Phosphodiesterase Isozymes Involved in Regulation of HCO3 Secretion in Isolated Mouse Stomach in Vitro

( )-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (NOR-3), a nitric-oxide (NO) donor, is known to increase HCO3 secretion in rat stomachs, intracellularly mediated by cGMP; yet, there is no information about the phosphodiesterase (PDE) isozyme involved in this process. We examined the effects of various isozyme-selective PDE inhibitors on the secretion of HCO3 in the mouse stomach in vitro and the type(s) of PDE isozymes involved in the response to NO. The gastric mucosa of DDY mice was stripped of the muscle layer and mounted on an Ussing chamber. HCO3 secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. NOR-3, 8-bromoguanosine 3 ,5 -cyclic monophosphate (8-Br-cGMP), and various PDE inhibitors were added to the serosal side. Vinpocetine (PDE1 inhibitor) or zaprinast (PDE5 inhibitor) was also added serosally 30 min before NOR-3 or 8-Br-cGMP. Both NOR-3 and 8-Br-cGMP stimulated HCO3 secretion in a dosedependent manner, and the response to NOR-3 was significantly inhibited by methylene blue. Likewise, the secretion induced by NOR-3 or 8-Br-cGMP was significantly attenuated by 6-((2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid (ONO-8711), the PGE receptor (EP)1 antagonist, as well as indomethacin and potentiated by both vinpocetine and zaprinast at doses that had no effect by themselves on the basal secretion, whereas other subtype-selective PDE inhibitors had no effect. NOR-3 increased the mucosal PGE2 content in a methylene blueinhibitable manner. These results suggest that NO stimulates gastric HCO3 secretion mediated intracellularly by cGMP and modified by both PDE1 and PDE5, and this response is finally mediated by endogenous PGE2 via the activation of EP1 receptors. The secretion of HCO3 from the surface epithelial cells is a key process that aids in preventing acid-peptic injury (Flemström and Garner, 1982; Flemström, 1987; Allen et al., 1993). The mechanisms that govern HCO3 secretion involve neurohumoral factors and luminal acid (Flemström and Garner, 1982; Takeuchi et al., 1991; Montrose et al., 2005); yet, both endogenous prostaglandins (PGs) and nitric oxide (NO) play a particularly important role in the local control of this secretion (Heylings et al., 1984; Takeuchi et al., 1986; Sugamoto et al., 2001, Aihara et al., 2005b). The stimulatory effect of PGE2 on HCO3 secretion in the stomach is known to be mediated by the activation of EP1 receptors and coupled intracellularly with Ca (Takeuchi et al., 1997b, 2005). In contrast, NO stimulates the secretion of HCO3 in the duodenum via the activation of soluble guanylate cyclase and an increase in the intracellular level of cGMP (Moncada et al., 1991; Furukawa et al., 1999; Sugamoto et al., 2001). We further showed that NOR-3, an NO donor, increased the secretion of HCO3 in the rat stomach, via endogenous PGE2 in a cGMP-dependent manner (Aihara et al., 2006). It is thus assumed that both PG/Ca and NO/cGMP are involved in the local regulation of HCO3 secretion in the stomach. Because the HCO3 stimulatory action of NO is intracellularly mediated by cGMP and because cGMP is degraded into inactive metabolites via hydrolysis by phosphodiesterase This research was supported in part by the Kyoto Pharmaceutical University’s 21st Century Centers of Excellence program and the Open Research Program from the Ministry of Education, Science and Culture of Japan. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.138941. ABBREVIATIONS: PG, prostaglandin; NO, nitric-oxide; EP, PGE receptor; NOR-3, ( )-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide]; PDE, phosphodiesterase; 8-Br-cGMP, 8-bromoguanosine 3 ,5 -cyclic monophosphate; L-NAME, N-nitro-L-arginine methylester; ONO-8711, 6-((2S,3S)-3-(4-chloro-2-methylphenysulfonylaminomethyl)-bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid; AE3-208, 4-(4-cyano-2-(2-(4-fluoronaphthalen-1-yl)propionylamino)phenyl)butyric acid; COX, cyclooxygenase; NOx, nitrite and nitrate; dbcAMP, dibutyryl 3 ,5 -cyclic adenosine monophosphate; IBMX, isobutyl methylxanthine; EHNA, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride; DMSO, dimethyl sulfoxide; AE5-599, 3-{2-({[(1R)-1-(3,5-dimethylphenyl)-3-methylbutyl]amino}carbonyl)-4-[(2-methylphenoxy)methyl]phenyl}propanoic acid. 0022-3565/08/3263-889–896$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 3 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 138941/3374841 JPET 326:889–896, 2008 Printed in U.S.A. 889 at A PE T Jornals on July 1, 2017 jpet.asjournals.org D ow nladed from (PDE), it is possible that PDE affects the response to NO by altering the levels of this nucleotide. At present, the PDE in mammalian tissues has been subdivided into 11 isozymes, each derived from separate gene families and having pharmacologically distinct roles (Francis et al., 2001). PDE1 to PDE5 have been well characterized, and selective inhibitors of these isozymes are used for the treatment of heart disease, depression, asthma, inflammatory disease, and erectile dysfunction (Thompson, 1991; Xu et al., 2000; Sung et al., 2003). To disclose the profiles of the subtype-selective PDE inhibitors would be helpful for eliminating the adverse influences of these agents in the body, including the gastrointestinal tract. We recently reported using the isolated mouse duodenum in vitro that the response of HCO3 to PGE2 is regulated by both PDE1 and PDE3, whereas the response to NO is modulated by only PDE1 (Hayashi et al., 2007). It remains, however, unexplored which PDE isozyme(s) is involved in the regulation of gastric HCO3 secretion. The regulatory mechanism of HCO3 secretion differs in many points between the stomach and the duodenum; in the latter, the secretion is mediated intracellularly with both cAMP, cGMP, and Ca , whereas that in the stomach is intracellularly mediated by Ca and cGMP but not cAMP (Flemström, 1977, 1987; Guba et al., 1996; Seidler et al., 1997; Takeuchi et al., 1997a; Furukawa et al., 1999; Rao et al., 2004; Sellers et al., 2005). Thus, there is a possibility that the PDE isozymes involved in the HCO3 response are different in these tissues. In the present study, we examined the effects of subtypeselective inhibitors of PDE1 to PDE5 on the secretion of HCO3 in response to NO as well as PGE2 in the isolated mouse stomach in vitro, and we investigated which isozymes of PDE are involved in the local regulation of gastric HCO3 secretion. In addition, we also examined the interactive role of NO and PGE2 in the stimulation of HCO3 secretion in the stomach. Materials and Methods Animals. Male DDY mice weighing 25 to 30 g (SLC Japan, Shizuoka, Japan) were used in all experiments. The animals, kept in stainless steel cages with raised mesh bottoms, were deprived of food but allowed free access to tap water for 18 h before the experiments. All experimental procedures used were carried out in accordance with the Helsinki Declaration and have been approved by the Committee for Animal Experimentation established by Kyoto Pharmaceutical University. Determination of HCO3 Secretion. Under deep diethyl ether anesthesia, the mouse was killed and the abdomen was opened by a midline incision. The entire stomach was removed and immediately placed in HCO3 Ringer’s solution containing indomethacin (10 6 M) to suppress trauma-induced PG release. The stomach was opened along the greater curvature and stripped off the muscular layers under a microscope (SZ-PT; Olympus, Tokyo, Japan). The tissues (the corpus mucosa) were then mounted between two halves of a lucite chamber, the exposed area being 12.5 mm, and they were bathed in unbuffered saline (154 mM Na and 154 mM Cl ) gassed with 100% O2 on the mucosal side and HCO3 Ringer’s solution (140 mM Na , 120 mM Cl , 5.4 mM K , 1.2 mM Mg , 1.2 mM Ca , 1.4 mM HPO4 2 , 2.4 mM H2PO4 , 25 mM HCO3 , 10 mM glucose, and 0.001 mM indomethacin) gassed with 95% O2, 5% CO2 on the serosal side (Seidler et al., 1997; Tuo et al., 2006; Hayashi et al., 2007). These solutions were warmed at 37°C and continuously circulated by a gas-lift system. The osmolalities for both solutions were approximately 308 mOsm/kg. The HCO3 secretion was measured by the pH-stat method (Comtite-980; Hiranuma industries, Ibaraki, Japan) using 2 mM HCl as the titrant to keep the mucosal pH at 7.0. To unmask HCO3 secretion, acid secretion had been completely inhibited by prior i.p. administration of omeprazole at a dose of 60 mg/kg. Omeprazole at this dose has been shown to have no influence on gastric HCO3 secretion in rats (Knutson and Flemström, 1989). Measurements were made every 5 min starting at least 1 h after the mounting of the tissues. After the rate of secretion had stabilized for 45 min, the following agents were added to the serosal solution; PGE2 (10 7 10 6 M), NOR-3 (NO donor; 10 3 3 10 3 M), 8-bromoguanosine 3 ,5 -cyclic monophosphate (8-Br-cGMP; cGMP analog; 10 5 10 3 M), dibutyryl 3 ,5 -cyclic adenosine monophosphate (dbcAMP; 10 3 M), forskolin (adenylyl cyclase stimulator; 5 10 6 M), vinpocetine (PDE1 inhibitor; 10 6 10 5 M), erythro-9-(2-hydroxy-3nonyl)-adenine hydrochloride (EHNA) (PDE2 inhibitor; 10 5 M), cilostamide (PDE3 inhibitor; 10 5 M), rolipram (PDE4 inhibitor; 10 5 M), and zaprinast (PDE5 inhibitor; 10 6 10 5 M). In some cases, the effects of vinpocetine and zaprinast on the response of HCO3 to PGE2, NOR-3, and cGMP were examined; the PDE inhibitors were added 30 min before the latter agents. In addition, the effects of the following inhibitors or antagonists on the response to PGE2, NOR-3, and 8-Br-cGMP as well as the PDE inhibitors were also examined: methylene blue (guanylate cyclase inhibitor; 1 10 4 M), N-nitro-L-arginine methylester (L-NAME; nonselective nitricoxide synthase inhibitor; 10 3 M), ONO-8711 (EP1 receptor antagonist; 10 5 M) (Takeuchi et al., 2006), AE5-599 (EP3 receptor antagonist; 3 10 6 M) (Aihara et al., 2007), AE3-208 (EP4 receptor antagonist; 10 6 M) (Aoi et al., 2004), or indomethacin [nonselective cyclooxygenase (COX) inhibitor; 10 5 M]. These agents were added serosally 30 min before the agonists. Determination of Nitrite and Nitrate Levels. The amount of NO generated in the mucosa was determined indirectly as nitrite/ nitrate (NO2 and NO3 ) in the normal mouse stomach and after treatment with L-NAME. After the stomach was rinsed with cold saline, the mucosa was scraped with glass slides and homogenized in 50 mM KHPO4 buffer. This was followed by centrifugation for 10 min at 10,000 rpm at 4°C. The nitrite and nitrate (NOx) levels of the supernatant were measured by the Griess reaction-dependent method described by Green et al. (1982), after the reduction of NO3 to NO2 with nitrate reductase. Nitrites were incubated with Griess reagent (0.1% naphthalene diamine dihydrochloride and 1% sulfanilamide in 2.5% H3PO4) for 10 min at room temperature, and the absorbance at 545 nm was measured. For the standard curve, sodium nitrite was used. L-NAME (10 3 M) was added to the serosal solution 1 h before the measurement of NOx. Measurement of Intracellular Levels of cGMP in Mouse Stomachs. The isolated mouse stomach (the corpus mucosa) mounted between two halves of a lucite chamber was treated with the serosal addition of NOR-3 (3 10 3 M) with or without vinpocetine (10 6 M) or zaprinast (10 6 M) for 10 min. The latter agents were added serosally 30 min before NOR-3. Ten minutes after the addition of NOR-3, the tissue was homogenized in 2 ml of 5% trichloroacetic acid in tissue homogenizer on ice (0 4°C). The precipitate was removed by centrifugation at 1500g for 10 min, and the supernatant was transferred to a test tube. The supernatant was then extracted 4 5 times with 2 volume of ether, and then the ether fractions were collected and removed from the aqueous layer by heating the sample to 70°C for 5 min. The levels of cGMP were measured using a cGMP enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Analyses for Gene Expression of PDE Isozymes by Reverse Transcription-Polymerase Chain Reaction. Mice were killed under deep ether anesthesia, and the stomachs were removed. Gastric tissue samples were pooled from three mice for extraction of total RNA, which was achieved by a single-step acid phenol-chloroform extraction procedure using TRIzol (Invitrogen, Carlsbad, CA). Total RNA primed by random hexadeoxy ribonucleotide was reverse-tran890 Kita et al. at A PE T Jornals on July 1, 2017 jpet.asjournals.org D ow nladed from scribed with the SuperScript preamplification system (Invitrogen). The sequences of sense and antisense primers for mouse PDE1 5, including the splicing variants in PDE1, -3, and -4, are listed in Table 1. An aliquot of the reverse transcription reaction product served as a template in 35 cycles of polymerase chain reaction with 1 min of denaturation at 94°C, 0.5 min of annealing at 58°C, and 1 min of extension at 72°C on a thermal cycler. A portion of the polymerase chain reaction mixture was electrophoresed in 1.8% agarose gel in TAE buffer (40 mM Tris buffer, 2 mM EDTA, and 20 mM acetic acid, pH 8.1), and the gel was stained with ethidium bromide and photographed. Preparation of Drugs. Drugs used were PGE2 (Funakoshi, Tokyo, Japan); NOR-3 (Doujindo, Tokyo, Japan); dbcAMP, 8-Br-cGMP, isobutyl methylxanthine (IBMX), vinpocetine, EHNA, cilostamide, rolipram, zaprinast (Aldrich Chemical Co., Milwaukee, WI); indomethacin, L-NAME (Sigma-Aldrich, St. Louis, MO); methylene blue, forskolin (Nacalai Tesque, Kyoto, Japan); ONO-8711, AE5-599, AE3208 (Ono Pharmaceutical Co., Osaka, Japan); and omeprazole (AstraZeneca, Möndal, Sweden). Omeprazole was suspended in 0.5% carboxymethylcellulose solution (Wako, Osaka, Japan), whereas other agents were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemicals, Osaka, Japan) and diluted with distilled water to desired concentrations. All agents were prepared before use and added to the nutrient solution. Statistical Analysis. Data are expressed as the mean S.E. for four to six mice. Statistical analyses were performed with a one-way analysis of variance followed by the Dunnett’s multiple comparison test or, when appropriate, Student’s t test. Values of P 0.05 are considered significant.