Although there was no increase in systolic blood pressure on day one, arteries from DOCA-high salt rats contracted to the 5-HT_2B receptor agonist BW723C86 (maximal contraction 52.5 ± 10.5 % of PE contraction). Arteries from DOCA-low salt, Sham-high salt and Sham rats did not respond to either BW723C86 or to the 5-HT_1B receptor agonist CP93129 (figures 3A and 3B). There was no enhanced contraction to 5-HT observed in any of the arteries from the treatment groups (figure 3C). These data suggest that while 5-HT_2B receptors are functional on day one, as indicated by contraction to BW723C86, this is not sufficient to result in an enhanced contraction to 5-HT. Additionally, the 5-HT_1B receptor agonist CP93129 was unable to stimulate contraction, suggesting that the 5-HT_1B receptor was not functionally coupled to contraction on day one.

On day three, BW723C86 contracted arteries from DOCA-high salt and Sham-high salt rats (figure 4A). There was also a significant contraction observed to CP93129 in arteries from DOCA-high salt rats (figure 4B). Arteries from DOCA-low salt and Sham rats did not contract to BW723C86 or CP93129 (figures 4A and 4B). Contraction to 5-HT was enhanced, as defined by an increased maximal contraction, decreased threshold for contraction and/or an increased potency, in arteries from Sham-high salt rats (figure 4C). The arteries from the DOCA-high salt rats showed no change in the maximal contraction to 5-HT but demonstrated enhanced 5-HT potency (-log EC₅₀ [M] values 6.02 ± 0.08, 5.74 ± 0.05 DOCA-high salt day 3 and Sham, respectively). Unexpectedly, arteries from DOCA-low salt rats showed an increased maximal response to 5-HT (maximal contraction 141.2 ± 11.8 % PE contraction). Since there was no response to CP93129 and very little response to BW723C86, this enhanced contraction to 5-HT was surprising. However, this enhancement of 5-HT-induced contraction in arteries from DOCA-low salt rats only occurred on day three. These findings suggest that the 5-HT_1B receptor requires the presence of elevated levels of DOCA, salt and pressure to become functionally coupled to contraction. 5-HT_2B receptors were able to mediate contraction in arteries from both DOCA-high salt and Sham-high salt rats, suggesting that the elevated levels of salt are required to observe functional changes in the 5-HT_2B receptor.

Experiments performed on day five of the time course demonstrated a similar profile of results. Arteries from DOCA-high salt and Sham-high salt rats contracted to both BW723C86 (maximal contraction 54.3 ± 11.9 % and 46.3 ± 6.6 % of PE contraction, DOCA-high salt and Sham-high salt, respectively) and CP93129 (maximal contraction 35.0 ± 8.6 % and 27.8 ± 7.4 % of PE contraction, DOCA-high salt and Sham-high salt, respectively) (figures 5A and 5B). Arteries from Sham-high salt rats also displayed an increased maximal contraction to 5-HT (maximal contraction 148.5 ± 11.4 % and 106.2 ± 8.7 % of PE contraction, Sham-high salt and Sham, respectively), a decrease in the threshold of activation of contraction and an increase in potency, and as compared to Sham arteries (-log EC₅₀ value [M] 6.06 ± 0.05 and 5.60 ± 0.07 Sham-high salt and Sham, respectively) (figure 5C). The arteries from DOCA-high salt rats also show a decrease in the threshold of activation of contraction (figure 5C). These results suggest that additional changes in the contractile mechanisms utilized by 5-HT, in addition to the 5-HT_1B and 5-HT_2B receptors being functionally coupled to contraction, are required in arteries from DOCA-high salt hypertensive rats to mediate hyperresponsiveness to 5-HT.

On day seven, arteries from DOCA-high salt rats contracted to both BW723C86 (maximal contraction 57.4 ± 5.9 % of PE contraction) and CP93129 (maximal contraction 63.8 ± 7.8 % of PE contraction) (figures 6A and 6B). Arteries from Sham-high salt rats also contracted to both BW723C86 (maximal contraction 24.5 ± 9.4 % of PE contraction) and CP93129 (maximal contraction 19.6 ± 8.1 % of PE contraction) (figures 6A and 6B). Interestingly, although the arteries from Sham-high salt rats were exposed to both increased levels of salt and pressure, these arteries did not contract to the same magnitude in the presence of BW723C86 (maximal contraction 24.5 ± 9.4 % and 57.4 ± 5.9 % of PE contraction, Sham-high salt and DOCA-high salt, respectively) and CP93129 (maximal contraction 19.6 ± 8.1% and 63.8 ± 7.8 % of PE contraction, Sham-high salt and DOCA-high salt, respectively) as those that are taken from DOCA-high salt rats on day seven. Additionally, arteries from DOCA-high salt and Sham-high salt rats both show hyperresponsiveness to 5-HT (figure 6C). These arteries demonstrate a characteristic increased maximal contraction (maximal contraction 130.3 ± 19.5 % and 119.5 ± 5.1 % of PE contraction, DOCA-high salt and Sham-high salt, respectively), the decreased threshold for contraction and the increased potency (- log EC₅₀ value [M] 6.41 ± 0.09, 6.13 ± 0.03 and 5.70 ± 0.07, DOCA-high salt, Sham-high salt and Sham, respectively). These data suggest that the combined presence of elevated levels of salt, DOCA and pressure all contribute to the functional changes in 5-HT_1B and 5-HT_2B receptors observed in the arteries from the DOCA-high salt hypertensive rats.

To determine if upregulation of 5-HT_1B and 5-HT_2B receptor proteins was responsible for the acquired contractility, we performed western analysis of total aortic homogenates. The authors acknowledge that the limitations of this technique do not speak to localization of the receptor proteins within the cell, only to the total amount of the receptor protein present. There was no increase in 5-HT_1B receptor protein levels in any of the treatment groups at any time point (figure 7), although there was a trend towards increasing levels of protein in the aortic homogenates from the DOCA-high salt rats. This was an unexpected finding as the arteries from both the DOCA-high salt and Sham-high salt rats contract to the 5-HT_1B agonist CP93129 starting on day three of the time course. These data suggest that although the receptor protein is upregulated by 28 days of DOCA-salt treatment (13), upregulation is not required to enable this receptor to participate in contraction.

In contrast, the 5-HT_2B receptor was upregulated significantly by day 3 of DOCA-high salt treatment (figure 8). However, 5-HT_2B receptor protein levels were not increased in any of the other treatment groups at any time point. Interestingly, the level of receptor protein was not significantly increased on day 1, even though there was a significant contraction to the 5-HT_2B receptor agonist BW723C86 on this day. There was also no significant increase in 5-HT_2B receptor levels in the aortic homogenates from the Sham-high salt rats in which contraction to BW723C86 occurred on days 3, 5 and 7. These findings suggest that upregulation of the 5-HT_2B receptor proteins is not absolutely required for this receptor to mediate agonist-induced contraction.

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-HT_2B and 5-HT_1B 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-HT_2B receptor protein on days 3, 5 and 7. Contrary to in vitro findings in which aldosterone alone increased 5-HT_2B receptor 13, the aortic homogenates from the DOCA-low salt rats showed no increase in the level of 5-HT_2B receptor. These findings suggest that in vivo an increase in mineralocorticoids alone is not sufficient to upregulate the 5-HT_2B receptor. Upregulation of the 5-HT_2B 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-HT_2B receptor protein.

Surprisingly, the in vivo time course experiments did not show any increase in the level of the 5-HT_1B receptor protein levels. These findings are also contrary to the in vitro data where aldosterone incubation produced an upregulation of these receptor protein levels 13. In contrast to the data obtained on days 1, 3, 5 and 7, after 28 days of DOCA-high salt hypertension the 5-HT_1B and 5-HT_2B receptors are upregulated approximately 2-fold in the aorta from DOCA-high salt hypertensive rats 13. 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-HT_1B or the 5-HT_2B 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-HT_1B and 5-HT_2B receptors to enable enhanced contraction. This is an intriguing possibility since the ability of the 5-HT_1B and 5-HT_2B receptors to mediate contraction does not appear to be solely dependent on the level of the receptor protein.

The physiological importance of the 5-HT_2B receptor is emphasized by the finding that administration of the 5-HT_2B receptor antagonist LY272015, in vivo, decreased the blood pressure of hypertensive DOCA-salt rats and LNNA hypertensive rats 416. Currently, studies using chronic in vivo administration of LY272015 to determine whether 5-HT_2B 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-HT_2B receptor was involved in the smooth muscle cell hypertrophy and hyperplasia observed in hypertension. In vivo studies with the 5-HT_1B 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-HT_1B receptor antagonist GR127935 (3 mg/kg/day) attenuated the increased right ventricular pressure, right ventricular hypertrophy and pulmonary vascular remodeling in the hypertensive rats 12. These findings suggest that both the 5-HT_2B and 5-HT_1B receptors and their involvement in cardiovascular diseases, such as hypertension, merit further study.

Additional support for the importance of 5-HT_2B receptors in the cardiovascular system comes from 5-HT_2B receptor knockout mice. 5-HT_2B receptor knockout mice show severe cardiovascular abnormalities 181920. Since 5-HT_2B receptors are required for morphogenesis and cell migration, one can speculate that 5-HT_2B receptors may be important to the vascular changes observed in arteries during the development of hypertension. No studies have been done in the 5-HT_1B receptor knockout mice examining the progression of systemic cardiovascular diseases.

These studies were the first and an important step into understanding whether an upregulation of the 5-HT_1B and 5-HT_2B receptor proteins was sufficient to enable contraction to agonists of these receptors. While the lack of increase in the level of the 5-HT_1B and 5-HT_2B receptors to correspond with the functional changes may suggest nonspecific actions for CP93129 and BW723C86, there is evidence to refute this idea. BW723C86 posseses pK_i values of 6.2 at the 5-HT_1B receptor, 6.6 at the 5-HT_2A receptor and 7.9 at the 5-HT_2B receptor (21). There is currently no published information about the ability of CP93129 to interact with the 5-HT_2B receptor, one possible nonspecificity. However, it is known that contractions mediated by CP93129 can be inhibited with the 5-HT_1B receptor antagonist GR55562 (11). The ability of the 5-HT_2B and 5-HT_1B 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-HT_2B and 5-HT_1B receptors under conditions of DOCA-salt hypertension. Currently, the ability to track changes in 5-HT_2A receptor is function is difficult due to limited tools available which includes a lack of 5-HT_2A 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.

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.

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.

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-5HT_2B receptor antibody (.5 μg/ml, Pharmingen, San Diego, CA) or guinea pig anti-5-HT_1B 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 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.