Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Abnormal vascular responsiveness to contractile agonists is often observed in forms of hypertension, including spontaneous, genetic, and experimental models. In particular, the augmented contractile response to serotonin (5-hydroxytryptamine, 5-HT) in isolated vascular preparations and whole animal studies can be profound (McGregor and Smirk, 1970; Hurley-Myers et al., 1985). In nondiseased conditions, 5-HT causes constriction in most vascular beds, although in coronary vascular tissues 5-HT reacts with endothelial cells to mediate relaxation of the smooth muscle cells (Martin, 1994; Glusa and Roos, 1996). 5-HT is produced in the enterochromaffin cells of the gut and released into the circulation where the majority of the monoamine is enzymatically degraded by the liver in the portal circulation (Vanhoutte, 1991). The small amount of 5-HT remaining is broken down by oxidases in the pulmonary endothelial cells or taken up by platelets. Ultimately, circulating levels of free 5-HT are low (12-150 nM) unless vascular damage or disease is present. If platelets are activated, this can result in a localized release of 5-HT from the dense granules. 5-HT can then interact with endothelial and smooth muscle cells in the vasculature. Considering the damage to the endothelium in cardiovascular disease, 5-HT may have direct access to receptors on smooth muscle cells.

Researchers have examined the role of 5-HT in the maintenance of hypertension or elevated total peripheral resistance because of the significant increase in arterial responsiveness to 5-HT (Thompson and Webb, 1987; Roson et al., 1990). Several mechanisms may contribute to this increase, including an increase in receptor number, a change in receptor subtype, more effective G protein-coupling mechanisms, or increased circulating levels of 5-HT. Our interpretation of past data and findings in a mineralocorticoid-based hypertension supports the notion of a switch in the predominant 5-HT receptor subtype mediating contraction.

Of the 5-HT₂ receptor family of proteins [5-HT_2A, 5-HT_2B (originally known as the 5-HT_2F receptor and cloned in the rat stomach fundus; Foguet et al., 1992; Kursar et al., 1992), and 5-HT_2C receptors], only the 5-HT_2A and 5-HT_2B receptors are found in the peripheral vasculature (Hoyer et al., 1994; Martin, 1994). Vascular contraction to 5-HT is primarily mediated by the 5-HT_2A receptor in the normotensive rat. Research in our laboratory and many others demonstrates that multiple different arteries from deoxycorticosterone acetate (DOCA) hypertensive rats have a significantly enhanced contraction to 5-HT (Watts et al., 1995, 1996; Watts, 1997; Watts and Harris, 1999). This includes the carotid artery, superior mesenteric artery, tail artery, femoral artery, and aorta. In the superior mesenteric artery and aorta, blockade of 5-HT-induced arterial contraction by 5-HT_2A receptor antagonists such as ketanserin or ritanserin was reduced in the DOCA hypertensive rats compared with normotensive rats. Moreover, arterial contraction to agonists of the 5-HT_2B receptor BW723C86, in particular, was markedly enhanced (Watts and Fink, 1999). These data support an up-regulation of the 5-HT_2B receptor in the arteries of the DOCA-salt rat. Recently, we have found in Western analyses that the 5-HT_2B receptor protein is in fact significantly up-regulated (~250% sham) (Banes and Watts, 2002).

Physiologically, this up-regulation is important because 5-HT has a 300-fold greater affinity for 5-HT_2B receptor, compared with the 5-HT_2A receptor (K_i = 3162.3 nM for 5-HT_2A; K_i = 10.2 nM for 5-HT_2B) (Wainscott et al., 1993). Moreover, the 5-HT_2B receptor antagonist LY272015 (Audia et al., 1996; Cohen et al., 1996) reduced blood pressure in severely hypertensive DOCA-salt rats (Watts and Fink, 1999), suggesting that the 5-HT_2B receptor is activated endogenously to maintain the hypertension. Recent evidence suggests that the promoter region of the 5-HT_2B receptor contains mineralocorticoid response elements, and mineralocorticoids can up-regulate the 5-HT_2B receptor directly (Banes and Watts, 2002). Thus, it is of concern whether the up-regulation of the 5-HT_2B receptor is specific to a mineralocorticoid-based form of hypertension.

The current studies use a hypertensive model based on inhibition of the enzyme nitric-oxide synthase. There is little evidence, to our knowledge, that this hypertension is based on elevated mineralocorticoid levels, and thus we use this model to contrast with the DOCA-salt rat. We hypothesize that the functional and molecular up-regulation of the 5-HT_2B receptor will also be observed in arteries from hypertensive LNNA rats. With the research presented herein, we hope to depict the N-nitro-L-arginine (L-NNA) model as an additional representation of experimental hypertension that supports the hypothesis that 5-HT_2B receptor up-regulation may be common and important to hypertension.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

LNNA Hypertension. Fifty-four male Sprague-Dawley rats (0.25 kg; Harlan, Indianapolis, IN) were divided into two groups, LNNA and control. Each day, two age-controlled rats began treatment in isolated stainless steel cages. The control rat received normal tap water, whereas the LNNA rat drank tap water mixed with L-NNA (0.5 g/l). Water intake was measured daily and both groups were fed normal rat chow ad libitum. On exactly day 14 of treatment, rats were weighed and systolic blood pressure was measured using a standard tail cuff method. Thereafter, rats were anesthetized with pentobarbital sodium (50 mg/kg) and sacrificed.

Isolated Tissue Bath Protocol. Thoracic aorta or stomach fundus was removed and placed in normal physiological saline solution containing 130 mmol/l NaCl, 4.7 mmol/l KCl, 1.18 mmol/l KH₂PO₄, 1.17 mmol/l MgSO₄ · 7H₂O, 1.6 mmol/l CaCl₂ · 2H₂O, 14.9 mmol/l NaHCO₃, 5.5 mmol/l dextrose, and 0.03 mmol/l CaNa₂ EDTA, pH 7.2. Vessels were trimmed of fat, cut into helical strips (1 × 10 mm in width), and, in most of the experiments, denuded of endothelium by gently rubbing a cotton swab along the lumen of the strip. An aortic strip from an LNNA hypertensive and a sham normotensive rat was mounted in the same tissue bath. Two longitudinal strips were made from each fundus. Tissues were attached to a fixed stainless steel rod at one end and to a force transducer at the other. Baths were filled with physiological saline solution, warmed to 37°C, and aerated with 95% oxygen and 5% carbon dioxide. Each strip was placed under optimum resting tension (previously determined 1500 mg for the rat aorta and 4000 mg for stomach fundus) and allowed to equilibrate for 1 h with frequent buffer changes. Tissues were then challenged with a maximal concentration of the ₁ adrenergic agonist phenylephrine (PE, for aorta, 10⁵ M) or KCl (for fundus, 67 mM) to initiate a maximal contraction, and washed repeatedly until tone returned to baseline. To examine the status of the arterial endothelium, tissues were contracted with a half-maximal concentration of PE (10⁸ M), and once the contraction plateaued, the muscarinic agonist acetylcholine (ACh, 10⁶ M) was administered. We observed less than a 5% relaxation to this concentration of ACh in rubbed tissues and thus included all the tissues in our analysis. Tissues were again washed until baseline was reached and then one of the following protocols was followed.

Agonists. Depolarizing KCl and two specific agonists of 5-HT receptors were used. First, in tissues from both LNNA hypertensive and sham normotensive rats, cumulative concentration-response curves were constructed for KCl (10-100 mM) and 5-HT (10⁹-10⁴ M). The 5-HT_2B agonist BW723C86 (10⁹-10⁵ M) was also examined. Each strip was exposed to two agonists, and the order in which the strips received these drugs was randomized.

Serotonergic Antagonists. One concentration of ketanserin (5-HT_2A/2C receptor-selective antagonist; 10 nM), LY272015 (5-HT_2B receptor-selective antagonist; 10 nM), or vehicle (0.1% dimethyl sulfoxide) was added. After 1 h of equilibration, a 5-HT concentration-response curve (10⁹-10⁴ M) was constructed in each bath.

6

2

6

Western Analyses. Aortae were dissected as described above and cleaned and denuded of endothelium before placing tissue in a liquid-nitrogen cooled mortar and pestle. In liquid nitrogen, tissues were ground to a powder and 1 ml of ice-cold homogenization buffer was added (125 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The homogenate was vortexed, sonicated briefly, and transferred to a plastic centrifuge tube and spun at 4°C to pellet debris. The supernatant was removed and total protein measured using the bicinchoninic acid method (Sigma-Aldrich, St. Louis, MO). Fifty micrograms of total protein from aortic homogenates (4:1 in denaturing sample buffer, boiled for 5 min) was separated on 10% SDS-polyacrylamide gels and transferred to Immobilon P membrane. Membranes were blocked for 3 h [Tris-buffered saline (TBS), 4% chick egg ovalbumin, and 2.5% sodium azide] and probed overnight with primary antibody (rocking at 4°C; 5-HT_2B receptor at 0.5 µg/ml; BD PharMingen, San Diego, CA). Blots were rinsed in TBS + Tween (0.1%) with a final rinse in TBS and incubated with the appropriate secondary antibody for 1 h at 4°C (rocking). Blots incubated with enhanced chemiluminescence reagents to visualize bands. All blots were reprobed with an antibody for smooth muscle cell -actin (Oncogene Research Products, Boston, MA) to ensure equal protein loading, and gels were also stained with Gel Code Blue (Pierce Chemical, Rockford, IL) to compare protein loading from lane to lane.

In Vivo Experiments. This surgery took place 8 days after LNNA treatment was begun. Catheters were constructed of polyvinyl chloride with silicone rubber tips and advanced to the abdominal aorta and vena cava via the left femoral artery and vein in rats anesthetized with pentobarbital (50 mg/kg i.p.). The ends of the catheters were tunneled subcutaneously to the head where the catheters were stabilized to the skull using jeweler's screws and dental acrylic. Catheter ends were passed through a stainless steel spring attached to a plastic swivel, through which infusions were given. Upon regaining consciousness, rats were housed singly in stainless steel cages in a climate-controlled room with a 12-h light/dark cycle.

Statistics. Contractile data are expressed as ± S.E.M. and reported as a percentage of the maximal contraction to phenylephrine (10⁵ M) or KCl (67 mM). Unpaired t tests were performed and a p value 0.05 was considered statistically significant. Agonist EC₅₀ values were calculated using a nonlinear regression analysis using the algorithm [effect = maximum response/1 + (EC₅₀/agonist concentration)] in the program GraphPad Prism (GraphPad Software, San Diego, CA). Apparent antagonist dissociation constants (K_B values) were calculating using the following equation: log (dr  1) = log [B]  log K_B, where dr is the EC₅₀ value of agonist in the presence of the antagonist divided by the EC₅₀ value of agonist in the absence of the antagonist, and [B] is the concentration of the antagonist tested. Quantitation of band density was performed on a G4 Macintosh computer using the public domain program NIH Image (version 1.62; written by Wayne Rasband at the National Institutes of Health and available from the Internet by anonymous ftp from zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA 22161, part number PB93-504868). Band density is reported in arbitrary densitometry units. Blood pressure is reported as a change in mean arterial blood pressure, and a repeated measures analysis of variance was used to ascertain statistical differences between groups.

Chemicals. All chemicals were made fresh on the day of use. Acetylcholine chloride, BW723C86, deoxycorticosterone acetate, 5-hydroxytryptamine hydrochloride, ketanserin tartrate, and phenylephrine hydrochloride were purchased from Sigma-Aldrich. PGF₂ was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA), and LY272015 was a generous gift from Eli Lilly & Co. (Indianapolis, IN).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Whole Animal Parameters. Figure 1 depicts whole animal parameters for the 22 pairs of sham and LNNA rats that were used in in vitro experiments. Not all parameters were measured in all 22 pairs. The rats receiving LNNA drank statistically similar amounts of fluid compared with sham rats per day, thus ensuring that the rats in fact received drug. The LNNA rats were significantly lighter (Fig. 1, middle) and possessed higher systolic blood pressures than sham rats (Fig. 1, bottom).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Average fluid consumption (top), body weight (middle), and systolic blood pressure (bottom) of sham and LNNA hypertensive rats. Data are reported as means ± S.E.M. Fluid for the sham rats was tap water and, for the LNNA rats, water containing LNNA (0.5 g/l). , statistically significant differences (p < 0.05) from sham rats.

2

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effect of 5-HT on PGF2-induced contraction in endothelium-intact rat aorta. Tissues were incubated with the 5-HT2A receptor antagonist to block the contractile 5-HT2A receptors. Points represent means ± S.E.M. for the number of rats in parentheses.

Response to Agonists. Aortic strips isolated from sham normotensive and LNNA hypertensive rats were pair mounted in isolated tissue baths and exposed to KCl (Fig. 3, top) or 5-HT (Fig. 3, middle). KCl-induced contraction was not altered in strips from LNNA hypertensive rats, but 5-HT-induced contraction was significantly enhanced in the lower concentrations of 5-HT; EC₅₀ values were not different (log EC₅₀ [M] sham = 5.99 ± 0.03; LNNA = 6.12 ± 0.05). Significantly enhanced contraction to the selective 5-HT_2B receptor agonist BW723C86 (Fig. 3, bottom) was also observed. Contraction to BW723C86 was virtually absent in tissues from sham animals (percentage of maximum PE contraction = 7 ± 4; no calculable EC₅₀ value) but was robust and concentration-dependent in arteries from LNNA hypertensive rats (percentage of maximum PE contraction = 61 ± 7, log EC₅₀ [M] = 6.70 ± 0.01). These data suggests that the function of the 5-HT_2B receptor is up-regulated under conditions of LNNA hypertension.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of KCl (top), 5-HT (middle), and the 5-HT2B receptor agonist BW723C86 (bottom) in aorta from sham and LNNA hypertensive rats. Data are reported as means ± S.E.M. and expressed as percentages of initial PE contraction. , statistically significant differences (p < 0.05) from sham rats.

Response to 5-HT in Presence of 5-HT Receptor Antagonists. The 5-HT_2B receptor antagonist LY272015 did not alter 5-HT-induced contraction in aorta from sham rats but reduced and shifted contraction to 5-HT 4-fold rightward in aorta from LNNA hypertensive rats (Fig. 4). In contrast, the 5-HT_2A receptor antagonist ketanserin caused a parallel and competitive shift (pK_B = 9.04 ± 0.05) in 5-HT-induced contraction in aorta from sham rats (Fig. 5, top). Ketanserin was unable to cause a clean parallel shift in 5-HT-induced contraction in aorta from LNNA hypertensive rats, and this is largely because of the relative insensitivity to ketanserin at low concentrations of 5-HT (10⁹-3 × 10⁷ M; Fig. 5, middle). A ketanserin-insensitive contraction to 5-HT is present in aorta from LNNA and not sham rats because when the response of the two groups of animals in the presence of ketanserin is compared, the curves remain significantly different (Fig. 5, bottom).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of 5-HT2B receptor antagonist LY272015 on aorta from sham (top) and LNNA (bottom) hypertensive rats. Points represent means ± S.E.M. , statistically significant differences (p < 0.05) from sham rats.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of 5-HT2A receptor antagonist ketanserin on aorta from sham (top) and LNNA hypertensive (middle) rats; comparison of ketanserin (10 nM)-resistant 5-HT-induced contraction in aorta of sham and LNNA hypertensive rats (bottom). Points represent means ± S.E.M. , statistically significant differences (p < 0.05) from sham rats.

Western Analysis. We next performed immunoblot analysis on homogenates from aorta of sham and LNNA hypertensive rats to ascertain whether 5-HT_2B receptor protein density was different. Equivalent amounts of total protein were immunoblotted with a 5-HT_2B receptor antibody. This antibody recognized a band at 55 and 110 kDa (Fig. 6, top), both of which are not observed if the primary antibody was removed from the experiment. When quantified by densitometry, the 5-HT_2B receptor immunoreactive bands were over 300% that found in the sham (Fig. 6, bottom). These findings support a molecular up-regulation of the 5-HT_2B receptor protein in LNNA hypertension.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Top, representative blot of the 5-HT2B receptor protein in aortic homogenates from sham and LNNA hypertensive rats. Blots were reprobed with an antibody directed against smooth muscle cell specific -actin to ensure equivalent total protein loading. Bottom, arbitrary densitometry units of the 5-HT2B receptor in aortic homogenates for the 55-kDa band. Bars represent means ± S.E.M. for the number of animals in parentheses. , statistically significant differences (p < 0.05) from sham rats.

Effect of in Vivo Administration of LY272015 to Sham and LNNA Rats. LY272015 was administered in increasing doses throughout a period of 90 min on day 14 of treatment, and changes in blood pressure followed. Sham rats had a resting mean arterial pressure of 123 ± 3 mm Hg and LNNA rats of 171 ± 9 mm Hg. In sham rats, LY272015 did not reduce blood pressure and, in fact, increased mean arterial blood pressure significantly at 3.0 mg/kg. In contrast, LNNA rats responded with a decrease in blood pressure. The maximum reduction produced by LY272015 was, on average, approximately 25 mm Hg (Fig. 7, bottom). Resting heart rates were not different in sham (354 ± 6 beats per minute) and LNNA rats (362 ± 9 bpm), and only the 3.0-mg/kg dose of LY272015 reduced heart significantly (sham = 29 ± 9 bpm; LNNA = 40 ± 7 bpm). We validated the ability of LY272015 to act as a 5-HT_2B receptor antagonist by demonstrating inhibition of 5-HT-induced contraction in the rat stomach fundus, the tissue in which the 5-HT_2B receptor was originally cloned (Fig. 7, top).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Top, representation of the effect of LY272015 (10 nM) on 5-HT-induced contraction in rat stomach fundus. Bottom, effect of in vivo administration of LY272015 (0.3-3 mg/kg) on mean arterial blood pressure where data are reported as a change in blood pressure calculated before and after LY272015 administration. Columns represent means ± S.E.M. for number of animals in parentheses. , statistical difference from baseline blood pressure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

These studies were originally undertaken to test the hypothesis that, as in the DOCA-salt model of hypertension, arterial 5-HT_2B receptors are up-regulated biochemically and functionally with a resultant hypersensitivity to 5-HT in LNNA-induced hypertension. The LNNA model is different from the DOCA-salt model in several ways. First, it may not to be a mineralocorticoid-driven model. Simmons and Freeman (1995) demonstrated that L-arginine analogs, including LNNA, reduced aldosterone secretion in rats with a likely result of reduced circulating aldosterone. However, we have been unable to find a study that tests the ability of a mineralocorticoid receptor antagonist to reduce LNNA-induced hypertension, and thus the pure independence of the LNNA model from mineralocorticoids is still not proven. Second, the LNNA model is not salt-dependent in that excess salt is not necessary to produce the hypertension. Third, the duration of treatment was 2 weeks for the LNNA model, whereas 4 weeks is the typical time to expose animals to DOCA. Fourth, the source of Sprague-Dawley rats used to generate each of these models is different. We used Sprague-Dawley rats from Charles River Laboratories, Inc. (Wilmington, MA) to generate DOCA-salt rats, whereas Sprague-Dawley rats from Harlan were used to generate LNNA rats. This may seem like a minor distinction, but we and others have found that rats obtained from Charles River Laboratories are relatively resistant to the hypertensive effects of the nitric-oxide synthase inhibition (S. W. Watts and A. Hongsakaphadana, unpublished observation; Pollock et al., 1998). These four differences are important to note as is the fact that even though these two models are different in mechanism, the magnitude of systolic blood pressures achieved with each intervention is similar (180-200 mm Hg systolic blood pressure). This specific similarity allows the testing of the hypothesis that up-regulation of the 5-HT_2B receptor may be general to hypertension.

Pharmacology of Contraction. Pharmacological experiments suggest an up-regulation of the 5-HT_2B receptor in aorta from LNNA hypertensive rats. First, the overall sensitivity of aorta to 5-HT from LNNA hypertensive rats is increased compared with that in the sham. This is reflected primarily as a decrease in the threshold concentration of 5-HT necessary to cause contraction. Most importantly, contraction to an agonist specific for the 5-HT_2B receptor, BW723C86, was significantly enhanced in aorta from LNNA hypertensive rats. Previously, we demonstrated that contraction to this agonist could be blocked by the 5-HT_2B receptor antagonist LY272015 but not the 5-HT_2A/2C receptor antagonist ketanserin (Watts and Fink, 1999). Unlike the jugular vein (Ellis et al., 1995; Glusa and Roos, 1996), the 5-HT_2B receptor does not seem to mediate endothelium-dependent relaxation in the aorta.

Biochemistry of Receptor Expression. One mechanism by which this can occur is through an increased 5-HT_2B receptor density. Because 5-HT has a higher affinity for the 5-HT_2B receptor compared with the 5-HT_2A receptor, one could envision that endogenous 5-HT would combine first with 5-HT_2B receptors; an increase in the density of these receptors increases the probability of activation. Thus, we performed Western analysis using aortic homogenates from sham and LNNA hypertensive rats. These experiments clearly demonstrated the presence of 5-HT_2B receptor in homogenates from both sham and LNNA aorta. In the same amount of total protein, aortic homogenates from the LNNA rats displayed approximately 3- to 4-fold higher amount of 5-HT_2B receptor. Thus, these findings are certainly consistent with our hypothesis. However, two points need to be made. First, the 5-HT_2B receptor is present in the sham homogenates. This receptor, from the standpoint of contractility, is virtually inactive because the pharmacology of the 5-HT receptor mediating contraction in aorta from normotensive rats is consistent with that of a 5-HT_2A receptor only. Why this receptor is present but not functional raises the possibility that the receptor may serve functions other than contractility such as growth. First, 5-HT is an arterial mitogen (Kavanaugh et al., 1988) and the 5-HT_2B receptor has been implicated in mitogenic pathways (Loric et al., 1995; Launay et al., 1996). Second, we have been unable to follow changes in 5-HT_2A receptor function and protein to determine whether these receptors, which are normally predominant, lose function or are down-regulated. Agonists that are selective for the 5-HT_2A receptor compared with the 5-HT_2B receptor are unavailable, and at this time antibodies to measure 5-HT_2A receptor immunohistochemistry or expression in Western analyses are unproven. Thus, it is possible that a change in the ratio of these two proteins rather than just a change in the 5-HT_2B receptor is the determining factor for the observed changes in contractility.

In Vivo Response to 5-HT_2B Receptor Blockade. Up-regulated 5-HT_2B receptors in LNNA rats has apparent physiological significance. LY272015, an antagonist of the 5-HT_2B receptor, was able to reduce but not normalize blood pressure in chronically instrumented LNNA hypertensive rats. This presented a different profile from the response of sham rats; these rats responded to LY272015 with an increase in blood pressure. A possible interpretation of this finding is that LY272015 is blocking vasorelaxant 5-HT_2B receptors. Such receptors would have to be in sites other than the aorta because we were unable to find evidence for 5-HT-mediated vasorelaxation in this vessel. In any case, the fall in blood pressure elicited by LY272015 in the LNNA rats suggests that the 5-HT_2B receptor is activated endogenously to, at least in part, maintain the elevated blood pressure observed in these rats. An acknowledged limitation of the current studies is that we have demonstrated the pharmacological and biochemical up-regulation of the 5-HT_2B receptor in the aorta, a conduit artery, as opposed to those vessels responsible for control of total peripheral resistance, the resistance arteries. For LY272015 to exert its antihypertensive effects, it is likely but not proven that the 5-HT_2B receptor is up-regulated functionally in small arteries. Alternatively, we have not discounted the possibility that LY272015 may act centrally to reduce blood pressure.

Speculation. Up-regulated and functional 5-HT_2B receptors have now been observed in two different models of hypertension. This begs the question as to whether up-regulation of the 5-HT_2B receptor results from the hypertension. In the DOCA-salt model, we previously performed time-course studies to determine when a pharmacological profile consistent with an up-regulation of the 5-HT_2B receptor (increased sensitivity to 5-HT, decreased threshold to 5-HT, increase in response to BW723C86, decreased ability of ketanserin to block 5-HT-induced contraction) occurred in relation to the rise in blood pressure. We have not yet done the same in the LNNA model, but in the DOCA-salt time-course experiments it is clear that the change in responses occur in parallel with or just slightly after a rise in blood pressure (Watts, 1998). Thus, it is unlikely that the up-regulated 5-HT_2B receptor is responsible for the rise in blood pressure. This is further substantiated by the inability of LY272015 to decrease blood pressure during the first 2 weeks of DOCA-salt treatment, a time when blood pressure is elevated (Watts and Fink, 1999). These findings are relevant to the human condition as the 5-HT_2B receptor has been cloned and localized in the arteries (Schmuck et al., 1994; Wainscott et al., 1996).