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Am J Physiol Heart Circ Physiol 294: H1745-H1752, 2008. First published February 8, 2008; doi:10.1152/ajpheart.91415.2007
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Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat

A. Elizabeth Linder, Jessica Diaz, Wei Ni, Theo Szasz, Robert Burnett, and Stephanie W. Watts

Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan

Submitted 7 December 2007 ; accepted in final form 6 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The handling of serotonin [5-hydroxytryptamine (5-HT)] depends on the serotonin transporter (SERT). A SERT knockout (KO) rat is a useful model to test the hypothesis that SERT is the primary mechanism for arterial 5-HT uptake and to investigate the impact of SERT removal on blood pressure. Wild-type (WT) and KO rats were used to measure 5-HT content (plasma, raphe, aorta, carotid, and mesenteric artery), aortic isometric contraction, and blood pressure. HPLC supported the lack of circulating 5-HT in plasma (ng/ml plasma, WT, 310 ± 96; and KO, 1.0 ± 0.5; P < 0.05). Immunohistochemistry and Western blot analyses validated the presence of the SERT protein in the WT rats and a lesser expression in the KO rat. The aorta isolated from KO rats had a normal contraction to phenylephrine and norepinephrine and a normal relaxation to the endothelium-dependent agonist acetylcholine compared with the aorta from WT. In contrast, the potency of 5-HT was increased in the aorta from KO rats compared with WT rats [–log EC50 (M); WT, 5.71 ± 0.08; and KO, 6.7 ± 0.18] and maximum contraction was reduced [%phenylephrine (10 µM) contraction, WT, 113 ± 6%; and KO, 52 ± 12%]. 5-HT uptake was reduced but not abolished in arteries of the KO compared with the WT rats. Diurnal mean arterial blood pressure, heart rate, and locomotor activity level of the KO rats were similar to the WT rats. These data suggest that there are other mechanisms of 5-HT uptake in the arteries of the rat and that although the absence of circulating 5-HT and/or SERT function sensitizes arteries to 5-HT, SERT dysfunction does not impair normal blood pressure.

vascular contraction; 5-hydroxytryptamine

SEROTONIN [5-hydroxytryptamine (5-HT)] alters vascular tone and promotes vascular smooth muscle cell growth. Thus protection from high levels of 5-HT is likely important. The serotonin transporter (SERT) provides such protection by operating to take up free 5-HT, bringing 5-HT back inside a cell (7). Platelet storage of 5-HT operates in just this way, and the platelet is the primary circulating source of 5-HT for the vasculature. SERT is recognized in its importance in neuronal function and is a target of the selective 5-HT reuptake inhibitors such as fluoxetine (Prozac) in the treatment of depression. Studies from multiple laboratories now suggest that SERT is not found just in neurons and that the gastrointestinal system (1, 3, 8), pulmonary system (5, 22), immune system (9), heart (18), and systemic vasculature (15–17) all possess a functioning SERT. SERT is one of the family members of the sodium chloride-dependent transporters that include the dopamine and norepinephrine (NE) transporters, large proteins (~60–80 kDa in mass) that possess 12 transmembrane domains (7, 21).

We previously published that arteries take up 5-HT, and the use of pharmacological inhibitors of SERT suggested that 5-HT was dependent on SERT for uptake (16). Thus the use of a genetically ablated SERT model enables us to test the hypothesis that SERT function is the primary mechanism by which blood vessels concentrate 5-HT. Cuppen and colleagues (11, 12) have created an important model for us to use in testing this hypothesis. Through N-ethyl-N-nitrosurea mutagenesis, this laboratory created a SERT knockout (KO) rat. Mutagenesis induced a premature stop codon in exon 3 of the rat (Wistar background), causing a truncation of the SERT protein within the second extracellular loop. Thus the first three transmembrane domains may be expressed, but the rest of the protein is presumably absent. In stark contrast with brain sections from the wild-type (WT) rats, the SERT radioligand [3H]citalopram was unable to visualize the SERT protein in sections from the KO rat, suggesting the absence of sites necessary for recognition. We have used these rats to investigate the expression of SERT in arteries, functional uptake of 5-HT and contractility to 5-HT, and blood pressure under normal conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal use. Male Sprague-Dawley (SD) rats (Charles River) or WT and SERT KO rats (Edwin Cuppen, Utrecht, Netherlands) were used and weighed between 250 and 400 g at the time of experimentation. All protocols were approved by the Michigan State University Institutional Animal Use and Care Committee.

Plasma 5-HT measurements. Five milliliters of blood were collected from the left cardiac ventricle and transferred into a EDTA anticoagulant vacutainer tube. Pargyline and ascorbic acid (10 µM each) were added. The tubes were centrifuged at 160 g for 30 min at 4°C to obtain platelet-rich plasma (PRP). Two milliliters of supernatant containing plasma and a buffy coat layer were 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. The tubes were centrifuged at 1,350 g for 20 min at 4°C for platelet-poor plasma (PPP). To the remaining pellet (platelet layer), 1 ml of platelet buffer, containing (in mM) 145 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, and 10 D-glucose and 1 µM ADP, was added. Pargyline and ascorbate were added. The tubes were vortexed and allowed to sit on ice for 15 min for platelets to become activated and degranulate. The tubes were centrifuged at 730 g for 10 min at 4°C. Trichloroacetic acid (10%) was added to deproteinate samples, and the samples sat on ice for 10 min. The samples were centrifuged at 4,500 g for 20 min at 4°C and then ultracentrifuged at 280,000 g for 2 h. 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentrations were measured using electrochemical detection in high-performance liquid chromatography (HPLC; ESA Biosciences, Chelmsford, MA) at 0.4 V and 0.9 ml/min flow rate compared with standards (including 5-HT and 5-HIAA) run daily.

Immunohistochemistry. Thoracic aortae were removed from animals anesthetized with pentobarbital sodium (60 mg/kg ip). Tissues were cleaned and formaldehyde fixed. Paraffin-embedded sections (8 µm) were cut, dewaxed, and taken through a standard protocol using a Vector kit. Aortic sections were incubated 24 h with SERT C-20 primary antibody (5 µg/ml; Santa Cruz Biotechnology), and the antibody was quenched with 5x competing peptide (CP) or no primary antibody. Sections were developed according to manufacturer's instructions using a diaminobenzidine developing solution (Vector, Burlingame, CA). Binding was observed as a dark brown/black precipitate, and specific binding was determined as that which is reduced in the presence of CP. All slides were counterstained with Vector hematoxylin for 30 s, with nuclei stained blue. The sections were dried, coverslipped, and photographed on a Nikon TE2000 inverted microscope using MetaMorph software.

Western blot analysis. Homogenates of the aorta were taken through standard Western blot analyses and transferred to Immobilon-P. The blots were probed overnight with goat C-20 or I-21 antibody (directed toward the COOH terminus) or goat N-14 antibody (directed toward the NH2 terminus) of SERT (Santa Cruz Biotechnology). The blots were reprobed with an antibody that recognizes smooth muscle {alpha}-actin (EMD Biosciences, La Jolla, CA) as a measure of equivalent protein loading. The appropriate secondary antibody was used, and the blots were developed using enhanced chemiluminescence reagents from Amersham (Piscataway, NJ).

Isometric contraction. Endothelium-intact thoracic aortic rings were isolated for measurement of isometric contractile force as previously described (16), using physiological salt solution (PSS) as the buffer containing (in mM) 103 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4·7H2O, 1.6 CaCl2·2H2O, 14.9 NaHCO3, 5.5 dextrose, and 0.03 CaNa2-EDTA. The integrity of the endothelium was tested by measuring relaxation to acetylcholine (10–5 M) in rings contracted with a half-maximal concentration of the adrenergic agonist phenylephrine (PE). Cumulative concentration response curves to NE, 5-HT, and acetylcholine were generated. In some experiments, vehicle or the SERT inhibitor fluvoxamine (1 µM) was added 1 h before the addition of 5-HT. Data were captured using chart software on a PowerLab 4/30 (ADInstruments, Colorado Springs, CO) connected to an Apple eMac computer.

5-HT content and uptake assay. At room temperature, arteries were placed in vehicle or 5-HT (1 µM, in 1.5-ml plastic centrifuge tubes for 15 min). In some experiments, tissues were incubated with vehicle or fluoxetine (1 µM) for 30 min before exposure to 5-HT (1 µM, 15 min). Tissues were briefly rinsed in drug-free PSS and placed in 75 µl of tissue buffer containing (in mM) 0.05 sodium phosphate and 0.03 citric acid buffer (pH 2.5) containing 15% methanol and kept at –80°C. The samples were thawed, sonicated for 3 s, and centrifuged at 10,000 g for 30 s. The supernatant was collected and transferred to new tubes. Tissue pellets were dissolved in 1.0 M NaOH and assayed for protein. Concentrations of 5-HIAA and 5-HT in tissue supernatants were determined by isocratic HPLC (ESA Biosciences).

Radiotelemetry. Under isoflurane anesthesia, radiotelemeter devices (PAC-40; Data Sciences, St. Paul, MN) with pressure-sensing tipped catheters were implanted subcutaneously through a 1–1.5-cm incision in the left inguinal area. Catheters were introduced into the left femoral artery 3–5 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 days of baseline measurements (mean arterial blood pressure, heart rate, and activity level) were made with a sampling schedule of 10 s every 10 min; meaned data for 1 h are reported. Blood pressure, heart, and activity measures were taken in the awake (1;00 AM) and sleep (1:00 PM) cycle. Activity is a gross measure of locomotor activity in the rat cage while over a telemetry receiver.

Data analysis. For in vivo data analysis, within-group differences were assessed by a one-way repeated-measures ANOVA with post hoc multiple comparisons using Dunnett's procedure (GraphPad Instat 3). 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 three or more groups, a one-way ANOVA with a Newman-Keuls post hoc was used. EC50 values are calculated within GraphPad Prism as the agonist concentration necessary to cause a half-maximal response. In all cases, a P value of <0.05 was considered significant. All results are presented as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Blood 5-HT. Whole blood was taken from WT and KO rats and separated into PPP (poor, representing free 5-HT) and PRP (rich). Figure 1 demonstrates that in the platelet-rich fraction of the WT rat blood, a substantial amount of 5-HT was measured. In contrast, the platelet-rich fraction from KO rat blood had virtually undetectable levels of 5-HT. In the free plasma (poor), 5-HT levels were significantly higher in the WT compared with the KO rats.


Figure 1
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  		Fig. 1. Separation of platelet-rich (rich) and -poor (poor) plasma for measurement of 5-hydroxytryptamine (5-HT). Bars represent means ± SE for number of animals in parentheses. KO, knockout; WT, wild-type. *Statistical difference from WT.

 
SERT localization and quantitation. Immunohistochemical detection of SERT in aortic sections from WT and KO animals was performed. We used an antibody directed toward the COOH terminus of SERT, a portion of SERT that should be missing in the KO rats. Staining, observed as brown/black precipitate, in the smooth muscle layers of the WT aorta could be reduced by CP (compare left and middle of Fig. 2A). There were no apparent aberrations in vessel structure since the hematoxylin staining in sections from the KO rat was qualitatively similar to that from the WT rat, with regular muscle layers aligned around the lumen (Fig. 2A, far right). In contrast, no such SERT staining was observed in sections from the KO rat, even when observed at a higher power as shown in Fig. 2A. Homogenates of the WT and KO aorta were also processed for Western blot analyses using antibodies that recognized both the NH2 terminus and COOH terminus of SERT. Figure 2B demonstrates that the NH2-terminus antibody recognized SERT protein in all samples (~70 kDa protein). Two different COOH-terminus antibodies (I-21 and C-20; only C-20 results shown) recognized a protein, although at a lower density in the KO versus WT samples (~25% WT). Differences in signal were not due to differences in protein loading since {alpha}-actin was similarly expressed in the aorta from WT and KO rats (Fig. 2B). A band in homogenate from a normal male Sprague-Dawley rats was readily detected, validating the use of this antibody.


Figure 2
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  		Fig. 2. A: immunohistochemical staining of serotonin transporter (SERT) protein in aortic sections from WT and KO rats. Competing peptide (CP), 5x excess of CP. No primary, sections exposed only to secondary antibody. Sections of KO rat were taken at a x40 vs. x20 objective so as to best show the lack of smooth muscle SERT, which is readily apparent in the WT. The arrow indicates specific staining in the WT and a lack of similar staining in the KO. B: profile of Western blot analyses for measuring SERT protein expression in homogenates from WT and KO rats. SD, normal male Sprague-Dawley aortic homogenate; MM, molecular mass marker; L, lumen. Smooth muscle {alpha}-actin was run as a loading control.

 
Vascular reactivity. Aortic rings were mounted in isolated tissue baths for measurement of isometric contractile force. Tissues were initially challenged with a maximal concentration of the {alpha}-adrenergic agonist PE, and the magnitude of this contraction was not different between WT and KO rings (Fig. 3A). Concentration-dependent contraction to another adrenergic agonist, NE, was also similar between WT and KO rings (Fig. 3B). Endothelial cell-dependent relaxation was intact in the rings from the KO rat since acetylcholine (10–9–10–5 M) caused a similar relaxation in PE-contracted rings compared with WT rings (Fig. 3C).


Figure 3
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  		Fig. 3. A: magnitude of maximal phenylephrine (PE)-induced contraction in aorta from WT and KO rats. B: cumulative concentration-dependent contraction to norepinephrine (NE) in aorta from WT and KO rats. C: relaxation of PE-contracted (half maximum) aorta to a maximal concentration of acetylcholine (ACh) in aorta from WT and KO rats. Points/bars represent means ± SE for number of animals in parentheses. C, control.

 
In contrast, 5-HT-induced contraction was altered in the KO versus WT rat. 5-HT was more potent in the aortic ring from the KO rat [–log EC50 (M), 5.71 ± 0.08; and KO, 6.70 ± 0.18; P < 0.05], and the efficacy of 5-HT was reduced (lower maximum contraction to 5-HT; 113 ± 6% vs. 52 ± 12% of maximum PE contraction; Fig. 4A). Importantly, a leftward shift to 5-HT was observed in the presence of the SERT inhibitor fluvoxamine in the aorta from the WT [–log EC50 (M), vehicle, 5.70 ± 0.10; and fluvoxamine, 6.40 ± 0.13; P < 0.05; Fig. 4B, left] but not the KO rat (vehicle, 6.72 ± 0.12; and fluvoxamine, 6.17 ± 0.10; Fig. 4B, right).


Figure 4
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  		Fig. 4. A: cumulative concentration-dependent contraction to 5-HT in aorta from WT and KO rats. B: effect of vehicle or SERT inhibitor fluvoxamine (1 µM) on 5-HT-induced contraction. Points represent means ± SE for number of animals in parentheses. *Statistical difference from vehicle WT.

 
5-HT uptake. Multiple tissues were taken from the WT and KO rats for measurement of basal content of 5-HT and the metabolite 5-HIAA. The raphe was removed for measurement of the basal synthesis of 5-HT since this tissue normally produces 5-HT. Measurements of 5-HT and 5-HIAA in the WT and KO rat were statistically similar in the raphe (in ng/mg protein, WT 5-HIAA, 2.29 ± 0.031, and KO 5-HIAA, 2.28 ± 0.83; WT 5-HT, 2.83 + 0.33, and KO 5-HT, 1.73 + 0.49; P > 0.05 for both comparisons). In contrast, the basal concentration of 5-HIAA was reduced in the carotid and mesenteric artery of the KO compared with WT rat, but basal 5-HT could still be measured in the tissues from the KO animal (Fig. 5). When tissues were exposed to exogenous 5-HT, levels of 5-HIAA (an index of 5-HT uptake in the absence of the monoamine oxidase A inhibitor pargyline) were elevated in all arteries from the WT, indicative of uptake since 5-HT is rapidly metabolized to 5-HIAA. This ability was largely lost in the carotid artery of the KO rat, reduced in the aorta, and slightly but not significantly reduced in the mesenteric artery.


Figure 5
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  		Fig. 5. Tissue concentration of 5-HT and 5-hydroxyindole acetic acid (5-HIAA) in basal and 5-HT-exposed conditions. Left: WT tissues. Right: KO tissues. Bars represent means ± SE for number of animals in parentheses. Statistically significant differences (P < 0.05) vs. *respective vehicle, #respective 5-HT-incubated tissue, and {dagger}respective WT value are shown. Fluo, fluoxetine.

 
ANOVA of aortic 5-HIAA values between WT and KO groups supported that the increase in the 5-HIAA level of the KO aorta with 5-HT incubation was reduced compared with that of the aorta from WT, but still measurable. Importantly, Fig. 5 demonstrates that the concentration of 5-HIAA in the aorta, when exposed to exogenous 5-HT, could not be inhibited by fluoxetine in the tissues from the KO animal, whereas fluoxetine effectively reduced this same measure in the aorta from the WT.

Blood pressure. The circadian patterns and magnitude of mean arterial blood pressure (Fig. 6A), heart rate (Fig. 6B), and locomotor activity (Fig. 6C) were not different between WT and KO rats.


Figure 6
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  		Fig. 6. Diurnal blood pressure (A), heart rate (HR; B), and activity level (C) of WT and KO rats. Bars/points represent means ± SE for number of animals in parentheses. MAP, mean arterial pressure; bpm, beats/min; C, cycle.

 

   DISCUSSION
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 MATERIALS AND METHODS
 RESULTS
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We have previously demonstrated that SERT is responsible for at least a portion of the uptake of 5-HT into isolated arteries. The present use of the SERT KO rat provided an excellent opportunity to determine how the absence of SERT modified contraction to 5-HT, blood pressure, and, most importantly, 5-HT uptake.

SERT in the KO rat. Multiple pieces of evidence suggest that SERT function is ablated in the KO rat. We confirmed the lack of blood 5-HT in the KO rat, both in platelet-rich and platelet-poor fractions, and a similar 5-HT content in the raphe of the WT and KO rat (11, 12). One might expect that free plasma 5-HT concentrations could be higher in the KO versus the WT rat, since platelets cannot store 5-HT. However, 5-HT needs SERT function in the intestine to leave this site of synthesis, so it is likely that 5-HT is trapped where it is synthesized. Taken together, these data suggest that SERT activity has been ablated because platelets rely on SERT for the uptake of 5-HT (4), but 5-HT synthesis is normal. Moreover, we were unable to observe specific staining for the SERT protein in the smooth muscle of the aorta. In Western blot analyses, the NH2-terminus SERT antibody recognized bands in both WT and KO tissues, as expected. The recognition of SERT in some of the KO aortic homogenates by the C-20 antibody (faint and irregular in KO samples but nonetheless unexpected) suggests two things. The antibody may not be purely specific for the COOH terminus of SERT and/or some vessels may process a protein with a COOH terminus, but the protein is dysfunctional. SERT proteins were not studied in this way in the original description of the SERT KO rat, so these are the first experiments investigating SERT protein expression in tissues from these animals. Real-time PCR of neuronal tissue was performed in the original studies characterizing the KO rat, demonstrating a lack of SERT mRNA. We debated doing similar measures but decided that the question of the expression and function of SERT protein was more important to answer. Whether or not the whole protein is expressed, SERT function has been abolished in the KO rat.

Importance of SERT to contractility and uptake. In rat arteries, 5-HT is primarily a vasoconstrictor through the activation of the 5-HT2A receptor. We hypothesized that arteries from the KO rat would be more sensitive to 5-HT given that these blood vessels were exposed to a significantly lower concentration of 5-HT in vivo. 5-HT2A receptors can undergo desensitization and resensitization, so this hypothesis is logical (10). This hypothesis was supported by the selective increase in potency of 5-HT in the aorta of the KO rat, compared with a lack of change in contraction to NE. One question we have not answered is why the maximum effect of 5-HT was reduced in the aorta of the KO rat compared with the WT rat. This was likely not because of a derangement of smooth muscle function since contraction to NE was normal and hematoxylin staining showed a vessel grossly normal. Thus one can speculate that potentially fewer but more sensitive/better-coupled 5-HT receptors are present. An examination of arterial contraction to a serotonergic agonist that is not a substrate for SERT would be useful, since this measure would purely speak to the ability of the receptor to respond to stimulation and would give insight into how 5-HT receptor dynamics might be changed in the SERT KO rat. However, we have not identified a 5-HT2 receptor agonist that is not, to some degree, a substrate for SERT. Alternatively, intracellular 5-HT, as facilitated by SERT, would have to be important to maximal contraction. This explanation is somewhat dubious given that the pharmacological inhibition of SERT caused a leftward (not a rightward) shift in 5-HT-induced contraction in aorta from the WT, and there was no reduction in maximal response. There is also the question as to why the removal of SERT function genetically (the KO rat) and pharmacologically (fluvoxamine) did not result in the same change in maximum arterial contraction to 5-HT, unlike the clear leftward shift in the 5-HT-induced contraction that both interventions caused. It is likely that these differences are based on an acute versus chronic effect of inhibiting/removing SERT function. Acutely, pharmacological SERT inhibition in the aorta alters 5-HT movement without a permanent vascular change, whereas SERT removal from birth is accompanied by other permanent changes, such as potential changes in receptor expression, coupling, and so on. The important finding in these experiments is that the leftward shift caused by fluvoxamine in the aorta from the WT rats was not observed in the aorta from the KO rats. These data support that SERT function was reduced in the KO arteries.

This was further supported by the profound reduction in 5-HT uptake/5-HIAA production in the carotid artery and, to a lesser extent, in the superior mesenteric artery and aorta of the KO compared with WT rat. 5-HIAA was used as an index of 5-HT uptake since we did not want to do these experiments in the presence of the monoamine oxidase A inhibitor pargyline, allowing for the observation of the most physiological response. When carotid arteries from the KO rat were exposed to exogenous 5-HT, the increase in 5-HIAA concentration was markedly reduced compared with that in the WT rat. However, an increase in 5-HIAA was still measurable. Both the mesenteric artery and aorta of the KO rat continued to take up 5-HT, evidenced by the increases in 5-HIAA concentration compared with those in vehicle. In the aorta of the KO rat, this uptake could not be blocked by the pharmacological inhibition of SERT. This was different from the reduction in 5-HIAA elevation exerted by fluoxetine in the aorta from the WT rat.

The qualitative difference between these blood vessels was unexpected. If we consider SERT function to be removed to an equivalent degree in all three vessels, this suggests that SERT is the primary mechanism in the carotid artery and that other non-SERT-dependent mechanisms of 5-HT uptake exist in the mesenteric artery and aorta.

The finding of SERT-independent uptake makes sense of puzzling observations made in the past. We were unable to abolish 5-HT uptake in the normal artery through pharmacological inhibition (16). In the aorta, at least, we have ruled out the endothelial cell, uptake 2 (corticosterone sensitive), the NE transporter, and sympathetic nerves as potential sites of additional uptake. Thus other mechanisms for 5-HT being inside the cell must exist. What might these mechanisms be, and are these mechanisms normally present? A considerable concern when using genetically modified animals is that the animal has somehow adapted to the genetic alteration and activated or even upregulated mechanisms that serve the function of the ablated gene, and thus uptake in these tissues might not be physiologically relevant. This, in fact, was observed in the intestine of the SERT-targeted mutated mouse, created by Murphy (14). The organic cation transporter (OCT) was upregulated in the intestine of the mutated mouse (3). We do not have experimental data that suggest any OCT is responsible for the 5-HT uptake observed in the mesenteric artery and aorta of the SERT KO rat. Other monoamine transporters, such as the dopamine transporter, represent the simplest and most reasonable alternative for 5-HT uptake independent of SERT. Zhou et al. (23) demonstrated a dopamine transporter-dependent uptake of 5-HT into the substantia nigra and ventral tegmental area of the SERT KO mouse. Similarly, the plasma membrane monoamine transporter that belongs to the equilibrative nucleoside transporter family was recently shown to handle 5-HT (6). Other mechanisms independent of protein transporters may also play a role in 5-HT uptake. 5-HT, as a small molecule that can alternate charge dependent on pH, could potentially diffuse through the plasma membrane and thus may move in and out of the cell without assistance from a transporter. Additionally, the internalization of 5-HT by 5-HT receptors represents another possibility not yet explored. This idea parallels the well-known ability of the endothelin type B receptor to act as a clearance receptor for endothelin-1 (13).

Importantly, 5-HT measured in blood vessels also may not rely on transport but be synthesized within the tissue itself. Our ability to measure both 5-HT and 5-HIAA in tissues of the KO rat suggests this as a possibility. Recent studies in multiple tissues including heart and skin have identified the rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase, in these tissues (17, 20).

Blood pressure. The studies presented here indirectly support the idea that 5-HT is not important to blood pressure, since the removal of SERT did not alter mean arterial blood pressure in the conscious, unrestrained animal. Additionally, circadian rhythms in heart rate and gross motor activity level were not different between KO and WT rats. These findings agree with a previous report stating that the systolic blood pressure of the female SERT KO was not different from control (11). The results of in vivo experiments performed in the present study were eagerly anticipated because of reports of the ability of pharmacological inhibitors of SERT to elevate blood pressure (17, 21). Collectively, these direct measures of blood pressure suggest that a lack of a functional SERT does not modify blood pressure. However, we recognize that our experiments only indirectly address the issue of the importance of 5-HT to blood pressure regulation since 5-HT was not depleted from the KO animal. Pharmacological inhibition of tryptophan hydroxylase previously resulted in a ~40-mmHg fall in the blood pressure of deoxycorticosterone acetate (DOCA)-salt hypertensive rats, suggesting that continued 5-HT synthesis supported the elevated blood pressure (2). However, there are multiple reports that suggest that inhibitors of 5-HT receptors do not lower blood pressure (20). More recently, a tryptophan hydroxylase 1 KO mouse was created (19), and this animal will be important to examine blood pressure in the absence of peripheral 5-HT.

Significance and conclusion. These studies indicate that 5-HT is taken up into arteries through mechanisms partially but not completely dependent on SERT. SERT function, globally, appears to have little impact on normal blood pressure and heart rate. These studies examine the contributions of SERT to normal, unchallenged levels of function. It may arguably be more important to examine how the lack of SERT functions under the condition of stress or disease or in situations in which an increase in SERT activity is necessary. Future studies are needed to examine some of these ideas. These experiments should include measuring blood pressure in SERT KO animals that are infused with 5-HT, a stimulus that would presumably drive SERT activity if it existed. The SERT KO animal would thus have to figure out how to respond/deal with excess 5-HT in the absence of SERT. Additionally, it would be important to determine whether animals that lack SERT have the ability to become hypertensive to DOCA and salt. This is a model well established in the rodent for creating hypertension, a stress placed on the animal. These studies will enable the next step forward in understanding how SERT and arterial 5-HT affect cardiovascular function.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by National Heart, Lung, and Blood Institute Grant HL-81115 (to S. W. Watts) and American Heart Association Postdoctoral Fellowship 0725729Z (to A. E. Linder).


   ACKNOWLEDGMENTS
 
We are extremely grateful to Dr. Edwin Cuppen (Utrecht, Netherlands) for allowing the use of the SERT KO rat.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. W. Watts, Dept. of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI 48824-1317 (e-mail: wattss{at}msu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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