JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 JPET FastThis articleForward. has not Publishedbeen copyedited on and February formatted. The 8, final2007 version as DOI:10.1124/jpet.106.114017 may differ from this version.
JPET #114017
The 5-HT2A receptor is involved in (+)-norfenfluramine- induced arterial contraction and blood pressure increase in DOCA-salt hypertension
Wei Ni, Gregory D. Fink and Stephanie W. Watts
Department of Pharmacology and Toxicology, Michigan State University, East Downloaded from Lansing, MI 48824-1317. (WN, GDF, SWW)
jpet.aspetjournals.org at ASPET Journals on September 30, 2021
1
Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 Running Title: (+)-Norfenfluramine in DOCA-salt hypertension
Corresponding Author:
Wei Ni, Ph.D.
B445 Life Sciences Building
Department of Pharmacology & Toxicology
Michigan State University Downloaded from East Lansing, MI 48824-1317
Telephone: (517) 353-3900
Telefax: (517) 353-8195 jpet.aspetjournals.org
E-mail: [email protected]
Number of page: 33 at ASPET Journals on September 30, 2021
Number of tables: 1
Number of figures: 4
Number of references: 20
Number of words in abstract: 235
Number of words in introduction: 351
Number of words in discussion: 1377
Recommended Section: Cardiovascular
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JPET #114017 Abbreviation list:
5-hydroxytryptamine 5-HT
Acetylcholine ACh
Deoxycorticosterone acetate DOCA
Mean arterial blood pressure MAP
Phenylephrine PE
Phosphate-buffered saline PBS Downloaded from Physiological salt solution PSS
Standard error of the mean SEM jpet.aspetjournals.org at ASPET Journals on September 30, 2021
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JPET #114017 Abstract
The highly effective anorexigen (+)-fenfluramine was widely used to control body weight until the association with primary pulmonary hypertension and valvular heart disease. (+)-Norfenfluramine is the major hepatic metabolite of
(+)-fenfluramine and is primarily responsible for the anorexic effect as well as side effects. We reported that (+)-norfenfluramine causes vasoconstriction and a blood pressure increase in rats with normal blood pressure via the 5- Downloaded from hydroxytryptamine (5-HT)2A receptor. With the knowledge of (+)-norfenfluramine also has affinity for 5-HT2B receptors and that arterial 5-HT2B receptor expression
is upregulated in deoxycorticosterone acetate (DOCA)-salt hypertension, we jpet.aspetjournals.org tested the hypothesis that (+)-norfenfluramine-induced vasoconstriction and pressor effects are potentiated in DOCA-salt hypertensive rats in a 5-HT2 receptor-dependent manner. Contractions of arteries were measured using an at ASPET Journals on September 30, 2021 isolated tissue bath system or myograph. Mean arterial blood pressure was measured in chronically-instrumented conscious rats. Effects of (+)- norfenfluramine in stimulating arterial contraction (leftward shift vs. SHAM, aorta=5.13-fold; renal artery=1.95-fold, mesenteric resistance artery=1.77-fold) and raising blood pressure were significantly enhanced in hypertension. In arteries from both normotensive and hypertensive rats, (+)-norfenfluramine- induced contraction in aorta was inhibited by 5-HT2A receptor antagonists
(ketanserin and LY53857) but not by the 5-HT2B receptor antagonist, LY272015.
Ketanserin (3 mg/kg) reduced (+)-norfenfluramine-induced pressor response in both SHAM and DOCA rats. Our results demonstrate that (+)-norfenfluramine- induced arterial contraction and blood pressure increases are potentiated in
4 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017
DOCA-salt hypertensive rats. However, it is the 5-HT2A receptor and not the 5-
HT2B receptor that participates in these effects.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
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JPET #114017
Introduction
The highly effective anorexigen (+)-fenfluramine (Redux) was taken off the market in 1997 because of the incidence of pulmonary hypertension
(Abenhaim et al., 1996) and aortic valvular disease (Connolly et al., 1997). The anorexic effect of (+)-fenfluramine is due to activation of 5-hydroxytryptamine (5-
HT) 2C receptor (Smith et al., 2006), while the 5-HT2B receptor plays an Downloaded from important role in the pathogenesis of (+)-fenfluramine induced pulmonary hypertension (Launay et al., 2002) and aortic valvular disease (Fitzgerald et al.,
2000). jpet.aspetjournals.org
Radioligand binding experiments show that (+)-fenfluramine has low affinity for the 5-HT2A, 5-HT2B and 5-HT2C receptors, but the hepatic deethylated at ASPET Journals on September 30, 2021 metabolite of (+)-fenfluramine, (+)-norfenfluramine has high affinity at the 5-HT2B receptor (Ki=11.2 nM), 5-HT2C receptor (Ki=324 nM) and a lesser affinity at the 5-
HT2A receptor (Ki=1516 nM) (Rothman et al., 2000). Consistent with binding results, two studies showed that (+)-norfenfluramine causes more severe vasoconstriction by activation of 5-HT2A receptors in pulmonary arteries (Hong et al., 2004) and systemic arteries (Ni et al., 2004 a) compared to effects of (+)- fenfluramine in arteries from normal rats. Studies in cells expressing recombinant human 5-HT2B receptors demonstrated that (+)-norfenfluramine is a 5-HT2B receptor agonist because (+)-norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increasing intracellular Ca2+ and activated the mitogen- activated protein kinase cascade (Fitzgerald et al., 2000). Our previous experiments showed that (+)-norfenfluramine-induced arterial vasoconstriction by
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JPET #114017 activating 5-HT2A receptors but not 5-HT2B receptors in normal rats. However, because the 5-HT2B receptor is upregulated and plays an important role in mediating 5-HT-induced arterial contraction and elevated blood pressure (Watts and Fink, 1999) in deoxycorticosterone acetate (DOCA)-salt hypertensive rats
(Watts et al., 1996; Watts, 2002), we hypothesized that (+)-norfenfluramine- induced vasoconstriction and pressor responses would be increased due to activation of an upregulated 5-HT2B receptor. Downloaded from In this study we first investigated whether (+)-norfenfluramine was a more potent contractile agonist in various isolated arteries from DOCA-salt rats when
compared to SHAM rats. We then investigated the receptor mechanism of (+)- jpet.aspetjournals.org norfenfluramine-induced contraction in aorta from normotensive and DOCA-salt hypertensive rats by using 5-HT receptor antagonists. Lastly, we performed in vivo experiments to compare an acute (+)-norfenfluramine-induced response in at ASPET Journals on September 30, 2021 normotensive and hypertensive rats.
7 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 Method:
Animal Use
Normal male Sprague-Dawley rats (0.225 – 0.300 kg; Charles River,
Portage, MI, USA) were used in the experiments.
Model of Hypertension
Rats were uninephrectomized and DOCA (200 mg/kg in silicone rubber) Downloaded from pellets were implanted subcutaneously. Postoperatively, the rats were given a solution of 1% NaCl and 0.2% KCl for drinking. SHAM rats were
uninephrectomized, received no DOCA and drank normal tap water. All rats jpet.aspetjournals.org were given free access to standard pelleted rat chow (Harlan/Teklad 8640 rodent diet). Animals remained on this regimen for four weeks prior to use.
at ASPET Journals on September 30, 2021
Blood Pressure Measurement for in vitro Studies
Systolic blood pressures of rats were determined in the conscious state by the tail cuff method (pneumatic transducer, Narco, TX, USA).
Isolated Tissue Bath Protocol
Thoracic aorta, renal artery and stomach fundus were removed and placed in normal, physiological salt solution (PSS) containing (mM): NaCl, 130;
KCl, 4.7; KH2PO4, 1.18; MgSO4•7H2O, 1.17; CaCl2•2H2O, 1.6; NaHCO3, 14.9; dextrose, 5.5; and CaNa2EDTA, 0.03 (pH 7.2). Vessels were trimmed of fat, cut into helical strips (aorta, 2 x 10 mm; renal artery, 1 x 5 mm). Two longitudinal strips were made from each fundus. In experiments comparing responses of
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JPET #114017 arteries from normotensive and hypertensive rats, an arterial strip from a DOCA- salt hypertensive and a SHAM normotensive rat were mounted in the same tissue bath.
Tissues were attached to a fixed, stainless steel rod at one end and to a force transducer at the other. Baths were filled with PSS, warmed to 37°C and aerated with 95% oxygen and 5% carbon dioxide. Each tissue was placed under optimum resting tension (previously determined 1500 mg for the rat aorta, 500 Downloaded from mg for renal artery and 4000 mg for stomach fundus) and allowed to equilibrate for one hour with frequent buffer changes. Arterial tissues were then challenged jpet.aspetjournals.org with a maximal concentration of the α adrenergic agonist phenylephrine (PE 10
µM) or fundi were challenged with 67 mM KCl to initiate a maximal contraction, and washed repeatedly until tone returned to baseline. To examine the status of at ASPET Journals on September 30, 2021 the arterial endothelium, tissues were contracted with a half-maximal concentration of PE (10-100 nM) and once the contraction plateaued, the muscarinic agonist acetylcholine (ACh, 1 µM) was administered. We considered a relaxation to ACh greater than 60% of the PE-induced contraction as endothelium intact tissue. Tissues were again washed until baseline was reached and then one of the following protocols was followed.
Protocol 1: Testing of (+)-norfenfluramine as contractile agonists in tissues from
SHAM and DOCA-salt rats
Concentration response curves to (+)-norfenfluramine (10 nM to 300 µM for rat aorta and superior mesenteric artery or 1 nM to 30 µM for renal artery) were performed in a cumulative manner. Each concentration was incubated a
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JPET #114017 minimum of three minutes; when contraction reached a maximum, the next higher concentration of agonist was added. Contraction to agonist was normalized to maximal contraction to PE in arterial tissues or KCl in fundi.
Protocol 2: Testing of effect of antagonists on (+)-norfenfluramine-induced contraction
Vehicle or an antagonist was added to the bath. Antagonists were Downloaded from incubated with the tissues for 1 hour at which time the cumulative response to
(+)-norfenfluramine in the presence of vehicle (control) or an antagonist was
examined. All experiments testing the effect of an antagonist were paired with jpet.aspetjournals.org control (+)-norfenfluramine concentration curves in tissues from same rats.
Tissues were not exposed twice to (+)-norfenfluramine.
at ASPET Journals on September 30, 2021
Myograph Protocol
The whole intestine was placed in a silastic-filled petri dish in cold PSS and pinned down. Second and third order arteries (200-300 micron, inner diameter) were carefully dissected and placed in cold PSS. Two tungsten wires
(California Wire, CA) were threaded through the lumen of the artery. One wire was mounted to a micrometer and the other to a microtransducer. Arteries from
SHAM and DOCA-salt animals were mounted at the same time. All arteries were examined with an intact endothelium. Baths, filled with PSS, were warmed by a water-circulated jacket around the bath and were aerated with 95% O2, 5% CO2.
Tissues equilibrated for 30 minutes before an optimal passive tension of 400 mg
(determined previously) was applied using the micrometer. Tissues equilibrated
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JPET #114017 another 30 minutes with frequent buffer changes prior to challenge with a maximal concentration of PE (10 µM). Experiment protocols were as described above.
Immunohistochemistry
Rat thoracic aortae were cleaned of fat and fixed 24 hours in 10% formalin. Tissues were paraffin embedded and 5-µm sections were collected onto Downloaded from clean glass slides. Sections were dried overnight at 37°C. Slides were dewaxed in xylenes (2 washes, 3 minutes each) and then were rehydrated by passing through graded alcohols (2 changes 100% ethanol, 3 minutes each, followed by jpet.aspetjournals.org
2 changes 95% ethanol, 3 minutes each). Slides were rinsed in deionized water and air dried before use. Slides were immersed in Antigen Unmasking Reagent at ASPET Journals on September 30, 2021 (Vector Laboratories, USA) and microwaved on high for 5-minute intervals 2–3 times and were then allowed to cool in the unmasking solution. Slides were rinsed twice in deionized water before proceeding. Endogenous peroxidases were blocked by incubating samples with 0.3% hydrogen peroxide in phosphate- buffered saline (PBS) for 30 minutes. Sections were blocked for non-specific binding in PBS containing 1.5% of competing serum. In a humidified chamber, samples incubated overnight with a 5-HT2A receptor antibody (Immunostar, WI; 4 o C, 5 µg/ml with 1.5% blocking serum in PBS), 5-HT2A receptor antibody plus 5X competing peptide or only blocking serum as a control. Then samples were washed 3 times with PBS and incubated with a peroxidase-conjugated secondary antibody (30 minutes, room temperature). Samples were washed and incubated with Vectastain Elite® for 30 minutes and then incubated with 3,3-
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JPET #114017 diaminobenzidine/H2O2. Reactions were stopped with washing, counterstained with Vector hematoxylin, and slides were air dried, mounted, cover-slipped, and slides were photographed using an inverted Nikon microscope with a digital camera.
Real Time RT-PCR
Two step real time RT-PCR was performed using a GeneAMP 7500 Real Downloaded from Time PCR machine (Applied Biosystems, Foster City, CA). Total RNA was isolated using standard TRIzol® procedures (GIBCO Life Technologies,
Rockville, MD), reverse transcribed and then taken through standard real time jpet.aspetjournals.org
RT-PCR. Rat β-actin primer was purchased from SuperArray Bioscience
Corporation (Frederick, MD). The primers for rat 5-HT2A receptor were designed at ASPET Journals on September 30, 2021 as follow: 5-HT2A receptor forward = 5'–CTG ATA TGC TGC TGG GTT TCC TTG
TC–3', 5-HT2A receptor reverse = 5'– AGG TGC ATG ATG GAT GCC GTA GAA–
3'. Product was 141 base pair amplicon. PCR conditions were: 95°C for 10 minutes for Amplitaq activation, 15 seconds 95°C, 60 seconds 60°C. Twenty microliters of the amplified products were run on a 3% agarose gel and stained with ethidium bromide. Bands on the gel were visualized using a Bio-Rad® Fluor-
S (Hercules, CA).
In vivo Experiments
This surgery took place 3 weeks after DOCA or SHAM surgery. 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
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JPET #114017 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 hour light/dark cycle.
At least one week recovery was allowed before experiments were begun. Downloaded from (+)-Norfenfluramine (10-300 µg/kg, i.v.) was given in a cumulative fashion at 6 minute intervals. Mean arterial blood pressure (MAP) and heart rate were monitored prior to, during and for 6 minutes after each injection with a jpet.aspetjournals.org computerized DigiMed® system. Ketanserin was given 30 minutes prior to (+)- norfenfluramine. at ASPET Journals on September 30, 2021
Statistics
Contractile data are expressed as ± standard error of the mean (SEM) and reported as a percentage of the maximal contraction to PE (10 µM)/ 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. Apparent antagonist dissociation constants (KB values) were calculating using the following equation (1):
(1) log (dr-1)= log [B]-log KB
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JPET #114017 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, [B] is the concentration of the antagonist tested. Blood pressure is reported as a change of mean arterial blood pressure.
Chemicals
Acetylcholine chloride, deoxycorticosterone acetate, 5-hydroxytryptamine Downloaded from hydrochloride, ketanserin tartrate, phenylephrine hydrochloride and LY53857 (4-
Isopropyl-7-methyl-9-(2-hydroxy-1-methylpropoxycarbonyl)4,6,6a,7,8,9,10,10a-
octahydroindolo[4,3-fg]quinoline) were purchased from Sigma Chemical Co. (St. jpet.aspetjournals.org
Louis, MO). LY272015 (6-chloro-5-methyl-N-(5-quinolinyl)-2,3-dihydro-1H-indole-
1-carboxamide) was a generous gift from Eli Lilly and Company (Indianapolis, IN) and (+)-norfenfluramine was graciously provided by SRI International (Menlo at ASPET Journals on September 30, 2021
Park, CA).
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JPET #114017
Results
Response of Arteries from Normotensive and Hypertensive Rats to (+)-
Norfenfluramine
SHAM rats had a systolic blood pressure 117± 4 mmHg and DOCA-salt
185± 7 mmHg. Figure 1 compares responses of thoracic aorta (a), renal artery
(b) and mesenteric resistance artery (c) from normotensive and DOCA-salt Downloaded from hypertensive rats to (+)-norfenfluramine. (+)-Norfenfluramine contracted arteries in a concentration-dependent manner with a dramatic decrease in threshold
(aorta and mesenteric resistance artery, Table 1, Figure 1 a, c), increase in jpet.aspetjournals.org potency (EC50 value decreased, all arteries) and increase in maximal response
(aorta and mesenteric resistance artery) in arteries from DOCA-salt rats compared with arteries from SHAM rats (Table 1). These data are reported as a at ASPET Journals on September 30, 2021 percentage of an initial PE (10 µM) contraction (Table 1); PE contraction in terms of maximal response and EC50 values was not significantly different
(P > 0.05, Student's t-test) between arteries from SHAM and DOCA rats (data not shown).
Existence of the 5-HT2A Receptor in Aorta from SHAM and DOCA-salt Rats
Immunohistochemical experiments using an antibody that recognized the
N-terminal synthetic sequence corresponding to amino acids 22-41 of rat 5-HT2A receptor, localized the 5-HT2A receptor protein to endothelial and smooth muscle layers of the aorta section from both SHAM and DOCA-salt rats (Figure 2A left panel, arrows). A lighter staining was observed in aorta incubated with primary
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JPET #114017 antibody plus competing peptide (middle), suggesting specific 5-HT2A receptor localization. The staining on adventitia was competed off by competing peptide in aorta from SHAM rats but not DOCA-salt rats. The right picture shows non- primary control. All slides were counterstained with Vector hematoxylin to indicate the nuclei (blue staining) of smooth muscle cells lying between bundles of collagen and elastin.
Figure 2B depicts ethidium bromide-stained products of real time RT-PCR Downloaded from for 5-HT2A receptor mRNA. We observed one product of the expected size (141 bp) in all of aorta samples from SHAM and DOCA rats. Beta-actin was used as a control. CT values were measured as the value at which measurable product was jpet.aspetjournals.org first observed in real time RT-PCR. We did not detect a difference in 5-HT2A receptor mRNA expression in aorta samples from SHAM and DOCA rats as the at ASPET Journals on September 30, 2021 delta CT values of 5-HT2A receptor mRNA were not significantly different [(5-HT2A receptor CT - β-actin CT), SHAM= 12.23±0.50, DOCA= 11.82±0.33; p=0.09; n=4-
5].
Effects of 5-HT Receptor Antagonists on (+)-Norfenfluramine-induced
Contraction in Aorta from SHAM and DOCA-salt Hypertensive Rats
To determine the receptor type mediating (+)-norfenfluramine-induced enhanced contraction in DOCA-salt rats, contraction in aorta was examined in the presence of 5-HT2A/2C receptor antagonist ketanserin and 5-HT2A/2B receptor antagonist LY53857. Ketanserin (Figure 3 a; 10 nM) competitively shifted the (+)- norfenfluramine concentration response curve rightward 29.5-fold in aorta from normotensive rats and 20.4-fold in DOCA-salt rats. The apparent dissociation
16 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 constant calculated from these shifts were 9.54±0.21 in SHAM rats and estimated as 9.25±0.21 in DOCA rats. LY53857 inhibited (+)-norfenfluramine- induced contraction in aorta from SHAM rats (-LogEC50, vehicle= 5.54±0.13,
+LY53857= 3.32±0.17) and DOCA-salt rats (-LogEC50, vehicle= 6.23±0.09,
+LY53857= 4.93±0.24) (Figure 3 b). (+)-Norfenfluramine-induced rat stomach fundus contraction was inhibited by 10 nM 5-HT2A/2B receptor antagonist,
LY272015. We then tested the effect of same concentration of LY272015 on (+)- Downloaded from norfenfluramine-induced aortic contraction in SHAM and DOCA-salt hypertensive rats. LY272015 (Figure 3 d) did not shift (+)-norfenfluramine-induced contraction jpet.aspetjournals.org in aorta from SHAM or DOCA-salt rats.
Response to (+)-Norfenfluramine in Whole Animal at ASPET Journals on September 30, 2021 We reported (+)-norfenfluramine increased blood pressure in a dose- dependent and 5-HT2A receptor-dependent manner in conscious normal rats with no concomitant change in heart rate (Ni et al., 2004 a). In this study we compared the (+)-norfenfluramine pressure effect in conscious SHAM and
DOCA-salt rats (basal mean arterial blood pressure, SHAM = 104±5 mmHg,
DOCA= 145±7 mmHg). Figure 4 depicts effects of ketanserin on the (+)- norfenfluramine-induced pressor response in conscious SHAM and DOCA-salt rats (change in MAP at 300 µg/kg, mmHg, SHAM vehicle= 36±4, SHAM ketanserin=7±2, DOCA= 51±5, DOCA ketanserin= 19±1). The pressor effect of
(+)-norfenfluramine was significantly increased in DOCA-salt hypertension compared with SHAM rats. The pressor response to (+)-norfenfluramine in
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JPET #114017
SHAM and DOCA rats was inhibited by the 5-HT2A receptor antagonist
(ketanserin, figure 4). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
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JPET #114017
Discussion
Our results showed that effects of (+)-norfenfluramine both in vitro and in vivo were significantly enhanced in mineralocorticoid hypertension and these effects are dependent on activation of the 5-HT2A receptor.
Enhanced Arterial Contraction to (+)-Norfenfluramine Downloaded from (+)-Norfenfluramine caused a concentration-dependent contraction in multiple arteries from SHAM and DOCA-salt hypertensive rats (Figure 1 a b c).
We observed enhanced (+)-norfenfluramine-induced contraction in both conduit jpet.aspetjournals.org
(aorta, renal artery) and resistance arteries (mesenteric resistance artery) (Figure
1 a, b, c), thus potentiated response to (+)-norfenfluramine in DOCA-salt rats arteries is not vessel-specific. In the following experiments, we used aorta as a at ASPET Journals on September 30, 2021 model to investigate the role of 5-HT receptors in (+)-norfenfluramine-induced contraction in DOCA-salt hypertension.
To investigate whether (+)-norfenfluramine-induced contraction in arteries from DOCA-salt rats depends on 5-HT2A receptor and 5-HT2B receptor, we first tested for the existence of the 5-HT2A receptor in aorta. The upregulated expression and function of 5-HT2B receptors (Banes and Watts, 2002; Watts et al., 1996) in aorta from DOCA-salt rats has been reported. However, without the use of a specific 5-HT2A receptor agonist, it has been difficult to prove this receptor is still present and functional in arteries of hypertensive animals. Our immunohistochemistry experiment used an antibody that recognizes the 5-HT2A receptor and localized this receptor to endothelium, smooth muscle and possibly
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JPET #114017 adventitial layers, confirmed by using competing peptide and no primary negative control (Figure 2 a). Though the staining of 5-HT2A receptor is lighter in aorta from DOCA-salt rats compared to SHAM, it is not proper to draw the conclusion that less 5-HT2A receptor expressed by the pictures from immunohistochemistry experiments. These pictures are intended to demonstrate qualitatively the existence of the 5-HT2A receptor in arteries of DOCA-salt rats. We tried western analysis to compare the amount of 5-HT2A receptor expressed in aorta from Downloaded from
SHAM and DOCA-salt rats. Unfortunately, the many 5-HT2A receptor antibodies available are not useful in quantifying the 5-HT2A receptor protein with this
technique. Due to this reason, we performed a real time RT-PCR to test the jpet.aspetjournals.org expression of 5-HT2A receptor mRNA. Our results showed an expression of 5-
HT2A receptor mRNA in aorta from SHAM and DOCA rats suggesting the at ASPET Journals on September 30, 2021 possibility of a local 5-HT2A receptor protein synthesis. The expression levels of mRNA were similar in aorta from SHAM compared to that in DOCA rats, which may or may not indicate a similar amount of 5-HT2A receptor protein expression, because there could be post-transcriptional regulations.
We then examined the pharmacology of (+)-norfenfluramine with 5-HT2A and 5-HT2B receptor antagonists in arteries from DOCA-salt hypertensive rats.
We have previously demonstrated that the 5-HT2A receptor but not the 5-HT2B receptor is the primary receptor mediating (+)-norfenfluramine-induced arterial contraction and pressor effects in normal rats (Ni et al., 2004 a). In this study, our observation of rightward-shift of (+)-norfenfluramine-induced aortic contraction in
DOCA-salt rats by ketanserin (10 nM) indicates the 5-HT2A receptor still plays an important role in (+)-norfenfluramine vasoconstriction because ketanserin has a
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JPET #114017 low affinity for the 5-HT2B receptor. The apparent dissociation constant calculated from our experiments are consistent with what we reported before in normal rats
(Ni et al., 2004a) and consistent with antagonism of the 5-HT2A receptor.
The involvement of the 5-HT2A receptor in (+)-norfenfluramine-induced aortic contraction was further supported by the 5-HT2A/2B receptor antagonist
LY53857 (30 nM). Different from ketanserin-induced inhibition, (+)- norfenfluramine-induced maximal contraction was significantly reduced by Downloaded from LY53857 in aorta from DOCA rats (%PE contraction, vehicle=116.9±10.5,
+LY53857=38.6±5.0). In contrast, ketanserin shifted the (+)-norfenfluramine concentration response curve to the right with no change of maximal response to jpet.aspetjournals.org
(+)-norfenfluramine. We do not know whether LY53857 inhibited the maximal, response to (+)-norfenfluramine in aorta from SHAM rats because at the highest at ASPET Journals on September 30, 2021 concentration of (+)-norfenfluramine being used in our experiment, we still did not observe the vasoconstriction plateau. According to studies from two different groups (Pertz and Eich, 1992; Szarek et al., 1995), LY53857 can be a non- competitive antagonist at 5-HT2 receptors. This might explain the different inhibition profiles of ketanserin and LY53857 on (+)-norfenfluramine-induced contraction.
Nevertheless, in this study, we showed that the 5-HT2A receptor was still involved in (+)-norfenfluramine-induced aortic contraction in DOCA-salt hypertension. This is important, because it has not yet been proven that the 5-
HT2A receptor is functional in arteries from DOCA-salt rats. The response to low concentrations of 5-HT (1 to 300 nM) was not inhibited by ketanserin (30 nM) in aorta (Watts, 2002) and superior mesenteric artery (Watts et al, 1996) from
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JPET #114017 DOCA-salt rats. Arterial contraction to low concentrations of 5-HT is likely due to
5-HT2B receptor activation. Thus, the lack of ability of ketanserin to block 5-HT- induced contraction in aorta from DOCA-salt rats could be because of dysfunction of the 5-HT2A receptor or masking by 5-HT2B receptor activation.
Here, we performed experiments suggesting that the 5-HT2A receptor protein was preserved and functional in aorta from DOCA-salt rats.
(+)-Norfenfluramine can function as a 5-HT2B receptor agonist as we Downloaded from observed a reduced (+)-norfenfluramine-induced contraction in rat stomach fundus by the 5-HT2B receptor antagonist LY272015 (10 nM, Figure 3 c).
Previous studies showed that 5-HT2B receptor was upregulated in DOCA-salt rats jpet.aspetjournals.org
(Banes and Watts, 2002; Watts et al., 1996; Watts 2002) as we observed that the
5-HT2B receptor antagonist LY272015 reduced 5-HT-induced contraction in aorta from and blood pressure (Watts and Fink, 1999) in DOCA rats but not SHAM at ASPET Journals on September 30, 2021 rats. We expected to observe that 5-HT2B receptors played a role in (+)- norfenfluramine-induced contraction in vessels from DOCA-salt rats. Contrary to our hypothesis, we did not observe inhibition of (+)-norfenfluramine-induced contraction in DOCA-salt hypertensive rats by the selective 5-HT2B receptor antagonist LY272015. Then, we tried using higher concentrations of LY272015
(30 nM and 50 nM). At these concentrations, LY272015 inhibited (+)- norfenfluramine-induced contraction in aorta from both SHAM and DOCA rats
(data not shown). The affinities of LY272015 at the human 5-HT2A and 5-HT2B receptors have been reported as: 5-HT2A= 28.7±2.3 nM, 5-HT2B= 0.75+0.06 nM
(Cohen et al., 1996). Thus, we do not believe that LY272015 at these higher
22 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 concentrations (30 nM and 50 nM) exert functions specifically to inhibit 5-HT2B receptors.
It is possible that the abundance of the 5-HT2B receptor, even though increased in DOCA-salt rats, is not sufficient for (+)-norfenfluramine to induce contraction. Thus, the data do not support the idea that 5-HT2B receptors play a role in (+)-norfenfluramine-induced contraction in arteries from SHAM or from
DOCA-salt hypertensive rats. Downloaded from
(+)-Norfenfluramine as a pressor agent
(+)-Fenfluramine has been reported to increase blood pressure in many jpet.aspetjournals.org studies (Mabadeje 1974; Michelakis et al., 1999). As the active metabolite of (+)- fenfluramine, (+)-norfenfluramine has a significantly longer half-life (20 hours vs.
32 hours) (Oeser, 1997). Thus it is logical to speculate that the (+)-fenfluramine at ASPET Journals on September 30, 2021 pressor responses in the above reports were caused, at least partially, by (+)- norfenfluramine. More importantly, we reported previously that (+)- norfenfluramine (10-300 µg/kg, i.v.) caused a dose-dependent increase in blood pressure in conscious normal rats without a concomitant change in heart rate
(data not shown) via activation of 5-HT2A receptor (Ni et al., 2004 b). In this study, we observed an increased pressor effect of (+)-norfenfluramine in hypertensive animals, which is consistent with the increased contractile response to (+)- norfenfluramine in arteries from DOCA-salt hypertensive rats. Thus it is possible that increased sensitivity to (+)-norfenfluramine in systemic arteries (Figure 1 a b c) leads to increased peripheral resistance and blood pressure. Although ketanserin (3 mg/kg) reduced (+)-norfenfluramine-induced pressor responses to
23 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 a similar extent in SHAM and DOCA-salt rats, ketanserin abolished blood pressure increases in SHAM but not in DOCA-salt rats. This is consistent with our in vitro observation of less rightward shift of (+)-norfenfluramine concentration response curve by ketanserin in aorta from DOCA-salt rats (Figure 3). Our in vivo and in vitro data indicated that in SHAM rats, the 5-HT2A receptor is the primary receptor responsible for (+)-norfenfluramine-induced vasoconstriction and blood pressure increase, while in DOCA-salt rats, in addition to 5-HT2A receptor, other Downloaded from receptors or mechanisms are also involved in these effects.
In summary, our study suggests that (+)-fenfluramine or (+)- jpet.aspetjournals.org norfenfluramine as a weight control drug might have a greater peripheral artery vasoconstriction and pressor effect in individuals who already have high blood at ASPET Journals on September 30, 2021 pressure. We also proved that the 5-HT2A receptors are preserved and functional in arteries from DOCA-salt hypertensive rats. As a 5-HT2A receptor agonist without activating arterial 5-HT2B receptor, (+)-norfenfluramine might be a useful tool for serotonin research in peripheral vasculature.
24 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 Acknowledgements
We are grateful to the pharmaceutical companies Eli Lilly (LY272015) and SRI
International ( (+)-norfenfluramine) for donating compounds. We thank Dr.
Xiaoling Dai and Dr. I. Theo. Szasz for their technical support. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
25 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 Reference:
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JPET #114017 Hong Z, Olschewski A, Reeve HL, Nelson DP, Hong F and Weir EK (2004)
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29 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 Footnotes:
This research was supported by grant from the National Institutes of Health
(HL081115).
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
30 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017
Legends for Figures
Figure 1. Effect of (+)-norfenfluramine on contraction of aorta (a), renal artery (b) and mesenteric resistance artery (c) from SHAM normotensive and DOCA-salt hypertensive rats. Data are reported as a percentage of an initial PE (10 µM) contraction; PE contraction was not significantly different (P > 0.05, Student's t- test) between the two groups of animals. Points represent means and vertical Downloaded from bars the SEM for the number of animals indicated in parentheses. PE = phenylephrine.
jpet.aspetjournals.org
Figure 2: a: Immunohistochemical staining of the 5-HT2A receptor in rat aorta smooth muscle between cables of elastin/collagen. Parallel sections were at ASPET Journals on September 30, 2021 incubated with antibody alone (left), quenched with a 5X excess competing peptide (middle) or no primary (right). Red arrows indicate the location of staining. Representative of four different experiments. CP=competing peptide.
b: Final product of real time RT-PCR for detection of 5-HT2A receptor (top) and β-actin (bottom) mRNA in aorta from SHAM and DOCA rats. Each lane represents sample from different rats.
Figure 3. a: Effect of 5-HT2A/2C receptor antagonist ketanserin (10 nM) on (+)- norfenfluramine-induced contraction in aorta from normotensive and hypertensive rats. b: Effect of 5-HT2A/2B receptor antagonist LY53857 (30 nM) on
(+)-norfenfluramine-induced contraction in aorta from normotensive and hypertensive rats. c: Effect of 5-HT2B receptor antagonist LY272015 (10 nM) on
31 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017 (+)-norfenfluramine-induced contraction in fundus from normal rats. d: Effect of 5-
HT2B receptor antagonist LY272015 (10 nM) on (+)-norfenfluramine-induced contraction in aorta from normotensive and hypertensive rats. Points represent means and vertical bars the SEM for the number of animals indicated in parentheses. *indicate statistically significant differences from SHAM rats (p <
0.05). #indicate statistically significant differences from control response (vehicle)
(p < 0.05). PE = phenylephrine. Downloaded from
Figure 4. Effect of ketanserin (3 mg/Kg) on (+)-norfenfluramine-induced pressor jpet.aspetjournals.org response on SHAM and DOCA-salt hypertensive conscious rats. Points represent means and vertical bars the SEM for the number of animals indicated in parentheses. *indicate statistically significant differences from SHAM rats (p < at ASPET Journals on September 30, 2021 0.05). #indicate statistically significant differences from control group (p < 0.05).
MAP = mean arterial blood pressure.
32 JPET Fast Forward. Published on February 8, 2007 as DOI: 10.1124/jpet.106.114017 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #114017
Table 1. Potency (-log EC50) and Maximum contraction elicited by (+)- norfenfluramine in arteries from SHAM normotensive and DOCA-salt hypertensive rats.
Mesenteric Aorta Renal Artery Resistance Artery SHAM 5.43 ± 0.08 6.28 ± 0.07 5.70 ± 0.06 Downloaded from -log EC50 [M] DOCA 6.10 ± 0.05* 6.55 ± 0.04* 5.95 ± 0.03*
SHAM 75.26 ± 3.96 122.5 ± 10.5 67.7 ± 16.7 % PE Contraction jpet.aspetjournals.org DOCA 101.28 ± 4.61* 145.4 ± 12.1* 107.5 ± 6.0*
SHAM 6.48± 0.14 7.34±0.24 6.59±0.35 -log Threshold [M]
DOCA 7.27± 0.15* 7.95±0.13* 7.51±0.22* at ASPET Journals on September 30, 2021
*= statistically different from SHAM Value (p <0.05), PE = phenylephrine,
EC50 (concentration of agonist necessary to produce a half-maximal
response). N=6-19.
33 Rat Aorta Rat Renal Artery a. b. 125 SHAM 200 SHAM DOCA DOCA * 100 (N=15-19) * * 150 (N=6-9) * This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
75 JPET FastForward.PublishedonFebruary8,2007asDOI:10.1124/jpet.106.114017 * 100 50 50 25 * * 0 0 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 Log (+)-norfenfluramine [M] Log (+)-norfenfluramine [M]
c. Rat Mesenteric Resistance Artery
125 Sham * DOCA * * 100 (N=6-7) *
75 * 50
25
0 -9 -8 -7 -6 -5 -4 -3
Log (+)-norfenfluramine [M] Figure 1
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at SHAM Rat Aorta
5-HT receptor antibody 5-HT2A receptor antibody No Primary 2A +CP This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonFebruary8,2007asDOI:10.1124/jpet.106.114017 Intima Adventitia
100 uM 100 uM 100 uM
DOCA Rat Aorta
5-HT receptor antibody 5-HT2A receptor antibody No Primary 2A +CP Intima Adventitia
100 uM 100 uM 100 uM
Figure 2a
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonFebruary8,2007asDOI:10.1124/jpet.106.114017
5-HT2A receptor 200 bp
100 bp
β-actin
200 bp 100 bp
SHAM Rat Aorta DOCA Rat Aorta
Figure 2 b
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at a. Rat Aorta b. Rat Aorta 150 SHAM vehicle 150 SHAM vehicle DOCA vehicle DOCA vehicle * * 125 SHAM ketanserin 125 SHAM LY53857 * DOCA ketanserin * * DOCA LY53857
* * * This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 100 (N=10-11) * 100 (N=9-11)
# * JPET FastForward.PublishedonFebruary8,2007asDOI:10.1124/jpet.106.114017 75 * 75 # # 50 # # 50 # # * # * # # 25 * 25 # # # * * # # # # # # # * # # # # 0 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3 Log (+)-norfenfluramine [M] Log (+)-norfenfluramine [M] c. Rat Stomach Fundus d. Rat Aorta 150 SHAM vehicle 150 Control DOCA vehicle LY272015 125 SHAM LY272015 125 DOCA LY272015 (N=5-6) 100 (N=11-12) * 100 * * * 75 75 * * 50 50 * * 25 25 * * 0 * 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3
Log (+)-norfenfluramine [M] Log (+)-norfenfluramine [M] Figure 3
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 30, 2021 30, September on Journals ASPET at Concious Rats This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 80 SHAM control JPET FastForward.PublishedonFebruary8,2007asDOI:10.1124/jpet.106.114017 70 SHAM ketanserin DOCA control 60 DOCA ketanserin * 50 (N=4-10) 40 30 * 20 # # 10 # # # 0 0 100 200 300 400 (+)-Norfenfluramine [ µg/Kg]
Figure 4
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