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Cardiovascular Research 2003 59(2):360-368; doi:10.1016/S0008-6363(03)00394-8
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Copyright © 2003, European Society of Cardiology

Diverse regulation of atrial natriuretic peptide secretion by serotonin receptor subtypes

Chunhua Cao, Jeong Hee Han, Sung Zoo Kim, Kyung Woo Cho and Suhn Hee Kim*

Department of Physiology, Institute for Medical Sciences and Basic Research Institute, Chonbuk National University Medical School, 2-20 Keum-Am-Dong-San, Jeonju 561-180, South Korea

* Corresponding author. Tel.: +82-63-274-9788; fax: +82-63-274-9892. shkim{at}moak.chonbuk.ac.kr

Received 13 January 2003; accepted 7 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Serotonin (5-hydroxytryptamine [5-HT]) receptors are located in peripheral tissues as well as in the central nervous system. Serotonin receptors mediate positive inotropic and chronotropic effects in atria. The aim of this study was to investigate physiological role of endogenous serotonin on the regulation of atrial natriuretic peptide (ANP) secretion from the atria. Methods: An isolated perfused nonbeating rat atrial model was used. Changes in atrial volume induced by increasing intra-atrial pressure were measured. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. Results: Serotonin, an endogenous 5-HT receptor agonist, caused concentration-dependent suppressions of stretch-induced ANP secretion, which were less pronounced than those caused by {alpha}-methyl-5-HT maleate, a 5-HT2 receptor selective agonist. The suppression of stretch-induced ANP secretion due to serotonin and {alpha}-methyl-5-HT maleate was attenuated by ketanserin, a 5-HT2 receptor antagonist, and accentuated by RS23597-190, a 5-HT4 receptor antagonist. The suppressive effect of serotonin on ANP secretion was attenuated by neomycin, staurosporine, and chelerythrine. In contrast, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, a 5-HT4 receptor selective agonist, caused an accentuation of stretch-induced ANP secretion, which was completely blocked by RS23597-190 and SB203186 HCl but not by ketanserin. This effect was not affected by MDL12330, KT-5720, or H-89. The intracellular Ca2+ concentration in single atrial myocytes was not changed by serotonin and agonist for either 5-HT2 or 5-HT4 receptor. Conclusions: These results suggest that atrial 5-HT2 and 5-HT4 receptor agonists have opposite actions on the regulation of ANP secretion and the suppressive effect of serotonin on the ANP secretion may act through 5-HT2 receptor and phospholipase C pathway.

KEYWORDS Atrial function; Calcium; Inotropic agents; Natriuretic peptide; Receptor; Serotonin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Serotonin (5-hydroxytryptamine [5-HT]) is an important biogenic amine that is found in high concentrations in specific regions of the central nervous system, platelets, and enterochromaffin cells throughout the gastrointestinal tract [1]. Circulating serotonin secreted mainly from enterochromaffin cells, is stored in platelets and activated platelets release serotonin, causing vasoconstriction [2]. Serotonin mediates its diverse physiologic effects through at least 15 different receptor subtypes belonging to seven classes of receptors [3,4]. The diversity of its functions is also related to the wide variety of receptors and effectors. The functional responses of the cardiovascular system mediated by the serotonin are known to be highly complicated because of large number of receptor subtypes and their different actions. In many aspects, serotonin appears to be a neurohumoral substance involved in cardiovascular regulation.

It is well known that 5-HT produces positive inotropic and chronotropic effects in heart muscles of various mammalian species. The inotropic effect of 5-HT has been reported to be direct (by the stimulation of 5-HT specific receptors) [5,6], indirect (by the release of noradrenaline from the sympathetic nerve terminals) [7] or both [8], depending on species. Some have reported that 5-HT4 receptors mediate the positive inotropic and chronotropic effects in human and porcine atria [9,10] and that ketanserin-sensitive 5-HT2A receptors mediate the positive inotropic effect of serotonin in rat atria [11]. Mertens et al. [12] have also demonstrated that cardiac hypertrophy is associated with impaired chronotropic response to serotonin mediated by the 5-HT2 receptor. Both 5-HT4 and 5-HT2A receptors are expressed in the rat heart [11,13], and the coexpression of these receptors coupled to distinct signaling pathways and their opposite actions have been demonstrated in human intestinal muscle cells [14].

The aim of the present study was to characterize pharmacology of atrial 5-HT receptor subtypes, which may be involved in the regulation of atrial natriuretic peptide (ANP) secretion [15]. There are a few reports about the relationship between serotonin and atrial ANP secretion. Therefore, experiments have been performed to investigate the effects of an endogenous agonist and a specific agonist for 5-HT2 and 5-HT4 receptors on the ANP secretion from isolated perfused nonbeating rat atria and to characterize their mechanisms pharmacologically with specific receptor antagonists.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male Sprague–Dawley rats weighing 300–350 g were used. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85.23, revised 1996).

2.2 Isolated perfused atrial preparation
Rats were sacrificed by decapitation, and an isolated perfused atrial preparation was made by using a previously described method [16,17]. Briefly, the heart was rapidly removed and placed in oxygenated sodium chloride solution, and the left atrium was dissected from the heart. A Tygon cannula containing three small catheters sealed within it was inserted into the atrium and secured by ligatures. The cannulated atrium was transferred into an organ chamber containing a buffer solution at 36.5°C; this was covered with watertight silicone rubber cap. The atrium was immediately perfused with oxygenated HEPES buffer solution at a rate of 0.25 ml/min by using a peristaltic pump. The composition of the HEPES buffer solution was as follows: NaCl 118 mmol/l, KCl 4.7 mmol/l, CaCl2 2.5 mmol/l, MgSO4 1.2 mmol/l, NaHCO3 25 mmol/l, HEPES 10 mmol/l, glucose 10 mmol/l, and bovine serum albumin 0.1%. The pericardial buffer solution, which contained [3H]inulin in HEPES buffer to measure the translocation of extracellular fluid (ECF), was also oxygenated by placing silicone tubing coils inside the organ chamber. The pericardial space of the organ chamber was sealed and connected to a calibrated microcapillary tube, with which changes in atrial volume were monitored. The atrium was perfused for 40 min to stabilize the secretion of ANP and to maintain a steady-state [3H]inulin level in the extracellular space. The perfusate was collected at 2-min intervals at 4°C. After two collection periods, atrial distension was induced for 2 min by elevating the outflow catheter tip to 6 cmH2O, and then reduced by lowering the catheter tip to the basal level. Atrial pressure was applied at 10-min intervals.

2.3 Experimental protocols
Experiments were performed with three groups.

Group 1 was the control group. In this group, HEPES buffer was perfused throughout the experiment (n = 7).

Group 2 included the agonist-perfused groups. In these experimental groups, the following were introduced into the atrial lumen after a 30-min control collection period: serotonin, an endogenous 5-HT receptor agonist, at doses of 1 (n = 4), 10 (n = 9), 30 (n = 8) or 100 µmol/l (n = 9); {alpha}-methyl-5-hydroxytryptamine maleate (methyl-5-HT), a 5-HT2 receptor agonist, at doses of 1 (n = 6), 10 (n = 7) or 30 µmol/l (n = 8); or 2-[1-(4-piperonyl)piperazinyl]benzothiazole (PPB), a 5-HT4 receptor agonist, at a dose of 10 (n = 6), or 30 µmol/l (n = 11).

Group 3 was the modifier-pretreated group. To modify the effects of serotonin or methyl-5-HT on ANP secretion, ketanserin (5-HT2 receptor antagonist, 100 µmol/l, n = 8), or RS23597-190 (5-HT4 receptor antagonist, 30 µmol/l, n = 7) was administered as a pretreatment at the start of the perfusion, and then serotonin (30 µmol/l) was simultaneously infused after a 30-min control collection period. To modify the effects of PPB on ANP secretion, ketanserin (100 µmol/l, n = 7), or RS23597-190 (30 µmol/l, n = 7), or SB203186 HCl (5-HT4 receptor antagonist, 100 µmol/l, n = 7) was also preincubated and then PPB (30 µmol/l) was simultaneously infused, as described the above.

In order to investigate the signal pathway involved in the regulation of ANP secretion by serotonin receptors, phospholipase C (PLC) inhibitors such as neomycin (1 mmol/l, n = 9) and U 73122 (3 µmol/l, n = 5), nonspecific protein kinase C (PKC) inhibitors such as staurosporine (1 µmol/l, n = 5) and chelerythrine chloride (1 µmol/l, n = 5), adenylyl cyclase (AC) inhibitor (MDL12330, 10 µmol/l, n = 5) or protein kinase A (PKA) inhibitors such as KT-5720 (3 µmol/l, n = 4) and H-89 (1–10 µmol/l, n = 5) were administered as a pretreatment at the start of the perfusion and then serotonin or PPB was simultaneously infused after a 30-min control collection period.

2.4 Radioimmunoassay of ANP
The concentration of immunoreactive ANP in the perfusates was measured by using specific radioimmunoassay, as described previously [16,17].

2.5 Measurement of ECF translocation
We previously reported a two-step sequential mechanism of ANP secretion from the atria: First, the atrial release of ANP into interstitial space occurs by means of stretching, and second, the released ANP is secreted into the atrial lumen, concomitant with ECF translocation due to reduction [18,19]. The radioactivity of [3H]inulin in the atrial perfusate was measured by using a liquid scintillation counter (Tris-Carb 23-TR; Packard Bioscience, Downers Grove, IL, USA). The amount of ECF translocated through the atrial wall was calculated by total radioactivity in perfusate divided by radioactivity in pericardial reservoir, and expressed as µl/min/g.

2.6 Measurement of the intracellular Ca2+ concentration in single atrial myocytes
Single atrial myocytes were isolated by using a previously described technique [20,21], with minor modifications. Changes in the intracellular Ca2+ concentration, or [Ca2+]i, in single atrial myocytes were measured by using a fluorescence digital imaging microscopic system (Attofluor; Atto Instruments, Rockville, MD, USA), as described elsewhere [20,21]. After loading 2 µmol/l fura2-AM (Molecular Probes, Eugene, OR, USA) for 20 min at room temperature, atrial myocytes were attached onto a perfusion chamber coated with matrix gel and placed on the stage of an inverted fluorescence microscope (Axiovert 135; Carl Zeiss, Jena, Germany) connected to the digital fluorescence microscopic system. Cells were stimulated at 1 Hz and perfused with HEPES buffer containing Ca2+ at a concentration of 1 mmol/l at a rate of 0.7 ml/min for 5 min. Serotonin, methyl-5-HT, or PPB (all 10, 30, 100 µmol/l) was perfused for 5 min and then cells were imaged with excitation wavelengths of 338 and 380 nm and an emission wavelength of 520 nm. The fluorescence images were captured with intensified charge-coupled device camera and analyzed with the Attofluor image processing software. Changes in [Ca2+]i were presented as a ratio of the fluorescence intensities (i.e., f338/f380).

2.7 Statistical analysis
The results are given as the mean±S.E.M. The statistical significance of the differences was assessed by using an analysis of variance (ANOVA) and the Duncan multiple range test. In some cases, Student's t test was also used. The critical level of significance was set at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of serotonin on ANP secretion
Fig. 1 shows the ANP secretion and ECF translocation in response to increased atrial volume induced by the elevation of intra-atrial pressure by 6 cmH2O before and after the exposure to serotonin. After 40 min for stabilization, intra-atrial pressure of 6 cmH2O was applied three times every 10 min to serve as a control period. Then, serotonin was infused at a concentration of 100 µmol/l. The intra-atrial pressure was increased by elevating the outflow catheter tip for 2 min; afterward, it was returned to the basal level (Fig. 1A). Increases in atrial volume caused by changing pressure (i.e., DRV), measured by using a microcapillary tip connected to the pericardial space, were similar (Fig. 1B). The basal rate of ANP secretion was constant throughout the experiment (Fig. 1C), and ANP secretion in response to stretching was markedly increased. Serotonin suppressed stretch-induced ANP secretion. The basal rate of ECF translocation was constant throughout the experiment (Fig. 1D), and ECF translocation in response to stretching was markedly increased after the intra-atrial pressure returned to the basal level from 6 cmH2O. Mechanically stimulated ECF translocations in the control period were not significantly different from those in the serotonin infusion period. Therefore, the ANP secretion in terms of ECF translocation, which means the interstitial ANP concentration, was dramatically suppressed by serotonin (Fig. 1E).


Figure 1
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  		Fig. 1 Effect of 100 µmol/l serotonin, an endogenous 5-HT receptor agonist, on distension-and-reduction volume (DRV), ANP secretion, and ECF translocation in isolated perfused nonbeating rat atria. The intra-atrial pressure was increased by 6 cmH2O for 2 min at 10-min intervals (A), and atrial perfusate was collected at 2-min intervals. After a 30-min control period, serotonin (100 µmol/l) was perfused into the atrial lumen. Changes in atrial volume (i.e. DRV) (B) induced by the elevation of intra-atrial pressure were measured by using a calibrated microcapillary tube connected to the pericardial space. Increases in atrial volume increased in ANP secretion (C) and ECF translocation (D); these changes were reproducible. Serotonin decreased stretch-induced ANP secretion but not the mechanically-stimulated ECF translocation and DRV. Therefore, the ANP secretion in terms of ECF translocation, i.e. interstitial ANP concentration, was markedly decreased by serotonin (E). CONT, control period; SER, serotonin infusion period. Values are the mean±S.E.M. (n = 9). *P<0.01 vs. the third peak value.

 
To show the dose–response curve of serotonin, changes in ANP secretion and ECF translocation were calculated by subtracting the previous two values from the two peak values at each pressure. Fig. 2A shows the relative changes in stretch-activated ANP secretion in terms of ECF translocation caused by serotonin. Low doses of serotonin, 1 and 10 µmol/l, did not cause any significant changes in ANP secretion. By increasing the doses from 10 to 30 and 100 µmol/l, stretch-induced ANP secretion in terms of ECF translocation was proportionately suppressed.


Figure 2
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  		Fig. 2 Relative changes in stretch-induced ANP secretion in terms of ECF translocation over time as a result of serotonin (A) or {alpha}-methyl-5-HT maleate (B). Serotonin and {alpha}-methyl-5-HT maleate caused dose-dependent decreases in stretch-induced ANP secretion, which reached a peak value in the 30-min infusion period. Dose–response curves of serotonin and {alpha}-methyl-5-HT maleate on stretch-induced ANP secretion in terms of ECF translocation are shown. The {alpha}-methyl-5-HT maleate-induced inhibition of ANP release was significantly different from serotonin-induced effect (C). CONT, control group; SER, serotonin infusion group; methyl-5-HT, {alpha}-methyl-5-HT maleate infusion group. Values are the mean±S.E.M. (n = 4–9). *P<0.05 vs. the group infused with 10 µmol/l; #P<0.05, ##P<0.01 vs. serotonin infusion group.

 
3.2 Effect of the 5-HT2 receptor agonist on ANP secretion
To define the 5-HT receptor subtypes that may be involved in the serotonin-induced suppression of ANP secretion, methyl-5-HT (a 5-HT2 receptor agonist) was administered. As shown in Fig. 2B, methyl-5-HT also caused dose-dependent decreases in stretch-induced ANP secretion in relation to ECF translocation. To compare the suppressive effects of serotonin and methyl-5-HT, the mean of the last three values was obtained from Fig. 2A and B. As shown in Fig. 2C, methyl-5-HT-induced suppression of ANP release was more potent than serotonin-induced effect. In order to define the mechanism for the suppressive effect of serotonin and methyl-5-HT, antagonist for 5-HT2 or 5-HT4 receptor was used. Pretreatment with ketanserin attenuated the relative suppressive effect of serotonin (Fig. 3A) on stretch-induced ANP secretion in terms of ECF translocation. Interestingly, with the pretreatment involving RS23597-190, the relative suppressive effect of serotonin was accentuated; values changed from 0.64±0.04 to 0.51±0.06 (P<0.05, Fig. 3A). The suppressive effect of methyl-5-HT on ANP secretion was also blocked by ketanserin and accentuated by RS23597-190 (Fig. 3A). Serotonin-induced suppression of ANP release was attenuated by neomycin, chelerythrine and staurosporine (Fig. 3B). However, U73122 [GenBank] has no significant effect.


Figure 3
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  		Fig. 3 Modification of the suppressive effect of stretch-induced ANP secretion due to serotonin and {alpha}-methyl-5-HT maleate in the presence of receptor antagonists (A) or inhibitors for phospholipase C or protein kinase (B). The antagonist or inhibitor was infused at the beginning of experiment, and serotonin or {alpha}-methyl-5-HT maleate was simultaneously infused after a 30-min control period. Serotonin and {alpha}-methyl-5-HT maleate decreased stretch-induced ANP secretion, which was blocked by ketanserin but accentuated by RS23597-190. Serotonin-induced suppression of ANP secretion was also attenuated by neomycin, staurosporine and chelerythrine but not by U73122. CONT, control group; KET, ketanserin; RS, RS23597-190; methyl-5-HT, {alpha}-methyl-5-HT maleate; NEO, neomycin; ST, staurosporine; CH, chelerythrine. Values are the mean±S.E.M. (n = 5–8). *P<0.05, **P<0.01, ***P<0.005 vs. control group.

 
3.3 Effect of the 5-HT4 receptor agonist on ANP secretion
To define the effect of 5-HT4 receptor agonist on ANP secretion, PPB was perfused into the atrial lumen. Fig. 4 shows the effect of PPB on ANP secretion and ECF. The protocol was the same as that described in Fig. 1. After the application of 30 µmol/l PPB, stretch-induced ANP secretion gradually increased with time (Fig. 4C), but mechanically-stimulated ECF translocation and the DRV were relatively constant (Fig. 4B and D). Therefore, PPB caused an increase in interstitial ANP concentration (Fig. 4E). Fig. 5 shows the relative changes in stretch-induced ANP secretion in terms of ECF translocation by PPB, 10 and 30 µmol/l; these changes are represented as ratio of the mean values to the control values obtained from Fig. 4. The effect of PPB on ANP secretion was accentuated over time (Fig. 5). Fig. 6 shows the comparison of relative changes in stretch-induced ANP secretion between the control group and the modifier-treated groups. Control value was the mean of three control values, and the experimental value was the peak value. The accentuation in ANP secretion in terms of ECF translocation by PPB was blocked by RS23597-190 or SB203186 HCl, but not by ketanserin (Fig. 6A). This effect was not attenuated by MDL12330, KT-5720, or H-89 (Fig. 6B).


Figure 4
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  		Fig. 4 Effect of 30 µmol/l 2-[1-(4-piperonyl)piperazinyl]benzothiazole (PPB) on distension-and-reduction volume (DRV), ANP secretion, and ECF translocation in isolated perfused nonbeating rat atria. The same protocol as in Fig. 1 was applied. PPB caused a gradual increase in stretch-induced ANP secretion with time (C), but ECF translocation (D) and atrial volume (B), were not significantly different from those observed in the control period. Therefore, the interstitial ANP concentration was significantly increased by PPB infusion (E). CONT, control period; PPB, PPB infusion period. Values are the mean±S.E.M. (n = 11). *P<0.05, **P<0.01 vs. third peak value.

 

Figure 5
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  		Fig. 5 Relative changes in stretch-induced ANP secretion with time by 2-[1-(4-piperonyl)piperazinyl]benzothiazole (PPB). PPB gradually increased stretch-induced ANP secretion in terms of ECF translocation. (bullet, {circ}) PPB-infused groups (PPB) (10 or 30 µmol/l, respectively); (Figure 5) control group (CONT). Values are the mean±S.E.M. (n = 6–11). *P<0.05, **P<0.01 vs. control group.

 

Figure 6
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  		Fig. 6 Modification of the accentuation of stretch-induced ANP secretion caused by 2-[1-(4-piperonyl)piperazinyl]benzothiazole (PPB) in the presence of receptor antagonists (A) or inhibitors for adenylyl cyclase or protein kinase A (B). PPB increased stretch-induced ANP secretion, which was completely blocked in the presence of SB203186 HCl and RS23597-190 but not in the presence of ketanserin (n = 7). PPB-induced accentuation of ANP secretion was not modified by MDL12330, KT-5720, or H-89 (n = 4 to 5). SB, SB203186 HCl; MDL, MDL12330; KT, KT-5720; PPB, 2-[1-(4-piperonyl)piperazinyl]benzothiazole. Legends are the same as in Fig. 3. Values are the mean±S.E.M.

 
3.4 Effects of 5-HT receptor agonists on [Ca2+]i
Changes in [Ca2+]i caused by the application of serotonin, methyl-5-HT or PPB were measured in single beating atrial myocytes. As shown in Fig. 7, serotonin, methyl-5-HT or PPB did not cause any significant changes in [Ca2+]i. However, [Ca2+]i was increased by isoproterenol.


Figure 7
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  		Fig. 7 Changes in [Ca2+]i concentration in single atrial myocytes stimulated by serotonin, {alpha}-methyl-5-HT maleate, or 2-[1-(4-piperonyl)piperazinyl]benzothiazole. Changes in [Ca2+]i were not significantly changed with serotonin, {alpha}-methyl-5-HT maleate or 2-[1-(4-piperonyl)piperazinyl]benzothiazole but markedly increased with isoproterenol, as a control. ISP, isoproterenol. Legends are the same as in Figs. 3 and 6Go. Values are the mean±S.E.M. (n = 8–12).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we have shown that serotonin and a specific 5-HT2 receptor agonist have concentration-dependent suppressive effects of stretch-activated ANP secretion. The suppressive effect of serotonin on the ANP secretion by 5-HT2 receptor was mediated through PLC and PKC pathway. In contrast, a specific 5-HT4 receptor agonist has an accentuated effect of stretch-activated ANP secretion mediated by 5-HT4 receptor.

4.1 Diverse regulation of ANP secretion through 5-HT2 and 5-HT4
Serotonin is present in the mammalian heart at a concentration of 0.4 µg/g and both 5-HT4 and 5-HT2A receptors are expressed in the rat heart [13,22]. The positive inotropic effect is mediated by either 5-HT4 receptor [9,10] or 5-HT2A receptor [11] and serotonin increases the calcium current in human atrial myocytes by 5-HT4 receptor [23]. In this study, we found diverse regulation of ANP secretion by serotonin receptor subtypes. Serotonin and 5-HT2 receptor agonist caused a decrease in ANP secretion. The potency of 5-HT2 receptor agonist was three times greater than serotonin. These effects were attenuated by 5-HT2 antagonist and accentuated by 5-HT4 antagonist. The accentuated effect of serotonin by the pretreatment of 5-HT4 antagonist can be explained by the binding of serotonin to 5-HT2 receptor more by blocking 5-HT4 receptor. Our results are consistent with other report showing the augmentation of 5-HT-induced contraction by a specific 5-HT4 receptor antagonist [14]. Interestingly, a specific 5-HT4 receptor agonist accentuated stretch-induced ANP secretion with time. This effect was blocked by specific antagonists for 5-HT4 receptor, but not by a 5-HT2 receptor antagonist. Therefore, we suggest that serotonin decreases ANP secretion by means of a 5-HT2 receptor and that 5-HT2 and 5-HT4 receptors may regulate the secretion of ANP in different ways. Our results are comparable with other report showing 5-HT2A receptors mediating contraction and 5-HT4 receptors mediating relaxation in human intestinal muscle cells [14].

4.2 Signal transduction of diverse effects through 5-HT2 and 5-HT4
It has been reported that serotonin increases cAMP and PKA activity by 5-HT4 receptors in the human atrium [5,9]. Serotonin also increases 1,4,5-inositol-triphosphate by activating 5-HT2 receptors and cAMP by activating 5-HT4 receptors [11,24–26]. Serotonin-induced suppression of ANP secretion was attenuated by neomycin, staurosporine and chelerythrine but not by U73122. [GenBank] The suppression of ANP secretion by serotonin appears to be partly mediated through PLC and PKC pathway. However, we failed to modify the accentuation of ANP secretion through 5-HT4 receptor with inhibitors for AC and PKA, such as MDL 12330, KT-5720 or H-89. We do not know the reason exactly. Because of differences in potency and specificity of inhibitors depending on tissues or species, further studies are needed to define their mechanism more clearly using specific inhibitors of PKA and PKC. Serotonin is known to increase the calcium current in human atrial myocytes by 5-HT4 receptor but not in rat, rabbit, guinea pig, or frog atrial myocytes [23]. The secretion of ANP is closely related to changes in [Ca2+]i even though controversy about their relationships still exists [27–29]. Neither receptor agonists caused any significant change in the value of [Ca2+]i. Therefore, changes in ANP secretion by 5-HT2 or 5-HT4 receptor agonist may be not closely related to changes in [Ca2+]i.

4.3 Experimental limitation
In this study, we used high concentration of serotonin to induce the suppression of ANP secretion by 5-HT2 receptor subtype. Serotonin plasma and serum levels of 0.3 and 15 µmol/l, respectively, have been reported in rats [11]. Thus, it is questionable that physiological humoral serotonin levels are sufficient to exert relevant effect on ANP secretion in vivo. However, since the local concentration of serotonin released from activated platelet may be much higher than expected, diverse effects of serotonin on the ANP secretion may partly contribute the regulation of blood pressure. Further in vivo studies allow us to prove the limitations.

4.4 Future directions
The regulation of cardiovascular function by serotonin is still controversial, because of the diverse distribution of 5-HT receptor subtypes in the cardiovascular system and their interrelationships. Recently, a compound acting on the 5-HT receptors was studied for use in the treatment of preeclampsia, hypertension, and some peripheral vascular diseases [30–32]. Our results show the possibility that the suppression of ANP secretion by 5-HT2 receptor as well as vasoconstriction [2] may relate to the development of hypertension and the accentuation of ANP secretion by 5-HT4 receptor may relate to the treatment of hypertension. The use of ketanserine and/or propranolol may allow the binding of circulating serotonin more to 5-HT4 receptor, which accentuates the ANP secretion followed by decreased blood pressure. More studies are needed to evaluate the beneficial effects of 5-HT4 receptor agonists on the treatment of hypertension. Additionally, the characteristics of serotonin binding sites as well as the signaling pathway involved in the different regulatory processes of ANP secretion by 5-HT receptor subtypes remain to be defined.

Time for primary review 35 days.


   Acknowledgements
 
This study was supported by a research grant from the Korea Science and Engineering Foundation (98-0403-10-01-5) and the Korea Research Foundation (2000-015-FP0023), Republic of Korea.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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