Arterial expression of 5-HT2B and 5-HT1B receptors during development of DOCA-salt hypertension
© Banes and Watts; licensee BioMed Central Ltd. 2003
Received: 25 June 2003
Accepted: 15 September 2003
Published: 15 September 2003
5-hydroxytryptamine (5-HT)2B and 5-HT1B receptors are upregulated in arteries from hypertensive DOCA-salt rats and directly by mineralocorticoids. We hypothesized that increased 5-HT2B and 5-HT1B receptor density and contractile function would precede increased blood pressure in DOCA-high salt rats. We performed DOCA-salt time course (days 1, 3, 5 and 7) studies using treatment groups of: DOCA-high salt, DOCA-low salt, Sham and Sham-high salt rats.
In isolated-tissue baths, DOCA-high salt aorta contracted to the 5-HT2B receptor agonist BW723C86 on day 1; Sham aorta did not contract. The 5-HT1B receptor agonist CP93129 had no effect in arteries from any group. On days 3, 5 and 7 CP93129 and BW723C86 contracted DOCA-high salt and Sham-high salt aorta; Sham and DOCA-low salt aorta did not respond. Western analysis of DOCA-high salt aortic homogenates revealed increased 5-HT2B receptor levels by day 3; 5-HT1B receptor density was unchanged. Aortic homogenates from the other groups showed unchanged 5-HT2B and 5-HT1B receptor levels.
These data suggest that functional changes of 5-HT2B but not 5-HT1B receptors may play a role in the development of DOCA-salt hypertension.
5-HT is an autocoid with a myriad of actions in the cardiovascular system. One of the important vascular effects of 5-HT is its ability to act as a vasoconstrictor. The 5-HT2A, 5-HT2B and 5-HT1B receptors have been implicated as mediators of 5-HT-induced contraction in vascular smooth muscle. 5-HT2A receptors mediate contraction in many arteries including the rat thoracic aorta  and pulmonary arteries . 5-HT2B receptors mediate 5-HT-induced contraction in the rat stomach fundus, the aorta and mesenteric arteries from hypertensive deoxycorticosterone acetate (DOCA)-salt rats [3–5]. The 5-HT2B receptor does not appear to mediate contraction in arteries taken from normotensive Sham rats [4, 5]. Involvement of 5-HT1B receptors in mediating 5-HT-induced arterial contraction in normal vessels has been described in the rat tail artery , human temporal artery , human umbilical artery , human pulmonary artery  and human coronary artery . Additionally, 5-HT1B receptors mediate 5-HT-induced contraction in mesenteric arteries from hypertensive DOCA-salt rats  and pulmonary arteries from rats with pulmonary hypertension [12, 2]. Furthermore, arteries from DOCA-salt hypertensive rats have approximately a 2-fold increase in the expression of the 5-HT2B and 5-HT1B receptor proteins . These findings suggest that the change in the function of the 5-HT2B and 5-HT1B receptors may be due to the alterations in the level of the receptor proteins.
Recent in vivo studies with the selective 5-HT2B receptor antagonist LY272015 have revealed that LY272015 lowered the blood pressure of DOCA-salt rats with established hypertension . These findings suggest that the 5-HT2B receptor is endogenously activated under conditions of hypertension to participate in the maintenance of the elevated blood pressure. No studies have examined whether 5-HT1B receptors are endogenously activated in established DOCA-salt hypertension. However, activation of 5-HT1B receptors have been implicated as a causative factor of pulmonary hypertension. [12, 14, 2]. Collectively, these studies suggest that 5-HT2B and 5-HT1B receptors can be endogenously activated and, by their ability to mediate 5-HT-induced vasoconstriction, may participate in the maintenance of the increased blood pressure.
We have recently found that aldosterone, in vitro, upregulates the 5-HT2B and 5-HT1B receptor proteins in endothelium-denuded aorta from rats with normal blood pressure . This upregulation was inhibited in the presence of the mineralocorticoid receptor antagonist spironolactone. Furthermore, studies in the Wistar-Furth rat model, a rat model which is relatively resistant to the hypertensive effects of DOCA and salt treatment , demonstrated that in blood pressure matched rats, the presence of elevated levels of DOCA and salt resulted in enhanced contraction to the 5-HT2B receptor agonist BW723C86 . These findings suggest that mineralocorticoids, such as DOCA, may be important independent regulators of these 5-HT receptors. A separation of the effects of DOCA, an increase in blood pressure, increased levels of salt and the combined effects of an increase in salt and DOCA on 5-HT receptor function and protein levels has never been performed. Separating the effects of these factors is necessary to understand their contribution to regulation of 5-HT2B and 5-HT1B receptors. Therefore, we proposed to determine when in the course of the DOCA-salt hypertension an increase in receptor density occurs and if the increase in the level of receptor proteins and functional response preceded the increase in blood pressure. We hypothesized that the increase in the 5-HT2B and 5-HT1B receptor density and functional responses would precede the increase in blood pressure observed in the DOCA-salt rats. We used the rodent selective 5-HT1B receptor agonist CP93129 and the 5-HT2B receptor agonist BW723C86 to test this hypothesis.
Contractile Studies on Day 1
Contractile Studies on Day 3
Contractile Studies on Day 5
Contractile Studies on Day 7
Protein Analysis Studies
Since the isolation of 5-HT, a role for it in systemic hypertension has been suggested. While the specific role which 5-HT plays in hypertension remains a controversial topic, there is no argument that 5-HT is both a vasoconstrictor and a mitogen. Currently, the mRNA for the 5-HT2A, 5-HT2B, 5-HT1B. 5-HT1D, 5-HT1F and 5-HT7 receptors have been localized to vascular smooth muscle cells. . However, only the 5-HT2A receptor has been shown to activate the mitogen-activated protein kinase (MAPK) pathway and participate in arterial contraction under conditions of normal blood pressure in the rat thoracic aorta. . Interestingly, the present studies suggest that 5-HT1B and 5-HT2B receptors are involved in mediating the arterial hyperresponsiveness to 5-HT observed in hypertension. This involvement appears to be due to changes independent of the level of receptor proteins as the functional response of contraction can be separated from an increase in the level of 5-HT2B and 5-HT1B receptor proteins.
Regulation of 5-HT Receptors
The studies presented here support the idea that mineralocorticoids combined with salt and increased blood pressure, but not mineralocorticoids alone in vivo, may be important mediators of the changes to both the level of 5-HT2B and 5-HT1B receptor protein levels and the signaling mechanisms utilized by these receptors. The aortic homogenates from DOCA-salt rats showed an increase in the level of the 5-HT2B receptor protein on days 3, 5 and 7. Contrary to in vitro findings in which aldosterone alone increased 5-HT2B receptor , the aortic homogenates from the DOCA-low salt rats showed no increase in the level of 5-HT2B receptor. These findings suggest that in vivo an increase in mineralocorticoids alone is not sufficient to upregulate the 5-HT2B receptor. Upregulation of the 5-HT2B receptor protein in vivo may require the elevated levels of salt, DOCA and pressure. On day one, arteries from the DOCA-salt rats with normal blood pressure contracted to BW723C86 but showed no increase in the level of the 5-HT2B receptor protein.
Surprisingly, the in vivo time course experiments did not show any increase in the level of the 5-HT1B receptor protein levels. These findings are also contrary to the in vitro data where aldosterone incubation produced an upregulation of these receptor protein levels . In contrast to the data obtained on days 1, 3, 5 and 7, after 28 days of DOCA-high salt hypertension the 5-HT1B and 5-HT2B receptors are upregulated approximately 2-fold in the aorta from DOCA-high salt hypertensive rats . These data suggest that in vivo regulation mechanisms of these receptors and their function maybe more complicated than the in vitro experiments suggested as elevated levels of mineralocorticoids alone in vivo was not sufficient to upregulate either the 5-HT1B or the 5-HT2B receptor proteins. However, as our studies did not address the issue of protein degradation, we can not rule out the possibility that there is an increase in the receptor proteins that is compensated for by an increase in protein degradation. Additionally, it is unknown at this point if the combination of elevated salt and mineralocorticoids affects the members of the signaling cascades used by the 5-HT1B and 5-HT2B receptors to enable enhanced contraction. This is an intriguing possibility since the ability of the 5-HT1B and 5-HT2B receptors to mediate contraction does not appear to be solely dependent on the level of the receptor protein.
The physiological importance of the 5-HT2B receptor is emphasized by the finding that administration of the 5-HT2B receptor antagonist LY272015, in vivo, decreased the blood pressure of hypertensive DOCA-salt rats and LNNA hypertensive rats [4, 16]. Currently, studies using chronic in vivo administration of LY272015 to determine whether 5-HT2B receptors are required for development of the increased blood pressure have not been done. Furthermore, the chronic administration of LY272015 would also address whether the 5-HT2B receptor was involved in the smooth muscle cell hypertrophy and hyperplasia observed in hypertension. In vivo studies with the 5-HT1B receptor antagonists in the model of DOCA-salt hypertension have not yet been performed. However, in the model of pulmonary hypertension, infusion of the 5-HT1B receptor antagonist GR127935 (3 mg/kg/day) attenuated the increased right ventricular pressure, right ventricular hypertrophy and pulmonary vascular remodeling in the hypertensive rats . These findings suggest that both the 5-HT2B and 5-HT1B receptors and their involvement in cardiovascular diseases, such as hypertension, merit further study.
Additional support for the importance of 5-HT2B receptors in the cardiovascular system comes from 5-HT2B receptor knockout mice. 5-HT2B receptor knockout mice show severe cardiovascular abnormalities [18–20]. Since 5-HT2B receptors are required for morphogenesis and cell migration, one can speculate that 5-HT2B receptors may be important to the vascular changes observed in arteries during the development of hypertension. No studies have been done in the 5-HT1B receptor knockout mice examining the progression of systemic cardiovascular diseases.
Speculations on Alternative Functional Changes
These studies were the first and an important step into understanding whether an upregulation of the 5-HT1B and 5-HT2B receptor proteins was sufficient to enable contraction to agonists of these receptors. While the lack of increase in the level of the 5-HT1B and 5-HT2B receptors to correspond with the functional changes may suggest nonspecific actions for CP93129 and BW723C86, there is evidence to refute this idea. BW723C86 posseses pKi values of 6.2 at the 5-HT1B receptor, 6.6 at the 5-HT2A receptor and 7.9 at the 5-HT2B receptor (21). There is currently no published information about the ability of CP93129 to interact with the 5-HT2B receptor, one possible nonspecificity. However, it is known that contractions mediated by CP93129 can be inhibited with the 5-HT1B receptor antagonist GR55562 (11). The ability of the 5-HT2B and 5-HT1B receptors to participate in contraction without a concomitant upregulation of the receptor protein levels suggest that changes in the signaling cascades and receptor/effector coupling are potentially involved in this enhanced contractility to 5-HT and agonists of the 5-HT2B and 5-HT1B receptors under conditions of DOCA-salt hypertension. Currently, the ability to track changes in 5-HT2A receptor is function is difficult due to limited tools available which includes a lack of 5-HT2A receptor specific agonists and antibodies.
The mechanisms by which elevated levels of salt and pressure participate in the pathogenic changes in receptor function observed in hypertension are still unclear and were not the focus of this study. They are, however, an area of future investigation. Future avenues of research also include investigation of the Rho-Rho-kinase pathway as well as the acute vs chronic effects of increased pressure on 5-HT receptor subtypes.
We initially speculated that upregulation of 5-HT1B and 5-HT2B receptor proteins were, at least partially, responsible for the increased contractility to 5-HT seen in the arteries from hypertensive rats. However, the results of the DOCA-salt time course studies do not support this idea. Instead, these data support the argument that an upregulation of 5-HT1B and 5-HT2B receptors are not necessary to observe an increased contractile response to agonists of these receptors. Additionally, there was an increased contractile response to the 5-HT2B receptor agonist BW723C86 prior to an increase in blood pressure in the DOCA-high salt and Sham-high salt treated rats. Interestingly, the hyperresponsiveness to 5-HT characteristic of hypertensive arteries was observed in Sham-high salt and DOCA-high salt arteries starting at day three. Collectively, the data presented herein suggest that 5-HT2B receptors may be involved in the development of DOCA-salt hypertension. However, the precise role of these receptors in the development of hypertension and possibly vascular remodeling are yet to be elucidated.
All procedures which involved animals were performed in accordance with the institutional guidelines of Michigan State University.
Surgical Procedures and Systolic Blood Pressure Measurement
All rats used were adult male Sprague-Dawley rats (0.20–0.25 kg; Charles River Laboratories, Inc., Portage, MI). The rats were separated into four treatment groups: DOCA-high salt, DOCA-low salt, Sham and Sham-high salt. The DOCA-high salt and DOCA-low salt were given a subcutaneous silastic implant impregnated with DOCA (200 mg/kg) and were uninephrectomized (left side, flank incision) under isoflurane (IsoFlo, Abbott Laboratories, N. Chicago, IL) anesthesia. Sham and Sham-high salt rats did not receive an implant but were uninephrectomized. Postoperatively, rats in the DOCA-high salt and Sham-high salt groups received drinking water containing 1.0% NaCl and 0.2% KCl. Sham and DOCA-low salt rats received normal tap water. Animals in the DOCA-high salt, Sham-high salt and Sham groups were fed a diet of standard rat chow and received ad libitum access to both food and water. Rats in the DOCA-low salt group were fed a diet of chow containing .002 μEq NaCl/g. A baseline blood pressure and on days 1, 3, 5 and 7 the systolic blood pressures were measured using the standard tail cuff method. Daily fluid intake was measured by giving a standard amount of fluid and using a graduated cylinder to ascertain the amount required to return to the initial amount.
Isolated tissue bath procedure
The thoracic aorta was removed, cleaned of debris and cut into helical strips. These strips were denuded of endothelial cells and placed in isolated tissues baths containing physiological salt solution consisting of (in mM) NaCl, 103; KCl, 4.7; KH2PO4, 1.18;MgSO4•7H2O, 1.17; CaCl2 •2H2O, 1.6; NaHCO3, 14.9; dextrose, 5.5; and CaNa2EDTA, 0.03. Tissues were mounted on stainless steel holders in tissue baths (50 ml) for isometric tension recordings and placed under optimum resting tension (1500 mg, determined previously). After a 1 hour equilibration, arteries were challenged with phenylephrine (10-5 M). Tissues were then washed and the lack of an intact endothelium was validated by observing the inability of acetylcholine (10-6 M) to relax arteries contracted with a half-maximal concentration of phenylephrine. Cumulative concentration response curves to agonists were performed. As there was no significant difference in the contractile response to PE (10-5 M) in any group at any day (data not shown) all the data is normalized and reported as a percentage of the PE (10-5 M) contraction.
The aorta was removed, cleaned, denuded of endothelial cells and cut into helical strips. The tissue was frozen in liquid nitrogen, pulverized in a liquid nitrogen-cooled mortar and pestle and solubilized in a lysis buffer [0.5 mol/L Tris HCl (pH 6.8), 10% SDS, 10% glycerol] with protease inhibitors (0.5 mM PMSF, 10 μg/ml aprotinin and 10 μg/ml leupeptin). Homogenates were centrifuged (11,000 g for 10 minutes, 4°C) and supernatant total protein measured (BCA, Sigma, St. Louis, MO).
Supernatant (50 μg total protein per lane, 4:1 in denaturing loading buffer, boiled 5 minutes) was loaded, separated on 10% denaturing SDS-polyacrylamide gels, and transferred to Immobilon-P membranes. Membranes were blocked for 3–4 hours in Tris buffer saline + Tween-20 (0.1%; TBS-T) containing 4% chick egg ovalbulmin and 0.025% sodium azide. Mouse anti-5HT2B receptor antibody (.5 μg/ml, Pharmingen, San Diego, CA) or guinea pig anti-5-HT1B receptor antibody (1:1000, Chemicon, Temecula, CA) were incubated with blots overnight (4°C). Following washes, secondary antibody linked to horseradish peroxidase [anti-mouse (1:10,000, Amersham Laboratories, Arlington Heights, IL) or anti-guinea pig (1:10,000 Chemicon, Temecula, CA)] was added for one hour and incubated with blots at 4°C. Enhanced chemiluminescence was performed using standard reagents (Amersham Laboratories, Arlington Heights, IL). Each blot was washed and redeveloped using an α-smooth muscle actin antibody (1:400, Oncogene Research Products, Boston, MA; anti-mouse secondary antibody, 1:5,000, Amersham Laboratories, Arlington Heights, IL). Equal lane loading of protein was ensured by comparing α-smooth muscle actin densitometry.
Data analysis and statistics
Data are presented as means ± standard error of the mean for the number of animals in parentheses. When comparing more than two groups, either a one-way ANOVA was performed followed by a Student-Newman-Keuls post hoc test or a two-way ANOVA was used, as was appropriate. The systolic blood pressure data was analyzed with repeated measures. In all cases, a p value less than or equal to 0.05 was considered statistically significant.
Acetylcholine chloride, phenylephrine hydrochloride, DOCA and 5-hydroxytryptamine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). BW723C86 was purchased from Tocris (Ballwin, MO). CP93129 was a gift from Pfizer (Groton, CT, USA).
This work was supported by the NIH grant HL58489 (SWW), an Atorvastatin Award (SWW) and a grant from the American Heart Association Award 0010194z (AKLB).
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