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CARDIOVASCULAR
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan
Received for publication
January 4, 2008
Accepted
February 26, 2008.
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Abstract |
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-nitro-L-arginine (0.5 g/l in water) prevented the fall in blood pressure to 5-HT. We conclude that chronic exogenous 5-HT reduces blood pressure in normotensive and hypertensive rats through mechanisms critically dependent on NOS.
) and by Irving Page (Rapport et al., 1948; Page and McCubbin, 1953a,b). In the periphery, 5-HT is made primarily in the enterochromaffin cells of the intestine. The circulatory system is exposed to 5-HT through aggregation of platelets (which take up and store a millimolar concentration of 5-HT), release from adrenergic nerves that have taken up 5-HT, and through direct exposure to 5-HT that is free in the blood. In many tissues, 5-HT is taken up and concentrated by the serotonin transporter (SERT) and is rapidly metabolized to an inactive metabolite, 5-hydroxyindole acetic acid (5-HIAA), by intracellular monoamine oxidase.
5-HT was originally described as a substance derived from serum (sero) that increased the tone of smooth muscle (tonin). Because of the close association of the platelet with the blood vessel, there has been a long-standing question as to the role of 5-HT in controlling vascular tone and modifying blood pressure under normotensive and hypertensive conditions. Several findings suggest that 5-HT contributes to systemic hypertension. These include the following findings: 1) plasma levels (free) of 5-HT are elevated in experimental and human models of hypertension (Fetkovska et al., 1990; Carrasco et al., 1998; Brenner et al., 2007); 2) SERT inhibitors such as fluoxetine cause an increase in blood pressure (Amsterdam et al., 1999; Lazartigues et al., 2000); 3) the ability of arteries to take up 5-HT through SERT is impaired in hypertension (Ni et al., 2006); and 4) arteries from hypertensive humans and experimental models are hyper-responsive to 5-HT (Watts and Fink, 1999; Russell et al., 2002; Watts, 2005). Thus, these findings suggest that the elevated plasma 5-HT observed in hypertensive subjects is at least associated with the disease and possibly a cause. In contrast, there have been equivocal studies as to the involvement of 5-HT in control of normal or elevated blood pressure (for review, see Watts, 2005).
Therefore, we undertook the study of mimicking the hypertensive condition by examining how an elevation in free systemic 5-HT would change blood pressure. We hypothesized that infusing 5-HT to chronically elevate free plasma 5-HT would increase blood pressure of normotensive rats and elevate blood pressure to an even greater extent in hypertensive rats. This hypothesis was largely guided by the substantial amount of literature demonstrating that systemic arteries are hyper-responsive to 5-HT. Hyper-responsiveness to an agonist can refer to a lower threshold (lower than normal concentration that initially elicits a contraction), a higher potency of the agonist, and/or a greater maximal contraction (higher efficacy). All of these changes have been observed in arterial response to 5-HT in hypertension.
A recent study by Gustaffson et al. (2005) presented the first model of chronic administration of 5-HT, supporting that this was a feasible undertaking. Rats were given 5-HT s.c. once daily for 3 months with 5-HT, which resulted in a 16-fold increase in free 5-HT. Heart valve function was impaired, but blood pressure was not measured. We used a different delivery of 5-HT (miniosmotic pump) but achieved similar elevations of free 5-HT in plasma. Our study was shorter than the 3 months originally planned because of the surprising results found within the 1st week. Contrary to our hypothesis, we show here for the first time that elevation in free plasma 5-HT resulted in a decrease in blood pressure of normotensive rats and the deoxycorticosterone acetate (DOCA)-salt hypertensive rat. The present studies support the interaction of 5-HT with NOS in causing this decrease in blood pressure, potentially at the level of the artery and/or the sympathetic nervous system.
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Materials and Methods |
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LNNA Model. After telemetry placement, male Sprague-Dawley rats (Harlan, Indianapolis, IN) were given 0.5 g/l LNNA, an inhibitor of NOS, in their drinking water for the duration of the experiment. Miniosmotic pumps were implanted 10 days after beginning administration of LNNA.
Telemetry and Pump Implantation. Under isoflurane anesthesia, radiotelemeter devices (Data Sciences International, St. Paul, MN) with attached catheters with pressure-sensing tips were implanted s.c. through a 1- to 1.5-cm incision in the left inguinal area. Catheters were introduced into the left femoral artery 3 to 5 mm distal to the level of the peritoneal wall, and the tip was advanced to the abdominal aorta. Rats were allowed 3 to 4 days to recover postoperatively, and then 3 to 4 days of baseline measurements were made. Mean arterial pressure, pulse pressure, and heart rate were recorded throughout the duration of the study. Seven to 10 days after radiotelemeter placement, osmotic pumps with a release rate of 10.0 µl/h and duration of 7 days (model 2ML1; Alzet, Cupertino, CA) were implanted s.c. between the scapulae. Two groups were used: 1) a group receiving vehicle or 2) a group receiving 5-HT (25 µg serotonin creatinine sulfate/kg/min s.c.). Vehicle consisted of 1% ascorbate (antioxidant) in sterile saline, pH balanced to between 6 and 7. In some experiments, hexamethonium (30 mg/kg i.p.) was given on day 4 after pump implantation. On day 7 postpump implantation, rats were sacrificed, and whole blood was drawn via cardiac puncture. Blood pressure data are reported only for those sham and DOCA-salt groups that were studied in parallel. Aortae were harvested for isometric contraction or Western blot analysis. Remaining volume of all pumps was measured to validate the following: 1) pumps had functioned, and 2) groups had similar volumes released during experimentation. This was not different between groups and thus is not reported.
Plasma 5-HT Measurements. Five milliliters of blood was collected from left cardiac ventricle and transferred into an EDTA anticoagulant Vacutainer tube. Pargyline and ascorbic acid (10 µM each) were added. Tubes were centrifuged at 160g (1000 rpm) for 30 min at 4°C for platelet-rich-plasma (PRP). Two milliliters of supernatant containing plasma and buffy coat layer were gently pipetted into EDTA-coated plastic tubes and mixed with a 1:1 dilution of 0.5 M EDTA. Pargyline and ascorbic acid (10 µM each) were added. Tubes were centrifuged for 20 min at 1350g at 4°C for platelet-poor-plasma (PPP). To the remaining pellet (platelet layer), 1 ml of platelet buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, and 10 mM D-glucose) and 1 µM ADP were added. Pargyline and ascorbate were added. Tubes were vortexed and allowed to sit on ice for 15 min for platelets to become activated and degranulate. Tubes were centrifuged at 730g for 10 min at 4°C. Trichloroacetic acid (10%) was added to deproteinate samples, and samples sat on ice for 10 min. Tubes were centrifuged at 4500g for 20 min at 4°C. Samples were ultracentrifuged at 280,000g for 2 h. 5-HT and 5-HIAA concentrations were measured using electrochemical detection high-performance liquid chromatography (HPLC/ESA Coulchem system) at 0.4 V and 0.9 ml/min flow rate. 5-HT and 5-HIAA standards were run daily, before experimental samples, to create a standard curve against which sample concentration could be calculated.
Isometric Contraction. Helical strips of endothelium-intact thoracic aortae were isolated for measurement of isometric contractile force as described previously (Ni et al., 2004). In brief, aortic strips were mounted into 50-ml tissue baths on Grass isometric transducers (FT03; Grass Instruments, Quincy, MA), placed under optimum passive force (1500 mg, determined previously), and allowed to equilibrate for 1 h before a challenge with a maximal concentration of phenylephrine (10 µM). After this initial challenge, tissues were washed until tone returned to baseline. Concentration response curves to acetylcholine, phenylephrine, and serotonin (10-9 to 3 x 10-5 M) were generated in all tissues because these agonists wash out readily from exposed tissues. When acetylcholine was investigated, tissues were first contracted with a half-maximal concentration of phenylephrine, and then acetylcholine (ACh) was added. Data were captured on a PowerLab (ADInstruments, Colorado Springs, CO) connected to an iMac computer using the program Chart.
Western Blot Analysis. Homogenates of aorta were taken through standard Western blot analyses and transferred to Immobilon F for visualization on the LiCor Odyssey (LiCor, Lincoln, NE). Blots were probed overnight with mouse IgG anti-endothelial cell nitric oxide synthase (eNOS)/NOS Type III primary antibody (1:1000; BD Biosciences, San Jose, CA), washed, and incubated with fluorescent anti-mouse 800 secondary antibody (1 h). Blots were reprobed with Tie-2 antibody (2 µg/ml; Calbiochem, San Diego, CA) for determination of relative endothelial cell content or smooth muscle 
-actin for comparative measures of protein loading and smooth muscle content (1:200; EMD Biosciences, San Diego, CA). Washed blots were directly detected and quantified on the LI-COR Odyssey Infrared Fluorescent Imaging System (LiCor).
Data Analysis. For blood pressure data analyses, within-group differences were assessed by a one-way repeated measures ANOVA with post hoc multiple comparisons using Dunnett's procedure (GraphPad Instat 3; GraphPad Software Inc., San Diego, CA). Between-group differences were assessed by a two-way mixed design ANOVA, and post hoc testing at each time point was performed using Bonferroni's procedure to correct for multiple comparisons (GraphPad Prism 4). For in vitro studies comparing two groups, a Student's t test was used. For in vitro studies comparing more than three groups, a one-way ANOVA with Dunnett's post hoc was used. In all cases, a p value < 0.05 was considered significant. All results are presented as means ± S.E.M.
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Results |
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Effect of 5-HT in the DOCA-Salt Model
Disposition of 5-HT. Three weeks after DOCA-salt surgery, telemeters were implanted. One week later, pumps were implanted and maintained in animals for 1 week. Figure 1A shows the concentration of 5-HT, as detected by high-performance liquid chromatography, in PPP (free 5-HT) or PRP (platelet-contained 5-HT) in blood taken from animals on the last (7th) day of administration. Three observations can be made. First, free plasma 5-HT in the DOCA-salt animals administered vehicle was higher than sham animals receiving vehicle. Second, 5-HT administration through the pump increased PPP 5-HT from 2.7 ± 0.29 to 47.1 ± 23 ng/ml plasma in the sham rat, whereas PPP was increased from 24.9 ± 5.06 to 137.9 ± 35.3 ng/ml plasma in the DOCA-salt rat. Platelets from both sham and DOCA-salt rats took up a significant amount of 5-HT (PRP fractions).
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Effect on Arterial Function. Aortic strips were mounted into tissue baths for measurement of isometric contractile force. All tissues were originally challenged with a maximal concentration of phenylephrine (PE; 10-5 M), and the magnitude of this response was not different among all groups (sham vehicle, 1018 ± 58 mg; sham 5-HT, 1093 ± 133 mg; DOCA vehicle, 1030 ± 136 mg; DOCA 5-HT, 1270 ± 109 mg; p > 0.05). Moreover, concentration response curves to PE (10-9–10-5 M) revealed no shift in sensitivity to PE with 5-HT administration. Classical hyper-reactivity to 5-HT was observed as an increase in the maximal response to 5-HT in aorta from vehicle-administered DOCA rats (143 ± 24% PE contraction) compared with vehicle-administered sham rats (90 ± 7%), and this change was smaller in the vessels of rats administered 5-HT (sham 5-HT, 84 ± 4%; DOCA-5-HT, 100 ± 13%) (Fig. 2).
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Next, tissues were contracted half-maximally with PE, and a cumulative ACh concentration response curve was performed. ACh-induced relaxation is almost completely dependent on activation of eNOS in this tissue and thus is a measure of endothelial cell function. Data in Fig. 3A are reported as a percentage of the contraction elicited by half-maximal (EC50) concentration of PE. This response in tissues from sham (vehicle- or 5-HT-administered) was robust. As expected, the maximal effect of ACh in aorta from the DOCA-salt rat given vehicle was reduced (39.7 ± 14.2% relaxation) when compared with the response in the sham rat receiving vehicle, demonstrating endothelial cell dysfunction (Fig. 3A). In contrast, relaxation was preserved in aortic strips from the DOCA-salt rats administered 5-HT (69.2 ± 9.1% relaxation; p < 0. 05). This difference was not based on an unequal contraction to half-maximal phenylephrine (
10 nM; sham vehicle, 638 ± 50 mg; sham 5-HT, 372 ± 95 mg; DOCA vehicle, 697 ± 94 mg; DOCA 5-HT, 641 ± 79; p > 0.05).
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140 kDa) in homogenates of cultured rat aortic endothelial cells, but we have not tested this same antibody in cultures of aortic vascular smooth muscle cells or fibroblasts. Tie2 was also probed in these homogenates to determine whether the endothelial cell content of the homogenate (and thus potentially eNOS) was different between samples. The endothelial cells comprise a small percentage of the whole homogenate, so the signal for Tie2 was weak but measurable (Fig. 3B). We did not observe differences in Tie2 expression between sham and DOCA samples, vehicle- or 5-HT-administered. In comparison, smooth muscle -actin expression for the same 50 µg of total protein was robust and not different between any groups (Fig. 3B). Thus, it is unlikely that the elevated eNOS expression in the 5-HT-administered DOCA-salt homogenates is because of a greater percentage of endothelial cell content. These findings implicate NO as a mediator of the 5-HT-induced hypotension in the normotensive and DOCA-salt hypertensive rat.
Effect on LNNA Model
Disposition of 5-HT. After 10 days of LNNA administration, minipumps were implanted, and either vehicle or 5-HT was administered (same concentration as in DOCA-salt animals). After 7 days, animals were sacrificed, and blood was procured for measurement of 5-HT. Figure 4A demonstrates that unlike the DOCA-salt rat, the LNNA hypertensive rat did not have a higher free 5-HT (PPP) when compared with sham. However, 5-HT administration raised free 5-HT similarly (200 ng/ml plasma) in both LNNA and sham rats. As expected, 5-HT was highly concentrated in the PRP. eNOS was at least partially inhibited in animals that received LNNA, as evidenced by the significant reduction in aortic maximal relaxation to acetylcholine (1 µM) after contraction with a half-maximal concentration of phenylephrine (10 nM). The percentage of PE-induced contraction remaining after ACh addition was 15.6 ± 7.1% in aorta from vehicle-treated rats and 57.0 ± 6.3% in aorta from LNNA-treated rats (p < 0.05, n = 5).
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Blood Pressure. LNNA administration rapidly elevated blood pressure (Fig. 4B). In sham animals given 5-HT, blood pressure fell similarly as to that which was observed in the shams that accompanied the DOCA-salt rats (Fig. 1B). In contrast, 5-HT-administration did not cause a reduction in blood pressure of the LNNA hypertensive rats. The LNNA rats were similarly hypertensive (168 mm Hg) to the DOCA-salt rats (166 mm Hg) that displayed a profound fall in blood pressure to 5-HT. Thus, inhibition of NOS prevented the 5-HT-induced fall in blood pressure. Heart rate was similarly elevated by 5-HT administration in sham and LNNA rats, recovering by the end of the protocol (Fig. 4C).
Role of Sympathetic Nervous System. We examined the potential of 5-HT to act as a sympatholytic agent. The ganglionic blocker hexamethonium (i.p.) was administered to DOCA-salt and LNNA hypertensive rats on day 4 of 5-HT administration. Peak fall in mean arterial blood pressure in DOCA 5-HT-administered rats was 43.4 ± 6.5 mm Hg, whereas that of DOCA rats administered vehicle was significantly greater, 90.6 ± 14.0 mm Hg (p < 0.05; Fig. 5). In LNNA vehicle-administered rats, peak fall was 67.0 ± 12.5 mm Hg and LNNA 5-HT-administered rats was 52.9 ± 6.21 mm Hg. In comparison, sham rat blood pressure (similar in both DOCA and LNNA sham rats) fell 32.6 ± 3.6 mm Hg. Thus, 5-HT administration reduced the hexamethonium-induced fall in blood pressure in DOCA-salt rats but not LNNA rats administered 5-HT.
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20–25 mm Hg) raised blood pressure from the already hypertensive levels caused by DOCA-salt. This made blood pressure comparable with the DOCA-salt rats used in experiments depicted in Fig. 1. 5-HT did not cause a fall in DOCA rats administered LNNA (Fig. 6B), and no heart rate elevation was observed, although basal heart rates of these animals were consistent with those of rats given DOCA-salt and LNNA alone (Fig. 6C). Sham data are not shown because they resemble all the other shams presented.
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Discussion |
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; Russell et al., 2002; Watts, 2005) and mitogenesis of arterial smooth muscle cells (Nemecek et al., 1986). Both of these endpoints, contraction and vessel growth, promote development of high blood pressure. Our laboratory has published a significant body of work that supports that the hyper-responsiveness to 5-HT in hypertension (DOCA-salt and LNNA forms) is served by up-regulation of a contractile 5-HT receptor for which 5-HT has a high affinity, the 5-HT2B receptor (Watts and Fink, 1999; Russell et al., 2002; Watts, 2005; Ni and Watts, 2006). However, the observation of vascular hyper-responsiveness to 5-HT in hypertension was made long ago (McGregor and Smirk, 1970). Subcontractile concentrations (low nanomolar) of 5-HT potentiate contraction to vasoconstrictors such as norepinephrine and endothelin-1 (Xiao and Rand, 1989; Yildiz et al., 1998; Watts, 2000). Moreover, we have recently discovered that a serotonergic system exists in isolated arteries. In this system, arteries are capable of synthesizing, releasing, taking up, and metabolizing 5-HT (Ni and Watts, 2006; Ni et al., 2006). Free systemic 5-HT concentrations are elevated in human and experimental forms of hypertension, and we presently report an elevated free 5-HT in the DOCA-salt rat compared with sham normotensive rat. Taken together, these data logically lead to the idea that 5-HT can function detrimentally in the cardiovascular system in hypertension. To this point, the long-term effects of 5-HT on blood pressure have not been well investigated. In contrast, the effects of 5-HT given acutely are well established. When given acutely (over minutes and in anesthetized animals), 5-HT causes a complex triphasic response consisting of an initial short lasting vasodepression, a vasopressor response, and then a late vasodepressor response (Saxena and Villalon, 1990). We present data that an elevated free plasma 5-HT concentration, in a more chronic situation, reduces blood pressure in normotensive and in the DOCA-salt hypertensive rats.
Chronic 5-HT Administration and Blood Pressure. This study is the first to measure blood pressure during relatively chronic 5-HT administration in a rat that is freely moving and conscious. This has been done in the broiler chicken, sheep, and dog, although administration of 5-HT was different (Emerson, 1968; Nelson et al., 1987; Chapman and Wideman, 2002). The dose of 5-HT we chose was based on a study by Gustafsson et al. (2005), in which they gave daily s.c. injections of 5-HT, measuring valvular dysfunction as their endpoint. Pilot studies using a dose 10 times lower also produced a fall in blood pressure of the DOCA-salt rats (data not shown). We had fully planned on conducting a study of similar length to Gustafsson (3 months) but truncated the study when we observed the fall in blood pressure caused by 5-HT administration. It is noteworthy that in this abbreviated protocol, we produced elevations of 5-HT in all animals using this protocol. Using the standard molecular weight of 5-HT, we estimated the nanograms per milliliter measures of 5-HT to formal concentration units. Plasma 5-HT concentration was 15 nM in sham rats and 140 nM in DOCA-salt rats without administration; both increased over 5-fold during 5-HT administration. It is noteworthy that platelets from both sham and DOCA-salt rats were effective in taking up 5-HT. Two points should be made. First, it is unclear whether the absolute magnitude or percentage increase in 5-HT is the biologically meaningful measure for effecting changes in blood pressure. Second, the plasma measurements were taken on the final day of 5-HT administration (day 7); thus, we do not know how 5-HT was handled at the nadir of the blood pressure response. In the future, it will be important to ascertain 5-HT distribution at the nadir of blood pressure and to carry out a longer protocol to determine the stability/duration of the fall in blood pressure to 5-HT in the DOCA-salt rat.
5-HT decreased blood pressure of the sham rats (20 mm Hg) and caused a profound drop in blood pressure in the DOCA-salt hypertensive rats (over 50 mm Hg). Heart rate was elevated, as expected through activation the baroreceptor reflex. However, this observation is complicated by the fact that 5-HT can stimulate the heart directly and/or through neuronal innervation to both stimulate and inhibit heart functions (Villalón and Centurion, 2007). The elevation in heart rate of the DOCA-salt rats was not greater than the sham rats, although the blood pressure fall was greater. One explanation for this is the observed depression of baroreflex sensitivity in the DOCA-salt model (Wang et al., 2005). Although blood pressure remained reduced in the sham and DOCA-salt rats administered 5-HT when compared with control, it seemed clear that blood pressure recovered to a degree. The mechanisms for this are unknown, but we speculate this may be due to clearance of 5-HT by multiple tissues and/or loss of 5-HT receptor sensitivity.
Given our original hypothesis, we anticipated 5-HT to increase modestly in the sham rats and to an even greater extent in DOCA-salt rats. This reasoning was based on the idea that in the rat, 5-HT is largely, although not solely, a vasoconstrictor. The 5-HT1B and 5-HT2A receptors mediate contraction in arteries from normotensive rats (Hoyer et al., 2002; Côté et al., 2004; Kaumann and Levy, 2006; Villalón and Centurion, 2007). Endothelial cell 5-HT2B receptors that mediate 5-HT-induced relaxation have been reported in the rat jugular vein (Ellis et al., 1995). 5-HT7 receptors, coupled to adenylate cyclase and mediating vasodilation, have been located in rat arteries (Terrón, 1997; De Vries et al., 1999; Jähnichen et al., 2005), and the 5-HT1B/1D receptor agonist 5-carboxoamidotryptamine causes a hypotension in the anesthetized rat (Terrón et al., 2007). Direct activation of these vasorelaxant 5-HT receptors might explain the 5-HT-induced reduction in blood pressure. Moreover, one could argue that the fall in blood pressure in the DOCA-salt animals was greater than the sham because the absolute value of free 5-HT was greater. However, there are several reasons that make the above unlikely. First, our laboratory has not been able to observe 5-HT-induced relaxation of a contracted artery (aorta, superior mesenteric artery, or mesenteric resistance artery when contractile 5-HT receptors are masked). Second, endothelial cell dysfunction exists in the DOCA-salt form of hypertension; thus, endothelial cell-dependent, 5-HT receptor-mediated responses would initially probably be reduced, not enhanced, in the DOCA-salt rat.
We have been unable to ascertain whether 5-HT receptor activation is necessary to 5-HT-induced reduction in blood pressure, and we recognize this as a limitation of this study. In initial studies, we administered the 5-HT receptor antagonist methiothepin at a dose that should interact with a majority of the 5-HT receptor subtypes that could be involved peripherally and centrally (5-HT1, 5-HT2, 5-HT3, 5-HT4, and 5-HT7). We observed a severe sedation in these animals, making interpretations of any effect 5-HT might have on blood pressure difficult. We continue to work on this important issue.
Role of Nitric Oxide. Several lines of evidence support the involvement of NO/NOS in 5-HT-induced hypotension in the DOCA-salt hypertensive rat. First, ACh-induced relaxation in the thoracic aorta was preserved in the 5-HT-administered DOCA rat, and aortic eNOS expression was elevated compared with vehicle-administered DOCA rats. ACh-induced relaxation is well established as being NO-dependent, being completely abolished with LNNA (100 µM). One possible explanation for the endothelial cell functional preservation and elevated levels of eNOS is because blood pressure was lower over the 7 days of 5-HT administration, and endothelial cells may be protected by the lower blood pressure. However, Western blot analyses of the endothelial cell marker Tie2 suggest this is not the case, although we recognize these blots are less than ideal. Alternatively, 5-HT may directly up-regulate or activate eNOS, an idea supported by work in bovine aortic endothelial cells (McDuffie et al., 1999; Richardson et al., 2003). We have initiated cultures of rat aortic endothelial cells to examine these ideas. Because we used a whole aortic homogenates in Western blot analyses, we cannot exclude the possibility that changes in eNOS are also in the vascular smooth muscle.
Second, 5-HT did not lower blood pressure in either LNNA hypertensive rats or DOCA rats given LNNA. Blood pressures of the hypertensive rats immediately before administration of vehicle or 5-HT were statistically equivalent, an important variable to be controlled. It is interesting to note that the heart rate of the LNNA rats receiving 5-HT increased even though blood pressure did not fall, suggesting a direct effect of 5-HT on the heart. Taken together, this work is consistent with the idea that 5-HT promotes/preserves endothelial cell function, either through protecting the cell from damage in high blood pressure or through direct actions. These ideas are line with other reports that the endothelial cell is capable of compensating for high blood pressure (King and Webb, 1988).
The 5-HT-induced fall of blood pressure in the DOCA-salt rat was rapid, and this hypertension model is well known to be dependent on the sympathetic nervous system (Ekas and Lokhandwala, 1980). Thus, we investigated whether the sympathetic nervous system activity was reduced in the 5-HT-administered DOCA rat. This is reasonable because 5-HT has been reported to both activate and inhibit ganglionic function (Villalón and Centurion, 2007). The ganglionic blocker hexamethonium reduced blood pressure in both DOCA and 5-HT-administered DOCA-salt rats, but the magnitude of fall in the DOCA rats receiving vehicle was nearly twice that of the DOCA rats receiving 5-HT. This suggests that sympathetic tone was reduced in DOCA rats given 5-HT, although still greater in the DOCA-salt versus sham rat. Moreover, 5-HT did not alter the hexamethonium-induced fall in blood pressure in the LNNA model, a model in which 5-HT did not reduce blood pressure. Thus, if 5-HT is exerting its effects through sympathoinhibition, this must be through a NOS-dependent, LNNA-sensitive pathway. We have not investigated the idea that elevations in central 5-HT resulted in the blood pressure reduction, although it is well known that introduction of 5-HT into brain areas result in increases and decreases in blood pressure, depending on the site of injection (Watts, 2005).
Limitations, Speculations, and Conclusions. Despite the controversy in the literature as to the role, if any, of 5-HT in blood pressure control, administering 5-HT by relatively chronic administration yielded the surprising finding that 5-HT reduced blood pressure in both DOCA-salt rats and sham rats. The ultimate response of the rat to 5-HT is most likely a combination of vascular and neuronal activation. We began these studies with the idea that the vasculature alone (or change in the vasculature) was responsible for changes in blood pressure in response to 5-HT. It is noteworthy that our data are not consistent with this idea, although this has been our focus of this manuscript because of our long-standing interest in vascular changes in hypertension. The findings with hexamethonium suggest that 5-HT elicits a complex response in the whole animal and that it is inappropriate to the view the vasculature alone in this response. We know that a 1-week administration of 5-HT reduced the hyper-reactivity to 5-HT in the aorta, restored relaxation to Ach in the aorta, but did not normalize blood pressure. Thus, changes in the vasculature alone cannot account for the fall in blood pressure upon administration of 5-HT.
We also recognize that we studied but one form of hypertension. Another limitation of this study is that we have not measured cardiac output or total peripheral resistance as a means to identify physiological mechanisms by which blood pressure was reduced by 5-HT.
These studies present the initial findings that suggest pursuing the interaction of 5-HT and NOS at the vascular level, and the ability of 5-HT to inhibit the sympathetic nervous system would be a good direction in which to proceed. These findings also suggest that the elevation in free plasma 5-HT observed in hypertensive humans and experimental models may be an adaptive response to hypertension, a physiological attempt to lower blood pressure to normotensive levels.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; SERT, serotonin transporter; 5-HIAA, 5-hydroxyindole acetic acid; DOCA, deoxycorticosterone acetate; LNNA, N-nitro-L-arginine; PRP, platelet-rich plasma; PPP, platelet-poor plasma; ACh, acetylcholine; eNOS, endothelial cell nitric oxide synthase; NOS, nitric oxide synthase; PE, phenylephrine.
Address correspondence to: Dr. Stephanie W. Watts, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824-1317. E-mail: wattss{at}msu.edu
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, difference from sham value. B, effect of chronic administration of vehicle or 5-HT on mean arterial blood pressure. Days on x-axis are control (C) or 5-HT-administered (S). *, statistically significant differences from control period C2; #, from vehicle. C, effect of chronic administration of vehicle or 5-HT on heart rate. *, statistically significant differences from control period. Bars/points, means ± S.E.M. for number of animals indicated in parentheses.
