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(Circulation Research. 2004;94:936.)
© 2004 American Heart Association, Inc.

Cellular Biology

High and Low Gain Switches for Regulation of cAMP Efflux Concentration

Distinct Roles for Particulate GC- and Soluble GC-cGMP-PDE3 Signaling in Rabbit Atria

Jin Fu Wen, Xun Cui, Jing Yu Jin, Soo Mi Kim, Sung Zoo Kim, Suhn Hee Kim, Ho Sub Lee, Kyung Woo Cho

From the Department of Physiology, Institute for Medical Sciences, Jeonbug National University Medical School (J.F.W., X.C., J.Y.J., S.M.K., S.Z.K., S.H.K., K.W.C), Jeonju; Department of Herbal Resources, Wonkwang University Professional Graduate School of Oriental Medicine (H.S.L.), Iksan, Korea.

Correspondence to Kyung Woo Cho, MD, PhD, Department of Physiology, Jeonbug National University, Medical School, 2-20, Keum-Am-Dong-San, Jeonju 561-180, Republic of Korea. E-mail kwcho{at}moak.chonbuk.ac.kr

	Abstract

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This study tests the hypothesis that particulate (p) guanylyl cyclase (GC) and soluble (s) GC are involved in the distinct roles for the regulation of cGMP-PDE-cAMP signaling and of mechanical and secretory functions in the heart. Experiments were performed in perfused beating rabbit atria. C-type natriuretic peptide (CNP) and SIN-1, an NO donor, or BAY 41-2272 (BAY), a direct activator for sGC, were used to activate pGC and sGC, respectively. CNP and SIN-1 increased cGMP and cAMP efflux in a concentration-dependent manner. Increase in cAMP was a function of cGMP. The changes in cAMP efflux concentration in terms of cGMP were much more prominent in the atria treated with CNP than in the atria treated with SIN-1. Increase in cAMP efflux concentration was blocked by milrinone but not changed by EHNA. BAY increased cGMP but not cAMP in a concentration-dependent manner. CNP and SIN-1 decreased atrial stroke volume and myocytic ANP release. The decreases in terms of cGMP efflux concentration were much more prominent in the atria treated with CNP than in the atria treated with SIN-1 or BAY. Milrinone accentuated GC agonist–induced decreases in atrial stroke volume and ANP release. In the presence of ODQ, SIN-1 or BAY induced effects were not observed. These data suggest that pGC and sGC activations have distinct roles via cGMP-PDE3-cAMP signaling in the cardiac atrium: high and low gain switches, respectively, for the regulation of cAMP levels and contractile and secretory functions.

Key Words: cGMP • cAMP • guanylyl cyclase • phosphodiesterase 3 • atrial natriuretic peptide

	Introduction

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Both particulate (p) guanylyl cyclase (GC)- and soluble (s) GC-cGMP systems are known to be involved in the regulation of cardiac contractile and secretory functions. cGMP-phosphodiesterase (PDE)-cAMP signaling is one of the mechanisms responsible for the function. Both systems are present in cardiac myocytes.^1,2 However, significance of the presence of the dual systems in the regulation of cardiac function has to be defined. This study tests the hypothesis that pGC and sGC are involved in the distinct roles for the regulation of cGMP-PDE-cAMP signaling and of mechanical and secretory functions in the heart.

Nitric oxide (NO) is an endogenous activator for sGC activity. The most important and main second messenger for NO is cGMP. The effects of NO on myocardial contractility and Ca²⁺ current are variable.³ In human atrial myocytes, NO donor (sodium nitroprusside, SNP) induced positive inotropic effect.⁴ In rabbit atrial cells, NO donor increased Ca²⁺ current.⁵ In contrast, in human atrial and ventricular myocardium, SNP induced negative inotropic effect via cGMP signaling.⁶ Also, it was shown that NO donors exhibited biphasic effects: an increase in contractile response of rat ventricular myocytes to a low increase in cGMP by NO donor and a decrease in contractile response to a high increase in cGMP.^7,8 Biphasic effect of NO was also observed with SIN-1 in the human atrial myocytes⁴: an increase in Ca²⁺ current with a low dose and a decrease with a high dose. High dose of NO donors usually induced negative inotropic effect^9,10 or decreased Ca²⁺ current.¹¹ Taken together, published data on the effects of NO-cGMP signaling in the heart are variable. Recently, direct and NO-independent activators for sGC, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazol (YC-1) and more potent and selective compound 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272),¹² have been introduced. Cardiac NO system has been shown to inhibit atrial natriuretic peptide (ANP) secretion.^13–15

C-type natriuretic peptide (CNP) is a specific activator for the pGC-coupled natriuretic peptide receptor-B (NPR-B) in the heart. CNP was shown to inhibit atrial dynamics and myocytic ANP release via pGC-cGMP signaling.¹⁶ Also, it was shown that CNP has a negative inotropic effect in rat papillary muscle¹⁷ or positive chronotropic and inotropic effects in the canine atria.^18,19

It was proposed in the cardiomyocytes that cGMP-PDE pathway regulates cAMP signaling, although the change in cAMP levels has not yet been clearly presented.^{4,8,10,20–25} cGMP-stimulated PDE2 and cGMP-inhibited PDE3 are involved in this signaling. Biphasic effect of cGMP on the cardiac Ca²⁺ current was shown to be related to the roles of PDE2 and PDE3.²⁵ It is possible that both sGC and pGC regulate the cGMP-PDE-cAMP signaling. However, why cardiomyocytes use two pathways to regulate cAMP levels is not known. sGC and pGC possibly be related to the functional compartmentalization of cGMP due to localized accumulation of the second messenger within the cardiomyocytes. Recently, it was shown that cAMP is compartmentalized in the regulation of atrial myocytic ANP release, in that PDE3 but not PDE4 is involved in the cAMP-induced decrease in ANP release.²⁶ The purpose of the present study was to define the comparative roles for the pGC-cGMP-PDE2/PDE3 and sGC-cGMP-PDE2/PDE3 signaling in the regulation of cAMP levels, atrial contractile, and secretory functions.

	Materials and Methods

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Beating Perfused Atrial Preparation
New Zealand White rabbits were used. All experiments were performed under approval of the Ethics Committee in the Institute for Medical Sciences of Jeonbug National University. An isolated perfused atrial preparation was prepared by the method described previously^27,28 allowing atrial pacing (at 1.3 Hz) and measurements of changes in atrial volume during contraction (stroke volume), pulse pressure, transmural extracellular fluid (ECF) translocation, cAMP efflux, cGMP efflux, and ANP secretion. The atrium was perfused with HEPES-buffered solution by means of a peristaltic pump (1 mL/min).

Experimental Protocols
The beating atria were perfused for 60 minutes to stabilize ANP secretion. [³H]Inulin was introduced to the pericardial fluid 20 minutes before the start of the sample collection.²⁷ The perfusate was collected at 2-minute intervals at 4°C for analyses. Experiments were performed using 21 groups of atria. Control period (four 12-minute periods) was followed by an infusion of CNP, an activator of pGC (30 nmol/L, group 1, n=8, Figures 1A, 3, and 7; 100 nmol/L, group 2, n=14, Figures 2, 3, and 7 ; 300 nmol/L, group 3, n=6, Figures 3 and 7), SIN-1, an NO-donor for sGC activation (30 µmol/L, group 4, n=6, Figures 3 and 7; 100 µmol/L, group 5, n=9, Figures 2, 3, 4, and 7 HREF="#FIG3">; 300 µmol/L, group 6, n=10, Figures 3, 5, 6, and 7 HREF="#FIG5">), BAY 41-2272 (BAY), a direct and selective activator for sGC (1 µmol/L, group 7, n=4, Figures 3, 4, and 7; 10 µmol/L, group 8, n=5, Figures 2, 3, 6, and 7), or vehicle (group 16, n=6, Figures 3, 4, and 6; vehicle ethanol for BAY control, group 17, n=3, Figures 3 and 6) for three cycles (36 minutes). BAY was a generous gift from J.-P. Stasch and Andreas M. Knorr (Bayer, Wuppertal, Germany). To analyze the effects of pGC and sGC activators, 36 minutes of milrinone, an inhibitor of PDE3, or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA),²⁵ a selective inhibitor of PDE2, was followed by an infusion of CNP, SIN-1 or vehicle [milrinone (1 µmol/L) plus CNP (30 nmol/L), group 9, n=6, Figures 1B and 3; milrinone plus CNP (100 nmol/L), group 10, n=7, Figure 3; milrinone plus SIN-1 (100 µmol/L), group 12, n=6, Figure 3; milrinone alone, group 11, n=6, Figure 3; EHNA (30 µmol/L) plus CNP (100 nmol/L), group 18, n=7, Figure 3; EHNA plus SIN-1 (100 µmol/L), group 19, n=7, Figure 3; EHNA plus SIN-1 (300 µmol/L), group 20, n=6, Figures 3 and 5; EHNA alone, group 21, n=3, Figure 3]. To analyze the effects of SIN-1 or BAY, 36 minutes of 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a specific inhibitor of sGC, was followed by an infusion of SIN-1, BAY, or vehicle [ODQ (10 µmol/L) plus SIN-1 (300 µmol/L), group 13, n=8, Figure 6; ODQ plus BAY (10 µmol/L), group 15, n=3, Figure 6; ODQ alone, group 14, n=6, Figure 6]. BAY was dissolved in ethanol and the final concentration of ethanol in the perfusate was less than 0.1% (v/v). Milrinone, EHNA, and ODQ were dissolved in DMSO and the final concentration of DMSO was less than 0.1%. The effects were evaluated after two cycles (24 minutes) of administration of the agent. For the time-matched control, vehicle was introduced and values obtained during the periods corresponding to the control and experimental observations were compared.

View larger version (31K): [in this window] [in a new window]   Figure 1. Effects of CNP and its modulation by milrinone on the levels of cGMP and cAMP and contractile and secretory functions in beating rabbit atria. A, CNP-induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), stroke volume (c), pulse pressure (d), and ANP concentration (e) (n=8). B, Modulation by milrinone of CNP-induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), atrial dynamics (c and d), and ANP concentration (e) (n=6). **P<0.01, ***P<0.001 vs the values before the addition of CNP; +P<0.05, ++P<0.01 vs control period.

View larger version (29K): [in this window] [in a new window]   Figure 3. Comparative analysis of the changes in cGMP efflux concentration, cAMP efflux concentration, atrial stroke volume, and ANP release induced by CNP, SIN-1, and BAY. CNP, 0.03 µmol/L (n=8), 0.1 µmol/L (n=14), 0.3 µmol/L (n=6); SIN-1, 30 µmol/L (n=6), 100 µmol (n=9), 300 µmol/L (n=10); BAY, 0 µmol/L (vehicle, n=3), 1 µmol/L (n=4), 10 µmol/L (n=5); milrinone (Mil, 1 µmol/L)+CNP (0.03 µmol/L) (n=6); Mil+CNP (0.1 µmol/L) (n=7); Mil+SIN-1 (100 µmol/L) (n=6); Mil alone (n=6); EHNA (30 µmol/L)+CNP (0.1 µmol/L) (n=7); EHNA+SIN-1 (100 µmol/L) (n=7); EHNA+SIN-1 (300 µmol/L) (n=6); EHNA alone (n=3); control (CONT) (n=6). Insets, Relations between atrial cGMP or cAMP content and cGMP or cAMP efflux, for cGMP, y=0.177x–0.609, R=0.938, P<0.001; for cAMP, y=0.0024x–0.031, R=0.826, P<0.001; cGMP or cAMP efflux in pmol/min per atrium; cGMP or cAMP content in the atrium, pmol/atrium. indicates control; , CNP; , SIN-1; , Mil+SIN-1; , EHNA+SIN-1. #P<0.05, *P<0.01 vs corresponding control; +P<0.05 vs the lower concentration. Open bar, control or activator alone; filled bar, milrinone or milrinone+ activator; and hatched bar, EHNA or EHNA+activator.

View larger version (11K): [in this window] [in a new window]   Figure 7. High and low gain effects of CNP and SIN-1 or BAY, respectively, on the cAMP levels, atrial contractile, and secretory functions in terms of cGMP efflux concentration. A, High and low gain effects of CNP and SIN-1 on cAMP levels. B, Comparative effects of CNP, SIN-1, and BAY on the stroke volume. C, Comparative effects of CNP, SIN-1, and BAY on the atrial myocytic ANP release. Gain of cAMP efflux concentration=%difference in cAMP efflux concentration over the control/%difference in cGMP efflux concentration over the control. indicates CNP (30 to 300 nmol/L); , SIN-1 (30 to 300 µmol/L); , BAY (1 to 10 µmol/L).

View larger version (11K): [in this window] [in a new window]   Figure 2. Time-dependent changes in the levels of cGMP and cAMP by CNP, SIN-1, and BAY. A, Differences in percent changes over the control in cGMP efflux concentration after the addition of activators. B, Differences in percent changes over the control in cAMP efflux concentration. CNP, 100 nmol/L, n=14; BAY, 10 µmol/L, n=5; SIN-1, 100 µmol/L, n=9.

View larger version (41K): [in this window] [in a new window]   Figure 4. Effects of SIN-1 and BAY on the levels of cGMP and cAMP and atrial contractile and secretory functions. A, SIN-1–induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), stroke volume (c), pulse pressure (d), and ANP concentration (e) (n=9). B, BAY-induced changes in the same parameters (n=4). C, Time-matched control in the same parameters (n=6).

View larger version (29K): [in this window] [in a new window]   Figure 5. Effects of SIN-1 and its modulation by EHNA on the levels of cGMP and cAMP and atrial contractile and secretory functions. A, SIN-1–induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), stroke volume (c), and ANP concentration (d) (n=10). B, Modulation by EHNA of SIN-1–induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), atrial dynamics (c), and ANP concentration (d) (n=6). **P<0.01, ***P<0.001 vs the values before the addition of SIN-1; ++P<0.01 vs control period.

View larger version (20K): [in this window] [in a new window]   Figure 6. Blockade by ODQ of SIN-1– and BAY-induced effects on the levels of cGMP and cAMP and atrial contractile and secretory functions. A, Effects of ODQ (10 µmol/L) on SIN-1–induced changes in cGMP efflux concentration (a), cAMP efflux concentration (b), stroke volume (c), and myocytic ANP release (d) (n=8). B, Effects of ODQ on BAY (10 µmol/L)-induced changes in the same parameters (n=3). *P<0.05, **P<0.01, ***P<0.001. NS indicates not significant.

Additional experimental details, detailed description of radioimmunoassay for ANP, cAMP, cGMP, and its expressions, and statistical analysis are in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.

	Results

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Effects of pGC Activation With CNP on the Levels of cGMP and cAMP, Atrial Dynamics, and Myocytic ANP Release
Atrial efflux of cGMP and cAMP, expressed as efflux concentration (µmol/L) of cGMP and cAMP in perfusate in terms of the ECF translocation, which reflects the concentration of cGMP and cAMP in the interstitial space fluid, were steady and stable during the control periods (Figures 1Aa, 1Ab, 4Ca, and 4Cb). Atrial dynamics, stroke volume and pulse pressure, and ANP secretion, the concentration of ANP in perfusate in terms of the ECF translocation, which reflects the rate of atrial myocytic ANP release, were also steady and stable (Figures 1Ac through 1Ae and 4Cc through 4 Ce). pGC activation with CNP (30 nmol/L) increased cGMP and cAMP efflux concentrations (Figures 1Aa and 1Ab). CNP decreased atrial dynamics and myocytic ANP release concomitantly with an increase in cGMP and cAMP levels (Figures 1Ac through 1Ae). CNP (30 nmol/L) increased cGMP efflux levels from 0.039±0.004 µmol/L (mean of two periods, fraction number 23 and 24) by 136.13±23.50% to 0.087±0.008 µmol/L (fraction number 29 and 30) at the first 12-minute cycle of an infusion of agent and then maintained the levels thereafter up to third cycle (36 minutes) without significant decrease (Figure 1Aa). With CNP, cAMP efflux concentration increased from 0.049±0.013 µmol/L by 14.27±6.37% to 0.055±0.014 µmol/L at first 12-minute cycle and then slightly further increased by 37.30±6.49% to 0.062±0.013 µmol/L (P<0.001) up to 36 minutes. A higher concentration of CNP (100 nmol/L) increased cGMP levels by 449.64±23.23% at the first 12-minute cycle and then declined to 314.98±28.53% at the third cycle of infusion (P<0.001) (Figure 2A). CNP increased cAMP levels by 57.44±6.35% at the first 12-minute cycle and then maintained the levels thereafter up to 36 minutes (Figure 2B). With CNP (300 nmol/L), the pattern of responses was similar to that of CNP (100 nmol/L) in terms of time. The effects of CNP on the levels of cGMP and cAMP, atrial dynamics and ANP release were concentration-dependent (Figure 3). cGMP or cAMP efflux increased in proportion to the atrial cGMP or cAMP content, respectively, which was not different between atria treated with CNP or SIN-1 (for cGMP, y=0.177x–0.609, R=0.938, P<0.001; for cAMP, y=0.0024x–0.031, R=0.826, P<0.001; Figures 3A and 3B, insets). For the time-matched control, changes in the parameters were constant and stable (Figure 4C). The responses were reproducible during the periods corresponding to the control and experimental observations (differences between the periods were not significant).

Effects of PDE3 Inhibition With Milrinone or PDE2 Inhibition With EHNA on pGC-cGMP–Induced Increase in cAMP Concentration, Atrial Dynamics, and Myocytic ANP Release
Because the effects of CNP on the atrial dynamics and ANP release are mainly mediated by pGC-cGMP signaling^16,17 and also PDE2 and PDE3 are target molecules for the cGMP, experiments were performed to define the role of PDE2 and PDE3 as for the downstream of pGC-cGMP signaling pathway.

Milrinone increased basal levels of cGMP and cAMP efflux concentrations up to 24 minutes and maintained thereafter (third cycle of infusion; n=6; for cGMP, 0.050±0.005 versus 0.035±0.004 µmol/L, 49.13±21.53%, P<0.05, Student’s paired t test; for cAMP, 0.044±0.007 versus 0.032±0.005, 41.45±11.81%, P<0.01; Figures 1Ba and 1Bb). The milrinone responses were then maintained thereafter without significant changes (Figures 1Ba and 1Bb and 3A and 3B). Milrinone slightly and transiently increased atrial dynamics and the effect was stable after the third cycle of infusion (Figures 1Bc and 1Bd and 3 C). Myocytic ANP release was decreased by milrinone and was maintained after the third cycle of infusion (Figures 1Be and 3 HREF="#FIG3">D). An inhibition of PDE3 with milrinone completely blocked CNP-induced increase in cAMP levels (Figures 1Bb and 3B). In the presence of milrinone, CNP-induced increase in cGMP levels was attenuated in the atria treated with higher concentration of CNP (100 nmol/L; 175.92±13.09, n=7 versus 314.98±28.53%, n=14, P<0.01) but not in the atria treated with lower concentration of CNP (30 nmol/L) (Figure 3A). CNP-induced decreases in atrial stroke volume and ANP release were accentuated by milrinone (both P<0.05 versus CNP alone; Figures 1Bc, 1Be, 3C, and 3 HREF="#FIG3">D). This indicates that PDE3 is involved in the regulation of CNP-induced increase in cAMP levels and decreases in atrial dynamics and myocytic ANP release.

Next, to define the role of PDE2 in the CNP-induced increase in cAMP efflux levels, an inhibitor of PDE2 was applied. EHNA increased basal levels of cGMP and cAMP (third cycle of infusion; n=7; for cGMP, 0.074±0.017 versus 0.031±0.007 µmol/L, 152.64±32.36%, P<0.05; for cAMP, 0.055±0.008 versus 0.035±0.009, 80.37± 20.02%, P<0.001). The responses were maintained thereafter without significant changes (EHNA alone; Figure 5). EHNA slightly decreased atrial dynamics and the effect was stable after the third cycle of infusion. ANP release was significantly increased by EHNA and was maintained after the third cycle of infusion. An inhibition of PDE2 with EHNA had no effect on the CNP (100 nmol/L)-induced increases in cGMP and cAMP levels and decreases in atrial dynamics and ANP release (Figures 3A through 3D). These results indicate that PDE2 is involved in the regulation of basal levels of cGMP and cAMP but not of the CNP-induced increase in cAMP levels.

Effects of sGC Activation With SIN-1 or BAY on the Levels of cGMP and cAMP, Atrial Dynamics, Myocytic ANP Release, and Its Modulation by PDE2 or PDE3 Inhibition
SIN-1 (30 µmol/L) increased cGMP efflux levels from 0.058±0.013 µmol/L by 35.26±14.91% to 0.078±0.018 µmol/L at the first 12-minute cycle and then gradually increased by 110.97±30.60% up to third cycle (36 minutes). SIN-1 increased cAMP levels from 0.065±0.011 µmol/L by 6.87±3.63% to 0.071±0.013 µmol/L at the first 12-minute cycle, and the increase was maintained up to 36 minutes. SIN-1 (100 µmol/L) increased the cGMP levels by 187.99±64.56% at the first 12-minute cycle and then gradually further increased to 401.11±90.83% at the third cycle of infusion (P<0.01, Figures 2A and 4Aa). SIN-1 (100 µmol/L) increased cAMP levels by 6.45±4.71% at the first 12-minute cycle and further increased by 28.31±3.83% up to 36 minutes (P<0.01; Figures 2B and 4Ab). With SIN-1 (300 µmol/L), the pattern of responses was similar to that of SIN-1 (100 µmol/L) in terms of time (Figure 5A). The effects of SIN-1 on cGMP and cAMP levels were concentration-dependent (Figures 3A and 3B). SIN-1 decreased ANP release at higher concentration (300 µmol/L) significantly but not atrial dynamics.

To further define the roles of sGC, sGC was directly activated with BAY. sGC activation with BAY (1 µmol/L) increased cGMP levels by 33.44±17.27% at the first 12-minute cycle and further increased to 209.90±17.78% up to 36 minutes (Figure 4Ba). BAY (10 µmol/L) further increased cGMP levels by 178.19±40.06% at first cycle to 1031.25±57.39% at third cycle of infusion (Figure 2A). BAY increased cGMP levels in a concentration-dependent manner (Figure 3A). BAY had no significant effects on cAMP levels (Figures 2B, 3B, and 4Bb) and ANP release (Figures 3D and 4Be). BAY slightly decreased atrial stroke volume (Figure 4Bc), which was relevant to the effect of vehicle ethanol (Figure 3C).

An inhibition of PDE3 with milrinone blocked SIN-1–induced increase in cAMP levels (Figure 3B). SIN-1–induced increase in cGMP levels was slightly but not significantly attenuated by milrinone (Figure 3A). SIN-1–induced decreases in stroke volume and ANP release were accentuated by milrinone (Figures 3C and 3D). This indicates that PDE3 is involved in the SIN-1–induced increase in cAMP levels and decreases in atrial dynamics and ANP release.

An inhibition of PDE2 with EHNA had no effects on SIN-1 (100 µmol/L)–induced increases in cGMP and cAMP efflux levels (Figures 3A and 3B). EHNA accentuated SIN-1 (300 µmol/L)–induced increase in cGMP levels (Figures 3A and 5Ba). EHNA slightly but not significantly accentuated SIN-1–induced increase in cAMP levels. SIN-1–induced decrease in ANP release was significantly accentuated by EHNA. This indicates that PDE2 is minimally involved in the SIN-1–induced increase in cAMP levels.

In the presence of ODQ, SIN-1–induced changes in cGMP and cAMP efflux levels and ANP release were not observed (Figure 6A). SIN-1 slightly but significantly increased atrial stroke volume in the presence of ODQ. The change in cAMP concentration by ODQ plus SIN-1 was not significantly different from that by SIN-1 or ODQ alone. ODQ alone increased cGMP and cAMP levels slightly but significantly during the periods corresponding to the experiment. Similarly, ODQ blocked BAY-induced increase in cGMP efflux concentration (Figure 6B).

High and Low Gain Effects of pGC-cGMP and sGC-cGMP Signaling on cAMP Levels and Atrial Function
Because both pGC- and sGC-activated increases in cAMP levels are closely related to the PDE3 activity, we analyzed the responses to define the relation between the levels of cGMP and cAMP. As shown in Figure 7A, increase in cAMP levels was a function of cGMP levels. The relation was different between pGC-cGMP-cAMP and sGC-cGMP-cAMP. The gain of cAMP concentration, ie, the ratio of %difference in cAMP efflux concentration over the control per %difference in cGMP efflux concentration over the control, in terms of changes in cGMP levels was significantly different between pGC and sGC signaling [0.206±0.024 for CNP (100 nmol/L), n=14, at 314.98±28.53% of cGMP versus 0.098±0.020 for SIN-1 (100 µmol/L), n=9, at 401.11±90.83% of cGMP, P<0.01]. This indicates that pGC-cGMP-PDE3 and sGC-cGMP-PDE3 are high and low gain switches for the regulation of cAMP levels, respectively. In the same line, both the changes induced by pGC and sGC activation in atrial stroke volume and ANP release were different in terms of changes in cGMP levels (Figures 7B and 7C). This indicates that pGC-cGMP signaling is much more prominent than sGC-cGMP in the regulation of atrial dynamics and ANP release.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study clearly shows for the first time the distinct roles for pGC-cGMP and sGC-cGMP signaling in the regulation of cAMP levels, atrial dynamics, and myocytic ANP release. In the present study, we have shown that pGC-PDE3 and sGC-PDE3 are high and low gain switches, respectively, in the regulation of cAMP levels, atrial dynamics, and secretary function. The present results show that PDE2 has little role in the regulation of cAMP levels via GC-cGMP-PDE-cAMP signaling in the rabbit atria. Mainstream of the effects of pGC or sGC activation was cGMP pathway.

High and Low Gain Effects of pGC- and sGC-cGMP Signaling on cAMP
pGC and sGC activation with CNP and SIN-1 or BAY, respectively, increased cGMP efflux levels in a concentration-dependent manner. An increase in cGMP levels by pGC activation was higher than that by sGC activation in terms of molar concentration of activators. Also, an increase in cGMP levels by BAY was higher than that by SIN-1. An increase in cGMP levels by pGC activation with CNP or sGC activation with SIN-1 was accompanied by an elevation in cAMP levels in a cGMP-dependent manner. cGMP-related increase in cAMP levels was not observed in the presence of a PDE3 inhibitor, milrinone. This finding indicates that an increase in cGMP levels by pGC or sGC activation increases cAMP levels via cGMP-PDE3 signaling in the rabbit cardiac atrium. This is consistent with previous reports.^{4,8,10,20–25} They proposed cGMP-PDE3 signaling in the regulation of cAMP levels. The present study shows distinct roles for pGC-cGMP-PDE3 and sGC-cGMP-PDE3 signaling in the regulation of cAMP levels. The pGC-cGMP-PDE3 signaling by CNP was much more prominent than sGC-cGMP-PDE3 by SIN-1. Despite sGC activation with NO donor, SIN-1, and direct activator, BAY increased cGMP efflux concentration, but BAY did not increase cAMP levels. These data suggest that pGC- and sGC-derived cGMP-PDE3 signaling is compartmentalized in the regulation of cAMP levels. It is not clear at present whether the compartmentalized functions are related to the different spatial confinement of GCs and/or PDE3s or not. Recently, it was shown that PDE3A is cytosolic and PDE3B is particulate.²⁹

As for the role of PDE2 in the regulation of cAMP levels via cGMP-PDE signaling, the present study shows that PDE2 is minimally involved in this pathway at high and low levels of cGMP. No difference in the cAMP levels was observed in the presence and absence of EHNA. Slight but not significant accentuation of SIN-1 (300 µmol/L)–induced increase in cAMP efflux concentration was related to the accentuation of cGMP levels by PDE2 inhibition with EHNA. The gain of cAMP efflux concentration in terms of cGMP levels was not different between the levels obtained by the presence and absence of EHNA [for SIN-1 (300 µmol/L), 0.036±0.002, n=6, in the presence of EHNA versus 0.045±0.006, n=10, in the absence of EHNA, P>0.05]. PDE2 inhibition with EHNA or PDE3 inhibition with milrinone increased basal levels of cGMP and cAMP efflux concentration (for cGMP, 138.34±22.73%, n=23, by EHNA, 50.22±10.28%, n=25, by milrinone, both P<0.05 versus time-matched control; for cAMP, 44.95±12.60% by EHNA, 45.59±7.12% by milrinone, both P<0.05). This indicates that both PDE2 and PDE3 are involved in the regulation of basal levels of cGMP and cAMP.

It was shown that PDE2 is involved in the regulation of Ca²⁺ current in human atrial myocytes^23,25 but not in rat atrial and ventricular myocytes.²³ This suggests that the role of PDE2 is species-specific. The role of PDE2 in the regulation of Ca²⁺ current of human cardiac myocytes^23,25 may be related to its localization at the periphery of the cells.³⁰ Taken together, the present study suggests that PDE2 and PDE3 are compartmentalized in the regulation of basal cAMP levels and activated cAMP levels via cGMP-PDE signaling in the rabbit atrium.

As shown in previous reports including ours,^16,17 cGMP signaling is mainstream for the CNP effects in the heart. In the same line, it was shown in the present study that SIN-1–induced cardiac effects including increase in cAMP levels were caused by cGMP signaling. Most effects caused by SIN-1 were not observed in the presence of ODQ, a selective inhibitor of sGC.

In summary, the present study shows distinct roles for the pGC-cGMP-PDE3 and sGC-cGMP-PDE3 signaling in the cardiac atrium: high and low gain switches for the regulation of cAMP levels, respectively (Figure 7).

Comparative Effects of pGC and sGC Activation on Atrial Dynamics and ANP Release
pGC activation with CNP decreased atrial dynamics via pGC-cGMP pathway. However, sGC activation with SIN-1 or BAY decreased atrial dynamics without significance. The results are not consistent with previous reports showing a negative inotropic effect by NO donor^6,9–11 and are consistent with reports on CNP^16,17 through cGMP signaling. In the presence of ODQ, SIN-1 rather increased atrial dynamics slightly but significantly. The response may be related to the previous finding that SIN-1 stimulates superoxide anion generation that is not blocked by ODQ.⁹ The atrial contractile response to pGC or sGC activation was accentuated by milrinone, in which cGMP-induced increase in cAMP was not observed. cAMP is well known to increase cardiac dynamics.³¹ The present data indicate that both cGMP and cAMP by cGMP-PDE3 signaling are involved in the regulation of cardiac dynamics (Figure 8).

View larger version (14K): [in this window] [in a new window]   Figure 8. Schematic diagram showing high and low gain signaling via pGC-PDE3 and sGC-PDE3 pathways, respectively.

In summary, pGC-cGMP signaling is more effective than sGC-cGMP to decrease atrial dynamics. It is suggested from these data that the role for cGMP-PDE3-cAMP signaling activated by pGC and sGC is compartmentalized in the regulation of cardiac dynamics.

pGC and sGC activation with CNP and SIN-1, respectively, decreased atrial myocytic ANP release. This is consistent with the previous reports.^13–16 Decrease in ANP release by pGC activation with CNP was much more prominent than that by sGC activation with SIN-1 in terms of cGMP levels. sGC activation with a direct activator, BAY, did not decrease ANP release. This means that cGMP is compartmentalized in the regulation of atrial myocytic ANP release. That is, sGC-induced increase in cGMP is not effective to decrease ANP release compared with pGC-induced cGMP. This may be related to the difference in pGC-cGMP and sGC-cGMP signaling in the regulation of cAMP levels.

Because both cAMP and cGMP decrease ANP release,^16,26,28 it was expected that in the presence of milrinone pGC or sGC activation–induced decrease in ANP release would be attenuated by blockade of increase in cAMP levels accompanied by an increase in cGMP levels. However, it was not the case. In the presence of milrinone, pGC or sGC activation-induced decrease in ANP release was rather accentuated. The mechanism for this unexpected response may be related to the hypothesis that a critical level of cAMP accentuates cGMP-induced decrease in ANP release. Although milrinone prevented an increase in cAMP levels induced by GC-cGMP-PDE3 signaling, this agent increased basal cAMP levels. This may be the case for the accentuation by PDE2 inhibition with EHNA of the SIN-1–induced decrease in ANP release. This indicates that increase in cAMP levels induced by pGC-cGMP-PDE3 signaling is involved in the regulation of ANP release (Figure 8). The present study indicates that GC-cGMP-PDE3 signaling activated by pGC or sGC is compartmentalized in the regulation of atrial myocytic ANP release.

In summary, our results demonstrate that pGC and sGC activations have distinct roles via cGMP-PDE3-cAMP signaling in the cardiac atrium: high and low gain switches, respectively, for the regulation of cAMP levels and contractile and secretory functions.

	Acknowledgments

 
This work was supported by research grants from the Korea Research Foundation (2000-015-FP0023), the Korea Science and Engineering Foundation (98-0403-10-01-5, R01-2003-000-10507-0), and the Ministry of Health and Welfare (HMP-00-PJ9-PG1-C003-0001). Jin Fu Wen was supported by a fellowship from Jeollabuk-do Province Foreign Scientist Program. The authors thank Kyong Sook Kim for secretarial work.

	Footnotes

 
Original received September 10, 2003; resubmission received December 1, 2003; revised resubmission received February 11, 2004; accepted February 11, 2004.

	References

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