA (type 2), milrinone Ro20–1724 caused concentration-dependent increases in (type 3), Ro20–1724 (type 4), or zaprinast (type 5) was the cAMP content of the gall bladder (Figs. 3A, C, and D), added cumulatively. and higher concentrations (100 µM) were required to cause Assay of cGMP or cAMP content: The cGMP and cAMP a significant increase in cAMP content for EHNA (Fig. 3B). content in the muscle strips was measured by enzyme There was a positive correlation between the relaxation of immunoassay, as reported previously [11]. After incubation the CCh-induced contractions and the increase in cAMP of the strips with each of the PDE inhibitors for 10 min in content elicited by vinpocetine, EHNA, milrinone, and the presence of carbachol (0.3 µM), the strips were rapidly Ro20–1724. However, at a concentration that induced half- frozen in liquid nitrogen, and stored at –80°C until homoge- maximum relaxation, the Ro20–1724-increased cAMP con- nized in 6% trichloroacetic acid (0.4 ml). The homogenate tent was smaller than those of the other inhibitors (Fig. 4A). was centrifuged at 3000 × g for 15 min, and the supernatant On the other hand, EHNA caused concentration-depen- was washed with 1.5 ml of water-saturated diethyl ether four dent increases in cGMP content of the gall bladder in the times. The cGMP and cAMP content of the strips was presence of CCh, and higher concentrations (100 µM) were assayed with an enzyme immunoassay kit (Amersham Phar- required to cause a significant increase in cGMP content macia Biotech, UK). The cGMP and cAMP content was (Fig. 3B). Zaprinast also caused a concentration-dependent expressed as picomole per gram wet weight. increase in cGMP content (Fig. 3E), but vinpocetine, mil- Chemicals: The chemicals used were milrinone, zapri- rinone, and Ro20–1724 did not significantly increase the nast, carbachol (Sigma, St. Louis, MO, U.S.A.), vinpocet- cGMP content above control (Figs. 3A, C, and D). There ine, erythro-9-(2-hydroxy-3-nonyl) adenine•HCl (EHNA) was a positive correlation between the relaxation and the (BIOMOL Research Laboratories, U.S.A.), and Ro20–1724 increase in cGMP content elicited by EHNA and zaprinast. (LC Laboratories New Boston U.S.A.). Vinpocetine crys- However, at a concentration that induced half-maximum tallizes if the concentration of vinpocetine exceeds 30 µM in relaxation, EHNA-increaesd cGMP content was less than PSS. Therefore, we used vinpocetine below 30 µM in the that of zaprinast (Fig. 4B). present experiment. Statistics: Values are expressed as mean ± SEM, and the DISCUSSION IC50 values (the concentration producing 50% relaxation) were determined by linear regression analysis. Statistical The functional role of PDE families has been examined in analyses were performed by Student’s t-test. several smooth muscle preparations as follows. It has been shown that PDE type 3 and type 4 inhibitors had more RESULTS potent effects than the other PDE inhibitors on the pre-con- tracted trachea [16], and that type 3 inhibitors had the most Effects of various selective PDE inhibitors on contractil- potent effects on the aorta [14]. In the guinea pig trachea, ity: Vinpocetine, EHNA, milrinone, Ro20–1724, and zapri- milrinone, a PDE type 3 inhibitor, and Ro20–1724, a PDE nast inhibited CCh-induced contractions in a concentration- type 4 inhibitor, inhibited CCh-induced contractions, and dependent manner in the gall bladder, trachea, and taenia their IC50 values were 30 µM [8]. Moreover, in the rat aorta, coli (Figs. 1A, and B, C and 2A, B, and C). Furthermore, milrinone and Ro20–1724 inhibited norepinephrine- these agents inhibited PE-induced contractions in a concen- induced contractions and their IC50 values were 6 µM and tration-dependent manner in the aorta (Figs. 1 D and 2D). 240 µM, respectively [18]. Our present results were consis- The concentrations of these agents producing 50% relax- tent with these reports. IC50 of milrinone and Ro20–1724 in ation (IC50) and the maximum relaxation of CCh- or PE- the trachea were similar, but IC50 of milrinone was larger induced contractions are presented in Table 1. IC50 and the than that of Ro20–1724 in the aorta. In the present study, maximum inhibition of vinpocetine in the gall bladder were selective PDE inhibitors inhibited CCh-induced contrac- similar to those in the other muscles. IC50 and the maximum tions in the trachea, gall bladder, and taenia coli, and the PDE INHIBITORS IN GALL BLADDER 661

Fig. 1. The effects of vinpocetine on contractions induced by 0.3 µM carbachol (CCh) in the gall bladder (A), trachea (B), and taenia coli (C), and on contractions induced by 10 µM phenylephrine (PE) in the aorta (D). The concentrations used were as follows: KCl, 65.4 mM; vinpocetine 0.03–30 µM. The traces represent typical results from 1 out of 4–6 preparations. rank order of inhibitory potency was Ro20–1724 > mil- guinea pig and rat ileal smooth muscle, and showed that rinone > vinpocetine > zaprinast > EHNA for the trachea, type 5 inhibitors were more potent than the other inhibitors. Ro20–1724 > vinpocetine > EHNA >milrinone > zaprinast These reports and our data showed that the inhibitory effect for the gall bladder, and zaprinast > vinpocetine > EHNA > of type 5 inhibitors in the gall bladder was similar to that in Ro20–1724 > milrinone for the taenia coli. For the aorta, the colon. We also showed that the effect of the inhibitors selective PDE inhibitors inhibited PE-induced contractions, in the taenia coli was similar to that in the ileum [10, 11]. and the rank order of inhibitory potency was milrinone > These results suggest that the differences in inhibitory vinpocetine > Ro20–1724 > zaprinast > EHNA. Further- potency on PDE inhibitors in the gastrointestinal tract is due more, IC50 of Ro20–1724 in the gall bladder was the lowest to the distribution of PDE families. in this experiment. There were a few reports that showed It has been reported that vinpocetine (type 1 inhibitor) the effects of selective PDE inhibitors on muscle contractil- induces relaxation by increasing the cGMP content in the ity in the smooth muscle of the gastrointestinal tract. Bar- rabbit aorta [1, 7]. However, in the rat urinary bladder [15] nette et al. [2] showed that PDE type 4 inhibitors were more and the guinea pig taenia coli [11], vinpocetine induces potent than PDE type 3 and type 5 inhibitors in canine relaxation through an increase in the cAMP content. In the colonic smooth muscle. Tomkinson and Raeburn [21] present study, there was a positive correlation between the investigated the inhibitory potency of PDE type 1, 3, 4, and inhibition of CCh-induced contractions and the increase in 5 inhibitors on methacholine-induced contractions in the cAMP content by vinpocetine in the guinea pig gall bladder. 662 T. KANEDA ET AL.

Fig. 2. The effects of vinpocetine ( ), EHNA ( ), milrinone ( ), Ro20–1724 ( ) and zaprinast ( ) on contractions induced by 0.3 µM CCh in the gall bladder (A), trachea (B), and taenia coli (C), and on contractions induced by 10 µM PE in the aorta (D). The contractions induced by 0.3 µM CCh or 10 µM PE just before the application of these PDE inhibitors were taken as 100%. Each point represents the mean of 4–7 preparations. Vertical bars indicate the SEM.

These results suggested that the relaxation induced by PDE This difference in potency of cGMP in these inhibitors is type 1 inhibitor was involved in the increase in cAMP con- due to the fact that EHNA increased both the cAMP and tent in the guinea pig gall bladder. PDE1 families are iden- cGMP contents, while zaprinast only increased the cGMP tified as PDE1A, 1B, and 1C. PDE1A and 1B have high content in the gall bladder. These results suggested that the affinity for cGMP, and PDE1C has a similar affinity for relaxation induced by PDE type 2 inhibitor was involved in cAMP and cGMP [3]. It is known that PDE1 hydrolyzes the increase in the cAMP and cGMP contents in the guinea cGMP in some smooth muscle cells [17]. However, vinpo- pig gall bladder. The present data showed that the relation- cetine might inhibit PDE1C, but not PDE1A and B in the ship between type 2 inhibitor-induced relaxation and the guinea pig gall bladder. increase in cyclic nucleotides in the guinea pig gall bladder It has been suggested that EHNA (type 2 inhibitor) was different from that in the guinea pig taenia coli [11]. relaxes the rat pulmonary artery tone by increasing the In the guinea pig taenia coli, the inhibition of CCh- cAMP and cGMP content [9]. Additionally, it has been induced contractions by milrinone (type 3 inhibitor) was reported that the relaxation induced by EHNA might be less than that of the other PDE inhibitors, and milrinone did involved in the increase in cAMP content in the urinary not significantly increase the cAMP and cGMP content bladder [15] and taenia coli [11]. In this paper, we showed [11]. In the present study, there was a positive correlation that there was a positive correlation between the inhibition between the inhibition of CCh-induced contractions and the of CCh-induced contractions and the increase in cAMP or increase in the cAMP content by milrinone in the guinea pig cGMP content caused by EHNA. However, EHNA-evoked gall bladder. These results suggested that the difference in increases of cGMP content at a concentration that induced a the inhibitory effect on PDE3 inhibitors is due to the distri- medium-sized relaxation, was lower than that of zaprinast. bution of PDE3 families in the gastrointestinal tract. PDE INHIBITORS IN GALL BLADDER 663

Table 1. IC50 and maximal inhibition (%) of various selective PDE inhibitors for contractions induced by CCh or PE Vinpocetine EHNA Milrinone Ro20–1724 Zaprinast Gall bladder IC50 (µM) 3.7 10.6 49.4 0.4 97.3 (3.4–4.0) (8.3–12.8) (39.1–52.6) (0.3–0.6) (78.7– >100) Maximal inhibition (%) 85.6 ± 3.2 90.8 ± 1.5 63.3 ± 4.0 83.3 ± 4.9 53.5 ± 2.3 n56676 Trachea IC50 (µM) 15.9 >100 6.8 2.0 98.4 (12.1–21.3) (5.5–8.5) (1.4–2.9) (61.1–>100) Maximal inhibition (%) 77.6 ± 3.2 53.9 ± 2.6 88.0 ± 2.5 78.1 ± 2.3 58.1 ± 4.5 n66864 Taenia coli IC50 (µM) 7.7 42.9 >100 >100 5.6 (7.4–8.0) (38.0–45.8) (3.6–7.6) Maximal inhibition (%) 72.6 ± 2.9 72.5 ± 3.6 12.4 ± 3.7 42.6 ± 3.8 86.4 ± 3.2 n6646 5 Aorta IC50 (µM) 18.1 >100 6.6 >100 >100 (15.2–21.7) (5.7–7.7) Maximal inhibition (%) 63.6 ± 3.8 1.8 ± 0.2 91.4 ± 0.1 25.8 ± 4.7 13.1 ± 4.3 n4 4545 Numbers in parentheses indicate 95% confidence limits. The inhibition induced by PDE inhibitors that fully recovered to the basal tension before the application of CCh (0.3 µM) or PE (10 µM) was taken as 100% inhibition.

Fig. 3. The effects of vinpocetine (A), EHNA (B), milrinone (C), Ro20–1724 (D) and zaprinast (E) on the cAMP and cGMP contents of the guinea pig gall bladder. Preparations were precontracted with 0.3 µM CCh, and were treated with these agents for 10 min. The control was treated with vehicle instead of these PDE inhibitors. Each point represents the mean of 4 experiments. Vertical bars indi- cate the SEM. * and **: Significant difference from each respective control, with P<0.05 and P<0.01, respectively. 664 T. KANEDA ET AL.

than that of the other inhibitors. We reported that MBCQ, a selective PDE 5 inhibitor, inhibits muscle contraction with- out changes in cAMP and cGMP content in ileal smooth muscle [12]. Therefore, it seems that Ro20–1724-induced relaxation may be partially involved in the cAMP- and cGMP-independent mechanism. Further study is needed to clarify the mechanisms of the cAMP- and cGMP-indepen- dent mechanism on Ro20–1724-induced relaxation in the guinea pig gall bladder. In the present study, there was a positive correlation between the inhibition of CCh-induced contractions and the increase in cGMP content elicited by zaprinast in the gall bladder. These data were consistent with the report that type 5 inhibitor relaxed the rat aorta by an increase in the cGMP content [20]. In conclusion, selective PDE inhibitors inhibited CCh- induced contractions in the guinea pig gall bladder, and the rank order of inhibitory potency was Ro20–1724 (type 4) > vinpocetine (type 1) > EHNA (type 2) > milrinone (type 3) > zaprinast (type 5). The gall bladder and taenia coli belong to the gastrointestinal tract, but the rank order of these PDE inhibitors in the gall bladder was different from that of tae- nia coli. Moreover, it was indicated that vinpocetine-, EHNA-, milrinone-, and Ro20–1724-induced relaxation has a correlation with cAMP, and that EHNA- and zaprinast- induced relaxation has a correlation with cGMP in the gall bladder.

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