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cGMP–PDE3–cAMP signal pathway involved in the inhibitory effect of CNP on gastric motility in rat
Regulatory Peptides 180 (2013) 43–49
Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
cGMP–PDE3–cAMP signal pathway involved in the inhibitory effect of CNP on gastric motility in rat Ying-Lan Cai a, Qian Sun a, Xu Huang b, 1, Jing-Zhi Jiang a, Mo-Han Zhang a, Li-Hua Piao a, Zheng Jin a,⁎, Wen-Xie Xu b,⁎⁎ a b
Department of Physiology, Yanbian University School of Medicine, 977 Gongyuan, Road, Jilin, Yanji 133002, China Department of Physiology, Shanghai Jiaotong University School of Medicine, 800 Dongchuan Road, 328 Wenxuan Medical Building, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 26 October 2012 Accepted 13 November 2012 Available online 24 November 2012 Keywords: Gastric smooth muscle CNP PDE2 PDE3 cGMP–PDE3–cAMP
a b s t r a c t In the present study, we investigated the mechanism of C-type natriuretic peptide (CNP)-induced inhibitory effect on spontaneous contraction of gastric antral smooth muscle to clarify CNP-NPR-B/pGC-cGMP downstream signal transduction pathway using organ bath and ELISA methods in rat. CNP significantly reduced the amplitude of the spontaneous contraction and increased the contents of cGMP and cAMP in the gastric antral smooth muscle tissue. In the presence of IBMX, a non-selective phosphodiesterase (PDE) inhibitor, the inhibitory effect of CNP on spontaneous contraction was significantly suppressed; however, the production of cGMP but not cAMP was still increased by CNP. EHNA, a PDE2 inhibitor, did not affect both CNP-induced inhibition of the contraction and CNP-induced increase of cGMP and cAMP generations in gastric smooth muscle tissue, while milrinone, a PDE3 inhibitor, similar to IBMX, attenuated the CNP-induced inhibitory effect on spontaneous contraction and increased the content of cGMP but not cAMP. The results suggest that cGMP–PDE3–cAMP signal pathway is also involved in the CNP-induced inhibition of gastric motility in rat. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cyclic nucleotide monophosphates cyclic AMP (cAMP) and cyclic GMP (cGMP) are important endogenous mediators of several processes, including smooth muscle motility [1,2]. Cyclic nucleotides are synthesized from the corresponding nucleotide triphosphates by the activity of adenylyl and guanylyl cyclases. The increase in cAMP or cGMP triggers a signal transduction cascade encompassing the activation of cyclic nucleotide-dependent protein kinases: cyclic adenosine-dependent protein kinase (cAK) or cyclic guanosine-dependent protein kinase (cGK), subsequent phosphorylation of the actinmyosin system, as well as Ca 2+ channels and ATP-driven Ca2+ pumps located in the outer cell membrane or the membrane of the sarcoplasmic reticulum [3,4]. This cascade leads to a reduction in cytosolic Ca 2+ and, finally, to smooth muscle relaxation. In the gastrointestinal tract, natriuretic peptides are expressed in specific regions of the stomach and intestine where there is evidence that they are involved in paracrine regulation of gastrointestinal motility [5–8]. Our previous studies have shown that CNP peptides relax
⁎ Corresponding author. Tel.: +86 433 2435122; fax: +86 433 2435104. ⁎⁎ Corresponding author. Tel.: +86 21 34205639; fax: +86 21 34204118. E-mail addresses: [email protected] (Z. Jin), [email protected] (W.-X. Xu). 1 Co-first author. 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.11.001
isolated gastric smooth muscle in different species, for example, guinea-pig, rat and humans, via CNP/natriuretic peptide receptor B (NPR-B)/cGMP pathway [8–11]; however, CNP-NPR-B/pGC-cGMP downstream signal transduction pathway is not clear in gastrointestinal smooth muscle. Cyclic nucleotides are degraded by phosphodiesterase (PDE) isoenzymes, a heterogeneous group of hydrolytic enzymes. PDE are classified according to their preferences for cAMP or/and cGMP, kinetic parameters of cyclic nucleotide hydrolysis, sensitivity to the inhibition by various compounds, allosteric regulation by other molecules and chromatographic behavior on anion exchange columns [3,4]. At least 11 PDE families and 18 subtypes have been identified in the mammal tissues by far, which play different roles [12]. And at least five PDE families have been found in the stomach, including Ca 2 +/calmodulin-dependent PDE (PDE1), cGMP-stimulated PDE (PDE2), cGMP-inhibited PDE (PDE3), cAMP-specific PDE (PDE4) and PKG-dependent PDE (PDE5) [13]. It has been reported that cGMP can play an intracellular role by regulating cGMP-stimulated PDE2 or cGMP-inhibited PDE3 [14], that is, cGMP can decrease the levels of cAMP and cGMP by activating PDE2 or increase both of them by inhibiting PDE3. So we can speculate that CNP-NPR-B/pGC-cGMP signal pathway may be modulated by PDEs in gastrointestinal smooth muscle. In the present study, we investigated the mechanism of CNP-induced inhibitory effect on spontaneous contractions of gastric antral smooth muscles to clarify
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CNP-NPR-B/pGC-cGMP downstream signal transduction pathway in rat. 2. Materials and methods 2.1. Tissue preparation Adult rats of either sex were killed by a sharp blow to the neck and exsanguination. The stomach was removed quickly and placed in aerated (95% O2 and 5% CO2) Krebs solution containing (mmol L −1): NaCl 118, KCl 4.75, NaHCO3 15.5, KH2PO4 1.19, MgSO4 1.19, D-glucose 10, and CaCl2 2.54 (pH was adjusted to 7.2–7.4 with HCl). The stomach was opened along the lesser curvature and the mucosa was removed. Muscle strips (2× 12 mm) of antrum 5 mm away from the pylorus were cut along the circular axis and placed in organ bath containing oxygenated Krebs solution maintained at 37 °C. One end of the strip was mounted on a platinum hook, and the other was fixed on an isometric force transducer. A tension of 1 g was applied to the tissues and they were equilibrated for 40 min before the experiments when rhythmic spontaneous contractions were recorded using RM6240 biological signal processing system (Sichuan, China). All experimental protocols performed were approved by the local Animal Care Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the Science and Technology Commission of P.R.C. (STCC Publication No. 2, revised 1988). 2.2. Isometric tension recording After equilibration, 10 −7 mol/L CNP was added to the bath solution, the effect of which was recorded for 5 min. The amplitude and frequency were measured for 5 min before and after the addition of the agents. After washout, the strips were pretreated with IBMX (3-isobutyl-1methylxanthine, a non-selective PDE inhibitor, 3 × 10 −6 mol/L), EHNA (an inhibitor of PDE2, 10−4 mol/L) and milrinone (an inhibitor of PDE3, 10−7 mol/L), respectively, and the effects of CNP were recorded in the presence of IBMX, EHNA and milrinone, respectively. The bath solution was collected and frozen by liquid nitrogen after addition of the agents, and then kept at − 80 °C to determine the content of cAMP and cGMP. The amplitude of the contraction was assessed in the experiment. The average amplitudes in 5 min before and after addition of the agents were considered as the control and the effect size, respectively. The change percentage of amplitude was expressed by (control − effect size) /control%. 2.3. cGMP and cAMP content The bath solution of 300 μL was dealt with 300 μL triethylamine (TCA), the final concentration of which was 6%, and then incubated for 15 min at room temperature. The samples were washed three times with water-saturated ethyl ether and lyophilized with lyophilizer (Savant, Farmingdale, NY). cGMP and cAMP in the samples were determined using cGMP or cAMP Direct Immunoassay Kit (BioVision Research Products, USA) after reconstituted in an assay buffer. The standards and samples were acetylated first according to the manufacturer's instructions to increase assay sensitivity for the analysis. 2.4. Drugs The drugs used in this experiment including C-type natriuretic peptide (CNP), 3-isobutyl-1-methylxanthine (IBMX), erythro-9-(2hydroxy-3-nonyl)adenine (EHNA) and milrinone were all purchased from Sigma.
2.5. Statistical analysis Data was analyzed using Origin 6.0 software and expressed as means ± SEM. Data recordings were evaluated using a Student's t test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Effect of CNP on the spontaneous contractions and generations of cGMP and cAMP in the gastric smooth muscles After equilibration, the gastric smooth muscles displayed spontaneous rhythmic contractions and CNP significantly inhibited the contractions (Fig. 1). The amplitude was reduced by 45.15± 0.71% after addition of CNP (10−7 mol/L) (Fig. 1B). The contents of cGMP and cAMP in perfusion solution of gastric smooth muscles were also changed significantly after addition of CNP. The content of cGMP was increased from 0.71±0.09 pmol/μL of control to 1.37± 0.05 pmol/μL by CNP and the percentage of increase was about 93.78% (n=8, P b 0.01, Fig. 2A). While the content of cAMP was increased from 0.15 ± 0.02 pmol/μL of control to 0.28± 0.04 pmol/μL after addition of CNP and the percentage of increase was about 47.86% (n= 8, P b 0.01, Fig. 2B). 3.2. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of IBMX The intracellular cGMP and cAMP are determined not only by their generation but also by their degradation by PDE. To determine the effect of PDE on CNP-induced inhibition of the contraction, a non-selective PDE inhibitor IBMX was employed. After equilibration, the spontaneous contractions of gastric smooth muscle were slightly inhibited by IBMX (3× 10−6 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of IBMX (Fig. 3A). The inhibition percentage of CNP on the amplitude was 49.97 ± 3.78% in the absence of IBMX and 25.73 ±2.46% in the presence of IBMX (Fig. 3B), respectively (P b 0.01, n = 8). These results suggest that IBMX inhibited the spontaneous contraction of the gastric smooth muscle and partially inhibited the effect of CNP on the contraction. The contents of cGMP and cAMP were increased slightly by IBMX, which were 0.78 ±0.04 pmol/μL (increase percentage was 9.61%, P b 0.05, n = 8) and 0.15 ± 0.01 pmol/μL (increase percentage was 5.48%, P b 0.05, n = 8) after addition of IBMX, respectively. However, in the presence of IBMX, the content of cGMP was 1.17 ± 0.04 pmol/μL and the increase percentage was 50.77% by CNP (n= 8, P b 0.01 vs IBMX, Fig. 4A). But the content of cAMP was not altered by CNP in the presence of IBMX, which was 0.16 ±0.01 pmol/μL after addition of CNP (n= 8, P > 0.05 vs IBMX, Fig. 4B). All these results indicate that PDE is involved in CNP-induced inhibitory effect on gastric motility. 3.3. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of EHNA CNP inhibits the gastric motility by increasing intracellular cGMP, and the increased cGMP can activate PDE2. To observe the role of PDE2 in inhibitory effect of CNP on gastric motility, a selective PDE2 inhibitor EHNA was employed. The spontaneous contractions of gastric smooth muscle were slightly inhibited by EHNA (10 −4 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of EHNA (Fig. 5A). The inhibition percentage of CNP on the amplitude was 47.90± 3.74% in the absence of EHNA and 51.63 ± 4.65% in the presence of EHNA, respectively (P > 0.05, n = 8, Fig. 5B). These results suggest that EHNA cannot block CNP-induced inhibition of the spontaneous contraction in the gastric antral smooth muscle although this agent can inhibit the contraction alone. The contents of cGMP and cAMP were increased slightly by EHNA, which were 0.97 ± 0.05 pmol/μL (increase percentage was 36.86%, P b 0.05, n = 8) and
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Fig. 1. Effect of CNP on the spontaneous contraction of the smooth muscle in gastric antrum. A. CNP inhibited the spontaneous contraction of smooth muscle in the gastric antrum. B. The panel shows a summary of the effect of CNP (*P b 0.05, n = 8).
0.19± 0.01 pmol/μL (increase percentage was 26.71%, P b 0.05, n = 8) after addition of EHNA, respectively. CNP increased the content of cGMP and cAMP further in the presence of EHNA, which were 1.24 ± 0.05 pmol/μL (increase percentage was 28.30%, P b 0.05 vs EHNA, n = 8, Fig. 6A) and 0.28± 0.02 pmol/μL (increase percentage was 50.81%, P b 0.01 vs EHNA, n = 8, Fig. 6B) after addition of CNP, respectively. All these results indicate that PDE2 is not involved in the inhibitory effect of CNP on gastric motility. 3.4. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of milrinone PDE3 can be inhibited by intracellular cGMP. To determine the role of PDE3 in inhibitory effect of CNP on gastric motility, a selective PDE3 inhibitor milrinone was employed. The spontaneous contractions of gastric smooth muscle were slightly inhibited by milrinone (10 −7 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of milrinone (Fig. 7). The inhibition percentage of CNP on the amplitude was 40.92 ±2.11% in the absence of milrinone and 19.87 ± 1.72% in the presence of milrinone, respectively
(P b 0.01, n = 8, Fig. 7B). These results suggest that milrinone can partially inhibit CNP-induced inhibition of the spontaneous contraction in the gastric antral smooth muscle. The contents of cGMP and cAMP were increased significantly by milrinone, which were 0.91 ± 0.03 pmol/μL (increase percentage was 28.80%, P b 0.05, n = 8) and 0.27± 0.03 pmol/μL (increase percentage was 80.0%, P b 0.05, n = 8) after addition of milrinone, respectively. CNP increased the content of cGMP further in the presence of milrinone, which was 1.18 ± 0.03 pmol/μL after addition of CNP, and the increase percentage was 28.90% (n= 8, P b 0.01 vs milrinone, Fig. 8A). But the content of cAMP was not altered significantly by CNP in the presence of milrinone, which was 0.17 ±0.01 pmol/μL after addition of CNP (n= 8, P > 0.05 vs milrinone, Fig. 8B). All these results indicate that PDE3 is involved in CNP-induced inhibitory regulation of gastric motility by blocking the generation of cAMP by CNP. 4. Discussion Like as other tissue in gastrointestinal smooth muscle PDE can modulate the levels of cAMP and cGMP by catalyzing their hydrolysis,
Fig. 2. Effects of CNP on the contents of cGMP and cAMP in the bath solution. A. The content of cGMP was increased significantly after addition of CNP (*P b 0.01, n = 8). B. The content of cAMP was increased significantly after addition of CNP (*P b 0.05, n = 8).
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Fig. 3. Effects of CNP on the spontaneous contraction in the presence of IBMX. A. CNP-induced inhibition of the spontaneous contraction was inhibited significantly in the presence of IBMX. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of IBMX (*P b 0.01, n = 8).
while cAMP and cGMP also modulate the activity of PDE. In the myocardial cells, NO plays a role in the regulation of cAMP level by generating cGMP and subsequent cGMP–PDE3–cAMP and cGMP–PDE2–cAMP signal pathway [15]. However, CNP regulates the cardiac muscle contractility by generating cGMP and subsequent cGMP–PDE3–cAMP signal pathway to increase cAMP, but the cGMP–PDE2–cAMP signal pathway plays a minor role [16,17]. It has been reported that NP-NPR-B/ pGC-cGMP signal pathway exists in the gastrointestinal tract, which inhibits the spontaneous contractions of the gastric smooth muscles in the guinea pig, rat and human [12,13,18]. However, the role of PDE in the CNP-NPR-B/pGC-cGMP downstream signal pathway is not clear in gastrointestinal smooth muscle.
It is well known that CNP plays an inhibitory role in regulation of gastrointestinal smooth muscle motility via CNP-NPR-B/pGc-cGMP signal pathway [7–10]. In the present study, CNP induced inhibitory effect of the spontaneous contraction of gastric antral smooth muscle together with the increase of intracellular cAMP and cGMP, in which different inhibitors of PDE played different roles. A non-selective PDE inhibitor IBMX significantly inhibited the spontaneous contraction of the gastric antral smooth muscle and increased the levels of cAMP and cGMP. We also found that IBMX significantly suppressed the inhibitory effect of CNP on the spontaneous contraction of gastric antral smooth muscle and blocked CNP-induced generation of cAMP but not cGMP. These results suggest that PDE is involved in the inhibitory
Fig. 4. Effects of CNP on the contents of cGMP and cAMP in the presence of IBMX. A. CNP significantly increased the content of cGMP in the presence of IBMX (*P b 0.01, n= 8). B. CNP had no significant effect on the content of cAMP in the presence of IBMX (P >0.05, n = 8).
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Fig. 5. Effects of CNP on the spontaneous contraction in the presence of EHNA. A. CNP-induced inhibition of the spontaneous contraction was not affected by EHNA. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of EHNA (P > 0.05, n = 8).
effect of CNP on the spontaneous contraction of gastric antral smooth muscle. Previous studies have shown that the increase of cAMP in the gastric smooth muscle can activate PKA to inhibit L-type calcium channel and subsequent decrease of intracellular calcium, which leads to inhibition of the spontaneous contraction in the gastric antrum [19]. In the present study, we consider that the suppressive effect of IBMX on the inhibitory effect of CNP on the spontaneous contraction may result from IBMX-induced blockade of intracellular cAMP generation by CNP. The results suggest that PDE is involved in CNP-induced inhibition of the gastric motility via regulating intracellular cGMP and cAMP level. In the present study we investigated which kind of PDE subtypes play a key role in CNP-induced inhibition of the gastric motility. In the present study, we also observed the effects of EHNA, a cGMP-stimulated PDE2 inhibitor, and milrinone, a cGMP-inhibited PDE3 inhibitor, on the inhibitory effect of CNP on spontaneous contraction of the gastric antral smooth muscle. We found that EHNA
did not affect the inhibitory effect of CNP on spontaneous contraction and productions of the cGMP and cAMP by CNP in gastric antral smooth muscle. The results suggest that PDE2 or cGMP–PDE2–cAMP signal pathway may be not involved in the inhibitory effect of CNP on spontaneous contraction of gastric antral smooth muscle in rat. However, milrinone similar to IBMX significantly suppressed the inhibitory effect of CNP on the spontaneous contraction of gastric antral smooth muscle and blocked CNP-induced generation of cAMP but not cGMP. In another hand, milrinone more significantly enhanced production of cAMP than that of cGMP. These results suggest that PDE3 is involved in CNP-induced inhibition of the spontaneous contraction. That is, intracellular cGMP generated by CNP-NPR-B/pGc-cGMP signal pathway inhibits the activity of PDE3 which is more potentiated for cAMP degradation, so leads to the inhibition of cAMP degradation and subsequent increase of cAMP to inhibit the spontaneous contraction. Our study is supported by previous investigation. Karnam et al. reported that there is a cross talk between cGMP and cAMP in the gastric smooth
Fig. 6. Effects of CNP on the contents of cGMP and cAMP in the presence of EHNA. A. CNP significantly increased the content of cGMP in the presence of EHNA (*P b 0.05, n = 8). B. CNP significantly increased the content of cAMP in the presence of EHNA (*P b 0.05, n = 8).
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Fig. 7. Effects of CNP on the spontaneous contraction in the presence of milrinone. A. CNP-induced inhibition of the spontaneous contraction was inhibited significantly in the presence of milrinone. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of milrinone (*P b 0.01, n = 8).
muscle. cGMP can increase cAMP by inhibiting PDE3, while cAMP can decrease cGMP by activating PKA-dependent PDE5 [20]. However, the mechanism of cAMP or cGMP-induced smooth muscle relaxation is not a simple pathway, for example, at least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration, (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the calcium sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation [21]. In conclusion, CNP-NPR-B/pGc-cGMP signal pathway in gastrointestinal smooth muscle is regulated by PDE and PDE3 plays a key
role in this process. Intracellular cGMP generated by CNP stimulates the generation of cAMP through cGMP–PDE3–cAMP signal pathway and cAMP activates PKA to inhibit L-type calcium channel and subsequent decrease of intracellular calcium, which results in the inhibition of spontaneous contraction in gastric antral smooth muscle. In addition cyclic nucleotides mediate direct and/or indirect regulation of gastrointestinal smooth muscle motility as above-mentioned.
Acknowledgment This study is supported by the National Natural Science Foundation of China (No. 81060036).
Fig. 8. Effects of CNP on the spontaneous contraction and the contents of cGMP and cAMP in the presence of milrinone. A. CNP significantly increased the content of cGMP in the presence of milrinone (*P b 0.05, n = 8). B. CNP had no significant effect on the content of cAMP in the presence of milrinone (P > 0.05, n= 8).
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Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
cGMP–PDE3–cAMP signal pathway involved in the inhibitory effect of CNP on gastric motility in rat Ying-Lan Cai a, Qian Sun a, Xu Huang b, 1, Jing-Zhi Jiang a, Mo-Han Zhang a, Li-Hua Piao a, Zheng Jin a,⁎, Wen-Xie Xu b,⁎⁎ a b
Department of Physiology, Yanbian University School of Medicine, 977 Gongyuan, Road, Jilin, Yanji 133002, China Department of Physiology, Shanghai Jiaotong University School of Medicine, 800 Dongchuan Road, 328 Wenxuan Medical Building, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 26 October 2012 Accepted 13 November 2012 Available online 24 November 2012 Keywords: Gastric smooth muscle CNP PDE2 PDE3 cGMP–PDE3–cAMP
a b s t r a c t In the present study, we investigated the mechanism of C-type natriuretic peptide (CNP)-induced inhibitory effect on spontaneous contraction of gastric antral smooth muscle to clarify CNP-NPR-B/pGC-cGMP downstream signal transduction pathway using organ bath and ELISA methods in rat. CNP significantly reduced the amplitude of the spontaneous contraction and increased the contents of cGMP and cAMP in the gastric antral smooth muscle tissue. In the presence of IBMX, a non-selective phosphodiesterase (PDE) inhibitor, the inhibitory effect of CNP on spontaneous contraction was significantly suppressed; however, the production of cGMP but not cAMP was still increased by CNP. EHNA, a PDE2 inhibitor, did not affect both CNP-induced inhibition of the contraction and CNP-induced increase of cGMP and cAMP generations in gastric smooth muscle tissue, while milrinone, a PDE3 inhibitor, similar to IBMX, attenuated the CNP-induced inhibitory effect on spontaneous contraction and increased the content of cGMP but not cAMP. The results suggest that cGMP–PDE3–cAMP signal pathway is also involved in the CNP-induced inhibition of gastric motility in rat. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cyclic nucleotide monophosphates cyclic AMP (cAMP) and cyclic GMP (cGMP) are important endogenous mediators of several processes, including smooth muscle motility [1,2]. Cyclic nucleotides are synthesized from the corresponding nucleotide triphosphates by the activity of adenylyl and guanylyl cyclases. The increase in cAMP or cGMP triggers a signal transduction cascade encompassing the activation of cyclic nucleotide-dependent protein kinases: cyclic adenosine-dependent protein kinase (cAK) or cyclic guanosine-dependent protein kinase (cGK), subsequent phosphorylation of the actinmyosin system, as well as Ca 2+ channels and ATP-driven Ca2+ pumps located in the outer cell membrane or the membrane of the sarcoplasmic reticulum [3,4]. This cascade leads to a reduction in cytosolic Ca 2+ and, finally, to smooth muscle relaxation. In the gastrointestinal tract, natriuretic peptides are expressed in specific regions of the stomach and intestine where there is evidence that they are involved in paracrine regulation of gastrointestinal motility [5–8]. Our previous studies have shown that CNP peptides relax
⁎ Corresponding author. Tel.: +86 433 2435122; fax: +86 433 2435104. ⁎⁎ Corresponding author. Tel.: +86 21 34205639; fax: +86 21 34204118. E-mail addresses: [email protected] (Z. Jin), [email protected] (W.-X. Xu). 1 Co-first author. 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.11.001
isolated gastric smooth muscle in different species, for example, guinea-pig, rat and humans, via CNP/natriuretic peptide receptor B (NPR-B)/cGMP pathway [8–11]; however, CNP-NPR-B/pGC-cGMP downstream signal transduction pathway is not clear in gastrointestinal smooth muscle. Cyclic nucleotides are degraded by phosphodiesterase (PDE) isoenzymes, a heterogeneous group of hydrolytic enzymes. PDE are classified according to their preferences for cAMP or/and cGMP, kinetic parameters of cyclic nucleotide hydrolysis, sensitivity to the inhibition by various compounds, allosteric regulation by other molecules and chromatographic behavior on anion exchange columns [3,4]. At least 11 PDE families and 18 subtypes have been identified in the mammal tissues by far, which play different roles [12]. And at least five PDE families have been found in the stomach, including Ca 2 +/calmodulin-dependent PDE (PDE1), cGMP-stimulated PDE (PDE2), cGMP-inhibited PDE (PDE3), cAMP-specific PDE (PDE4) and PKG-dependent PDE (PDE5) [13]. It has been reported that cGMP can play an intracellular role by regulating cGMP-stimulated PDE2 or cGMP-inhibited PDE3 [14], that is, cGMP can decrease the levels of cAMP and cGMP by activating PDE2 or increase both of them by inhibiting PDE3. So we can speculate that CNP-NPR-B/pGC-cGMP signal pathway may be modulated by PDEs in gastrointestinal smooth muscle. In the present study, we investigated the mechanism of CNP-induced inhibitory effect on spontaneous contractions of gastric antral smooth muscles to clarify
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CNP-NPR-B/pGC-cGMP downstream signal transduction pathway in rat. 2. Materials and methods 2.1. Tissue preparation Adult rats of either sex were killed by a sharp blow to the neck and exsanguination. The stomach was removed quickly and placed in aerated (95% O2 and 5% CO2) Krebs solution containing (mmol L −1): NaCl 118, KCl 4.75, NaHCO3 15.5, KH2PO4 1.19, MgSO4 1.19, D-glucose 10, and CaCl2 2.54 (pH was adjusted to 7.2–7.4 with HCl). The stomach was opened along the lesser curvature and the mucosa was removed. Muscle strips (2× 12 mm) of antrum 5 mm away from the pylorus were cut along the circular axis and placed in organ bath containing oxygenated Krebs solution maintained at 37 °C. One end of the strip was mounted on a platinum hook, and the other was fixed on an isometric force transducer. A tension of 1 g was applied to the tissues and they were equilibrated for 40 min before the experiments when rhythmic spontaneous contractions were recorded using RM6240 biological signal processing system (Sichuan, China). All experimental protocols performed were approved by the local Animal Care Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the Science and Technology Commission of P.R.C. (STCC Publication No. 2, revised 1988). 2.2. Isometric tension recording After equilibration, 10 −7 mol/L CNP was added to the bath solution, the effect of which was recorded for 5 min. The amplitude and frequency were measured for 5 min before and after the addition of the agents. After washout, the strips were pretreated with IBMX (3-isobutyl-1methylxanthine, a non-selective PDE inhibitor, 3 × 10 −6 mol/L), EHNA (an inhibitor of PDE2, 10−4 mol/L) and milrinone (an inhibitor of PDE3, 10−7 mol/L), respectively, and the effects of CNP were recorded in the presence of IBMX, EHNA and milrinone, respectively. The bath solution was collected and frozen by liquid nitrogen after addition of the agents, and then kept at − 80 °C to determine the content of cAMP and cGMP. The amplitude of the contraction was assessed in the experiment. The average amplitudes in 5 min before and after addition of the agents were considered as the control and the effect size, respectively. The change percentage of amplitude was expressed by (control − effect size) /control%. 2.3. cGMP and cAMP content The bath solution of 300 μL was dealt with 300 μL triethylamine (TCA), the final concentration of which was 6%, and then incubated for 15 min at room temperature. The samples were washed three times with water-saturated ethyl ether and lyophilized with lyophilizer (Savant, Farmingdale, NY). cGMP and cAMP in the samples were determined using cGMP or cAMP Direct Immunoassay Kit (BioVision Research Products, USA) after reconstituted in an assay buffer. The standards and samples were acetylated first according to the manufacturer's instructions to increase assay sensitivity for the analysis. 2.4. Drugs The drugs used in this experiment including C-type natriuretic peptide (CNP), 3-isobutyl-1-methylxanthine (IBMX), erythro-9-(2hydroxy-3-nonyl)adenine (EHNA) and milrinone were all purchased from Sigma.
2.5. Statistical analysis Data was analyzed using Origin 6.0 software and expressed as means ± SEM. Data recordings were evaluated using a Student's t test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Effect of CNP on the spontaneous contractions and generations of cGMP and cAMP in the gastric smooth muscles After equilibration, the gastric smooth muscles displayed spontaneous rhythmic contractions and CNP significantly inhibited the contractions (Fig. 1). The amplitude was reduced by 45.15± 0.71% after addition of CNP (10−7 mol/L) (Fig. 1B). The contents of cGMP and cAMP in perfusion solution of gastric smooth muscles were also changed significantly after addition of CNP. The content of cGMP was increased from 0.71±0.09 pmol/μL of control to 1.37± 0.05 pmol/μL by CNP and the percentage of increase was about 93.78% (n=8, P b 0.01, Fig. 2A). While the content of cAMP was increased from 0.15 ± 0.02 pmol/μL of control to 0.28± 0.04 pmol/μL after addition of CNP and the percentage of increase was about 47.86% (n= 8, P b 0.01, Fig. 2B). 3.2. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of IBMX The intracellular cGMP and cAMP are determined not only by their generation but also by their degradation by PDE. To determine the effect of PDE on CNP-induced inhibition of the contraction, a non-selective PDE inhibitor IBMX was employed. After equilibration, the spontaneous contractions of gastric smooth muscle were slightly inhibited by IBMX (3× 10−6 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of IBMX (Fig. 3A). The inhibition percentage of CNP on the amplitude was 49.97 ± 3.78% in the absence of IBMX and 25.73 ±2.46% in the presence of IBMX (Fig. 3B), respectively (P b 0.01, n = 8). These results suggest that IBMX inhibited the spontaneous contraction of the gastric smooth muscle and partially inhibited the effect of CNP on the contraction. The contents of cGMP and cAMP were increased slightly by IBMX, which were 0.78 ±0.04 pmol/μL (increase percentage was 9.61%, P b 0.05, n = 8) and 0.15 ± 0.01 pmol/μL (increase percentage was 5.48%, P b 0.05, n = 8) after addition of IBMX, respectively. However, in the presence of IBMX, the content of cGMP was 1.17 ± 0.04 pmol/μL and the increase percentage was 50.77% by CNP (n= 8, P b 0.01 vs IBMX, Fig. 4A). But the content of cAMP was not altered by CNP in the presence of IBMX, which was 0.16 ±0.01 pmol/μL after addition of CNP (n= 8, P > 0.05 vs IBMX, Fig. 4B). All these results indicate that PDE is involved in CNP-induced inhibitory effect on gastric motility. 3.3. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of EHNA CNP inhibits the gastric motility by increasing intracellular cGMP, and the increased cGMP can activate PDE2. To observe the role of PDE2 in inhibitory effect of CNP on gastric motility, a selective PDE2 inhibitor EHNA was employed. The spontaneous contractions of gastric smooth muscle were slightly inhibited by EHNA (10 −4 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of EHNA (Fig. 5A). The inhibition percentage of CNP on the amplitude was 47.90± 3.74% in the absence of EHNA and 51.63 ± 4.65% in the presence of EHNA, respectively (P > 0.05, n = 8, Fig. 5B). These results suggest that EHNA cannot block CNP-induced inhibition of the spontaneous contraction in the gastric antral smooth muscle although this agent can inhibit the contraction alone. The contents of cGMP and cAMP were increased slightly by EHNA, which were 0.97 ± 0.05 pmol/μL (increase percentage was 36.86%, P b 0.05, n = 8) and
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Fig. 1. Effect of CNP on the spontaneous contraction of the smooth muscle in gastric antrum. A. CNP inhibited the spontaneous contraction of smooth muscle in the gastric antrum. B. The panel shows a summary of the effect of CNP (*P b 0.05, n = 8).
0.19± 0.01 pmol/μL (increase percentage was 26.71%, P b 0.05, n = 8) after addition of EHNA, respectively. CNP increased the content of cGMP and cAMP further in the presence of EHNA, which were 1.24 ± 0.05 pmol/μL (increase percentage was 28.30%, P b 0.05 vs EHNA, n = 8, Fig. 6A) and 0.28± 0.02 pmol/μL (increase percentage was 50.81%, P b 0.01 vs EHNA, n = 8, Fig. 6B) after addition of CNP, respectively. All these results indicate that PDE2 is not involved in the inhibitory effect of CNP on gastric motility. 3.4. Effect of CNP on the spontaneous contractions and the generations of cGMP and cAMP in the presence of milrinone PDE3 can be inhibited by intracellular cGMP. To determine the role of PDE3 in inhibitory effect of CNP on gastric motility, a selective PDE3 inhibitor milrinone was employed. The spontaneous contractions of gastric smooth muscle were slightly inhibited by milrinone (10 −7 mol/L), and the inhibitory effect of CNP on the contractions still existed in the presence of milrinone (Fig. 7). The inhibition percentage of CNP on the amplitude was 40.92 ±2.11% in the absence of milrinone and 19.87 ± 1.72% in the presence of milrinone, respectively
(P b 0.01, n = 8, Fig. 7B). These results suggest that milrinone can partially inhibit CNP-induced inhibition of the spontaneous contraction in the gastric antral smooth muscle. The contents of cGMP and cAMP were increased significantly by milrinone, which were 0.91 ± 0.03 pmol/μL (increase percentage was 28.80%, P b 0.05, n = 8) and 0.27± 0.03 pmol/μL (increase percentage was 80.0%, P b 0.05, n = 8) after addition of milrinone, respectively. CNP increased the content of cGMP further in the presence of milrinone, which was 1.18 ± 0.03 pmol/μL after addition of CNP, and the increase percentage was 28.90% (n= 8, P b 0.01 vs milrinone, Fig. 8A). But the content of cAMP was not altered significantly by CNP in the presence of milrinone, which was 0.17 ±0.01 pmol/μL after addition of CNP (n= 8, P > 0.05 vs milrinone, Fig. 8B). All these results indicate that PDE3 is involved in CNP-induced inhibitory regulation of gastric motility by blocking the generation of cAMP by CNP. 4. Discussion Like as other tissue in gastrointestinal smooth muscle PDE can modulate the levels of cAMP and cGMP by catalyzing their hydrolysis,
Fig. 2. Effects of CNP on the contents of cGMP and cAMP in the bath solution. A. The content of cGMP was increased significantly after addition of CNP (*P b 0.01, n = 8). B. The content of cAMP was increased significantly after addition of CNP (*P b 0.05, n = 8).
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Fig. 3. Effects of CNP on the spontaneous contraction in the presence of IBMX. A. CNP-induced inhibition of the spontaneous contraction was inhibited significantly in the presence of IBMX. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of IBMX (*P b 0.01, n = 8).
while cAMP and cGMP also modulate the activity of PDE. In the myocardial cells, NO plays a role in the regulation of cAMP level by generating cGMP and subsequent cGMP–PDE3–cAMP and cGMP–PDE2–cAMP signal pathway [15]. However, CNP regulates the cardiac muscle contractility by generating cGMP and subsequent cGMP–PDE3–cAMP signal pathway to increase cAMP, but the cGMP–PDE2–cAMP signal pathway plays a minor role [16,17]. It has been reported that NP-NPR-B/ pGC-cGMP signal pathway exists in the gastrointestinal tract, which inhibits the spontaneous contractions of the gastric smooth muscles in the guinea pig, rat and human [12,13,18]. However, the role of PDE in the CNP-NPR-B/pGC-cGMP downstream signal pathway is not clear in gastrointestinal smooth muscle.
It is well known that CNP plays an inhibitory role in regulation of gastrointestinal smooth muscle motility via CNP-NPR-B/pGc-cGMP signal pathway [7–10]. In the present study, CNP induced inhibitory effect of the spontaneous contraction of gastric antral smooth muscle together with the increase of intracellular cAMP and cGMP, in which different inhibitors of PDE played different roles. A non-selective PDE inhibitor IBMX significantly inhibited the spontaneous contraction of the gastric antral smooth muscle and increased the levels of cAMP and cGMP. We also found that IBMX significantly suppressed the inhibitory effect of CNP on the spontaneous contraction of gastric antral smooth muscle and blocked CNP-induced generation of cAMP but not cGMP. These results suggest that PDE is involved in the inhibitory
Fig. 4. Effects of CNP on the contents of cGMP and cAMP in the presence of IBMX. A. CNP significantly increased the content of cGMP in the presence of IBMX (*P b 0.01, n= 8). B. CNP had no significant effect on the content of cAMP in the presence of IBMX (P >0.05, n = 8).
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Fig. 5. Effects of CNP on the spontaneous contraction in the presence of EHNA. A. CNP-induced inhibition of the spontaneous contraction was not affected by EHNA. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of EHNA (P > 0.05, n = 8).
effect of CNP on the spontaneous contraction of gastric antral smooth muscle. Previous studies have shown that the increase of cAMP in the gastric smooth muscle can activate PKA to inhibit L-type calcium channel and subsequent decrease of intracellular calcium, which leads to inhibition of the spontaneous contraction in the gastric antrum [19]. In the present study, we consider that the suppressive effect of IBMX on the inhibitory effect of CNP on the spontaneous contraction may result from IBMX-induced blockade of intracellular cAMP generation by CNP. The results suggest that PDE is involved in CNP-induced inhibition of the gastric motility via regulating intracellular cGMP and cAMP level. In the present study we investigated which kind of PDE subtypes play a key role in CNP-induced inhibition of the gastric motility. In the present study, we also observed the effects of EHNA, a cGMP-stimulated PDE2 inhibitor, and milrinone, a cGMP-inhibited PDE3 inhibitor, on the inhibitory effect of CNP on spontaneous contraction of the gastric antral smooth muscle. We found that EHNA
did not affect the inhibitory effect of CNP on spontaneous contraction and productions of the cGMP and cAMP by CNP in gastric antral smooth muscle. The results suggest that PDE2 or cGMP–PDE2–cAMP signal pathway may be not involved in the inhibitory effect of CNP on spontaneous contraction of gastric antral smooth muscle in rat. However, milrinone similar to IBMX significantly suppressed the inhibitory effect of CNP on the spontaneous contraction of gastric antral smooth muscle and blocked CNP-induced generation of cAMP but not cGMP. In another hand, milrinone more significantly enhanced production of cAMP than that of cGMP. These results suggest that PDE3 is involved in CNP-induced inhibition of the spontaneous contraction. That is, intracellular cGMP generated by CNP-NPR-B/pGc-cGMP signal pathway inhibits the activity of PDE3 which is more potentiated for cAMP degradation, so leads to the inhibition of cAMP degradation and subsequent increase of cAMP to inhibit the spontaneous contraction. Our study is supported by previous investigation. Karnam et al. reported that there is a cross talk between cGMP and cAMP in the gastric smooth
Fig. 6. Effects of CNP on the contents of cGMP and cAMP in the presence of EHNA. A. CNP significantly increased the content of cGMP in the presence of EHNA (*P b 0.05, n = 8). B. CNP significantly increased the content of cAMP in the presence of EHNA (*P b 0.05, n = 8).
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Fig. 7. Effects of CNP on the spontaneous contraction in the presence of milrinone. A. CNP-induced inhibition of the spontaneous contraction was inhibited significantly in the presence of milrinone. B. The panel shows a summary of the effect of CNP on the spontaneous contraction in the presence of milrinone (*P b 0.01, n = 8).
muscle. cGMP can increase cAMP by inhibiting PDE3, while cAMP can decrease cGMP by activating PKA-dependent PDE5 [20]. However, the mechanism of cAMP or cGMP-induced smooth muscle relaxation is not a simple pathway, for example, at least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration, (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the calcium sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation [21]. In conclusion, CNP-NPR-B/pGc-cGMP signal pathway in gastrointestinal smooth muscle is regulated by PDE and PDE3 plays a key
role in this process. Intracellular cGMP generated by CNP stimulates the generation of cAMP through cGMP–PDE3–cAMP signal pathway and cAMP activates PKA to inhibit L-type calcium channel and subsequent decrease of intracellular calcium, which results in the inhibition of spontaneous contraction in gastric antral smooth muscle. In addition cyclic nucleotides mediate direct and/or indirect regulation of gastrointestinal smooth muscle motility as above-mentioned.
Acknowledgment This study is supported by the National Natural Science Foundation of China (No. 81060036).
Fig. 8. Effects of CNP on the spontaneous contraction and the contents of cGMP and cAMP in the presence of milrinone. A. CNP significantly increased the content of cGMP in the presence of milrinone (*P b 0.05, n = 8). B. CNP had no significant effect on the content of cAMP in the presence of milrinone (P > 0.05, n= 8).
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