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(Hypertension. 1995;26:1056-1059.)
© 1995 American Heart Association, Inc.
Articles |
5-Hydroxytryptamine2B Receptor Mediates Contraction in the Mesenteric Artery of Mineralocorticoid Hypertensive Rats
From the Department of Pharmacology and Toxicology, Michigan State University, E Lansing (S.W.W.), and the Department of Physiology, The University of Michigan, Ann Arbor (L.G., R.C.W.).
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Abstract |
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Abstract Vascular responsiveness to 5-hydroxytryptamine (5-HT) is dramatically increased in hypertension. The hypothesis that augmented vasoconstriction to 5-HT in hypertension is due to a change in receptor subtype on vascular myocytes was tested. Mesenteric arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive (systolic blood pressure >180 mm Hg) and sham normotensive (systolic blood pressure <130 mm Hg) rats were mounted in isolated tissue baths for measurement of isometric contractile force. The receptor mediating contraction in isolated mesenteric arteries from sham and DOCA-salt hypertensive rats is a member of the 5-HT2 family based on rank order of agonist potency (5-HT=
-methyl-5-HT [5-HT2 receptor
agonist]>tryptamine>5-hydroxykynuramine). 5-HT was approximately
10-fold more potent in contracting mesenteric arteries from DOCA-salt
hypertensive rats compared with arteries from sham normotensive rats.
The tryptophan metabolite kynuramine, which possesses significant
contractile activity at the 5-HT2B receptor, contracted
hypertensive arteries significantly (50% of 5-HT maximum) but not sham
arteries. Ketanserin (5-HT2A antagonist)
competitively inhibited contraction to 5-HT in arteries from
normotensive rats (-log dissociation constant [mol/L];
pKB=8.54) but not from hypertensive rats
(pKB >6.5). Moreover, contraction to kynuramine
was not blocked by ketanserin. Thus, under normal conditions,
5-HT2A receptors mediate contraction to 5-HT. However, in
DOCA-salt hypertension, ketanserin-insensitive 5-HT2
receptors, possibly 5-HT2B receptors, mediate mesenteric
arterial contraction to 5-HT.
Key Words: serotonin • hypertension, experimental • vasoconstriction • receptors, serotonin
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Introduction |
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Hypertension is characterized by an increased responsiveness to vasoconstrictor agonists. Because the response to 5-hydroxytryptamine (5-HT, serotonin) is particularly enhanced,1 2 numerous investigators have examined the involvement of 5-HT in the initiation or maintenance of high blood pressure. These studies are conflicting; tests of 5-HT2A receptor antagonists (eg, ketanserin) result in variable changes in blood pressure and vascular reactivity in experimental and human hypertension.1 2 3 Thus, the role of 5-HT in hypertension is unclear.
We present data demonstrating that vascular reactivity to a tryptophan metabolite kynuramine is increased in deoxycorticosterone acetate (DOCA)-salt hypertension. This novel observation may provide one explanation as to why serotonergic responsiveness is dramatically enhanced in hypertension and why ketanserin is a relatively ineffective antihypertensive agent in some forms of hypertension. Kynuramine contracts the rat stomach fundus via the recently cloned 5-HT2B receptor.4 5 In contrast, kynuramine has little activity in preparations in which 5-HT2A receptors primarily mediate contraction.4 Thus, kynuramine may be a useful tool when 5-HT2B receptors are investigated. We tested the hypothesis that the receptor subtype subserving contraction to 5-HT receptor agonists in the rat mesenteric artery is altered in DOCA-salt hypertension.
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Methods |
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All animal procedures followed were in accordance with institutional guidelines of the University of Michigan.
Muscle Bath Protocol
Uninephrectomized, male Sprague-Dawley rats (weight,
300 to 350 g; Charles River, Portage, Ind) were made hypertensive by
subcutaneous implantation of 200 mg/kg DOCA as previously
described.6 Rats receiving DOCA were given water
containing 1.0% NaCl and 0.2% KCl. Sham rats received tap water. Rats
were given standard chow and were on therapy for 4 weeks.
Systolic blood pressures (tail-cuff method) were greater
than 180 mm Hg for DOCA-salt rats and less than 130 mm Hg for sham rats
(P<.05).
Rats were then killed (pentobarbital 80 mg/kg IP) and
superior mesenteric arteries were removed. Arteries were dissected into
helical strips (0.07x1 cm) and placed in
physiological buffer for measurement of isometric
contractile force by standard bath procedures.
Physiological salt solution contained the following
(mmol/L): NaCl 130, KCl 4.7, KH2PO4 1.18,
MgSO4-7H2O 1.17,
CaCl2-2H2O 1.6, NaHCO3 14.9,
dextrose 5.5, and CaNa2-EDTA 0.03 (pH 7.2). Vessels from a
DOCA-salt and a sham rat were placed in the same glass tissue bath
(University of Michigan, Ann Arbor). One end of the preparation
was attached to a stainless steel rod; the other was attached to a
force transducer (FT03, Grass Instruments) and placed under optimum
resting tension (
600 mg). Muscle baths were filled with warmed
(37°C), aerated (95% O2/5% CO2)
physiological salt solution. Changes in isometric
force were recorded on a Grass polygraph (Grass Instruments).
Endothelium was not removed because it does not affect
responsiveness to 5-HT receptor agonists (data not shown). After 1 hour
of equilibration, arteries were challenged with
norepinephrine (10 nmol/L). Tissues were washed, and
cumulative concentration response curves to an agonist were performed.
In some experiments, tissues contracted with agonist were washed and
ketanserin (30 or 300 nmol/L) or vehicle added to the bath. After 1
hour of incubation, agonist-induced contraction in the presence of
ketanserin or vehicle was examined. In investigating the effects of
ketanserin on kynuramine-induced contraction, a maximal contraction
to kynuramine was established and a high concentration of ketanserin (2
µmol/L) added directly to the bath.
Data Analysis
Data are presented as mean±SEM for 4 to 9 animals.
Unpaired or paired Student's t tests were used when
appropriate (P<.05 was considered statistically
significant). We calculated agonist EC50 values by
nonlinear regression analysis using the following algorithm:
Effect=Maximum Response/1+(EC50/Agonist
Concentration). Apparent antagonist dissociation constants
(KB values) were determined according to the
following equation:
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where B is the antagonist concentration and the dose ratio is the EC50 value of 5-HT in the presence of antagonist divided by the control EC50 of 5-HT. KB values are reported as the negative logarithm (pKB) of the mean of individual KB values.
Materials
Compounds were prepared in deionized water unless indicated
otherwise. The following materials were used: 5-HT hydrochloride,
tryptamine hydrochloride, kynuramine hydrochloride, DOCA (Sigma
Chemical Co); -methyl-5-HT, ketanserin (dimethyl sulfoxide;
RBI); and 5-hydroxykynuramine hydrochloride (a generous gift from Dr
D.E. Clarke).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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5-HT, the 5-HT2 receptor agonist
-methyl-5-HT,
tryptamine, 5-hydroxykynuramine, and kynuramine contracted mesenteric
arteries from normotensive and hypertensive rats (Fig 1). As evidenced by the leftward
placement of the concentration-response curves, agonists were
significantly more potent in arteries from DOCA-salt rats than sham
normotensive rats. The rank order of potency was similar in arteries
from DOCA-salt and sham normotensive rats. Notably, the tryptophan
metabolite kynuramine caused a significant contraction in hypertensive
arteries and only minimal contraction in the sham arteries. A strong
correlation (r=.783, P<.05) was found between
agonist EC50 values in arteries from DOCA-salt rats and
agonist binding affinities at the 5-HT2 receptor
(Ki values; rat brain cortex, n=7). Correlations
between agonist EC50 values and binding affinities for
other 5-HT receptors (5-HT1A,
5-HT1B, 5-HT1D,
5-HT3, and 5-HT4) were not significant
(EC50). Complete binding data on 5-HT5,
5-HT6, and 5-HT7 receptors are not yet
available to test similar correlations.
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);
); tryptamine (n=3; 
);
5-hydroxykynuramine (n=8; 
); and kynuramine (n=8; 
) in
mesenteric arteries from sham normotensive (left) and
deoxycorticosterone acetate (DOCA)-salt hypertensive (right) rats.
Points represent mean±SEM for the number of rats indicated.The 5-HT2A/5-HT2C receptor antagonist ketanserin (30 and 300 nmol/L) competitively shifted contraction to 5-HT in the sham mesenteric artery with a pKB of 8.54 (Fig 2), suggesting that 5-HT2A receptors mediate contraction. However, ketanserin was ineffective in shifting contraction to 5-HT in hypertensive arteries (Fig 2). Similarly, kynuramine-induced contraction in the mesenteric artery from the DOCA-salt rat was not reduced by ketanserin (Fig 3). At 300 nmol/L, ketanserin did reduce the maximal contraction to 5-HT, indicating that at a high concentration, 5-HT probably stimulates 5-HT2A receptors. However, the receptor mediating contraction to low concentrations of 5-HT in arteries from DOCA-salt rats cannot be a 5-HT2A receptor. Since the general agonist profile of the receptor in the mesenteric artery from DOCA-salt rats is in agreement with that of a 5-HT2 receptor, the receptor mediating contraction in the hypertensive mesenteric artery may be a 5-HT2B receptor.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Serotonergic and tryptophan metabolites have demonstrable contractile effects on smooth muscle. Pomfret et al4 demonstrated that 5-hydroxykynuramine but not kynuramine contracted the rat aorta or jugular vein, two preparations with documented 5-HT2A receptors. In contrast, kynuramine markedly contracted the rat stomach fundus in which the 5-HT2B receptor mediates contraction to 5-HT. Two characteristics of the 5-HT2B receptor include a high affinity for 5-HT5 7 and a relative insensitivity to ketanserin (5-HT2B Ki=3559 nmol/L5 ; 5-HT2A Ki=1.25 nmol/L7 ).
The data presented in this brief report are the first to
suggest that a 5-HT2B receptor may be involved in the
disease process of hypertension and that the receptor subtype(s)
mediating a functional vascular response to 5-HT can change. First,
serotonin is more potent in mesenteric arteries from
DOCA-salt rats than from normotensive rats. While the potency of
several agonists is increased in hypertension, that of 5-HT is
dramatically enhanced.1 2 5-HT is significantly potent in
the rat stomach fundus.4 8 Thus, activation of a
5-HT2B receptor may explain the significant and partially
selective increase in vascular reactivity to 5-HT in DOCA-salt
hypertension. Second, kynuramine contracts mesenteric arteries from
DOCA-salt but not sham normotensive rats. To this date, kynuramine
appears to cause significant contractions in those tissues in which
there are 5-HT2B receptors (ie, rat stomach fundus). The
contractile agonist profile in the mesenteric arteries is in agreement
with the 5-HT receptor being a member of the 5-HT2 receptor
family. Thus, these findings suggest that the 5-HT2
receptor in the DOCA-salt mesenteric arteries may be a
5-HT2B receptor. It must be noted that the absolute
selectivity of kynuramine is not clear. Binding data for kynuramine at
the 5-HT2 receptor subtypes are unavailable. In addition,
kynuramine can act as an indirect sympathomimetic in some
tissues9 ; this has not been examined in our experiments.
While kynuramine has some -adrenergic receptor
antagonist properties,9 it is unlikely that
this pharmacological activity can explain the altered activity of
kynuramine observed in the present experiments. Nonetheless, the
significant contraction produced by kynuramine in the rat stomach
fundus (5-HT2B receptor) and not in the rat aorta or
jugular vein (5-HT2A receptor) suggests but is not proof
that kynuramine may be a useful 5-HT2B receptor
agonist.
Finally, 5-HT induced a ketanserin-insensitive contraction in mesenteric arteries from DOCA-salt but not sham normotensive rats. If a 5-HT2A or 5-HT2C receptor were stimulated in the arteries from DOCA-salt hypertensive rats, ketanserin should shift the response curves in a parallel fashion with a calculable KB. This was not the case. The shift from a ketanserin-sensitive to relatively ketanserin-insensitive contraction to 5-HT has recently been observed in the aorta from the Watanabe hyperlipidemic rabbit.10 Furthermore, contraction to kynuramine in the mesenteric artery from DOCA-salt hypertensive rats also cannot be blocked by ketanserin.2 It should be noted that not all arteries from DOCA-salt rats display this ketanserin insensitivity since ketanserin was able to cause a parallel shift in 5-HT-induced contraction in the femoral artery.11 The reduction of the maximal contraction to 5-HT in mesenteric arteries from DOCA-salt rats suggests that 5-HT also stimulates 5-HT2A receptors. Thus, there are most likely multiple serotonin receptor subtypes mediating contraction to 5-HT. Collectively, these data suggest that the receptor subtype(s) serving contraction to 5-HT in the mesenteric arteries from Sprague-Dawley rats switches from a 5-HT2A to a mixed 5-HT2A/5-HT2B receptor under conditions of DOCA-salt hypertension. Once available, a 5-HT2B receptor antagonist such as SB 20474112 should provide further evidence toward this hypothesis.
The mechanism behind this switch in functional utilization of 5-HT receptors is unknown. Since we have examined this change only in the mineralocorticoid form of hypertension, it is inappropriate to extrapolate these findings to all forms of hypertension. Because the receptor switch occurs in DOCA-salt hypertension, it is tempting to speculate that mineralocorticoids may stimulate steroid responsive elements in the 5' flanking region of the promoter for the 5-HT2B receptor gene. However, the promoter region of the 5-HT2B receptor gene has not been cloned. Moreover, these studies do not dissect the separate impacts of DOCA and salt on changes. Thus, this possibility must remain a speculation.
These studies are significant for two reasons. First, these data provide one possible reason as to why ketanserin has proved relatively ineffective as an antihypertensive agent in some forms of hypertension. It must be considered, however, that the lack of antihypertensive activity of ketanserin in some forms of hypertensive may be because 5-HT is not involved in high blood pressure or that the receptor switch we observed plays no role in hypertension. Second, these data show that the 5-HT2B receptor may have a role in mineralocorticoid hypertension and possibly other diseases involving abnormal vascular smooth muscle growth.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-08892 and HL-18575. The authors thank Mary Lloyd for help with animal care.
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Footnotes |
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Reprint requests to Stephanie W. Watts, PhD, 7813 Medical Science Building II, Department of Physiology, The University of Michigan, Ann Arbor, MI 48109-0622. E-mail cans@umich.edu.
Received June 18, 1995; first decision August 18, 1995; accepted September 5, 1995.
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Pomfret DW, Schenck KW, Fludzinski P, Cohen ML.
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5. Wainscott D, Cohen ML, Schenck KW, Audia JE, Nissen JS, Baez M, Kursar JD, Lucaites VL, Nelson DL. Pharmacological characteristics of the newly cloned rat 5-hydroxytryptamine2F receptor. Mol Pharmacol. 1993;43:419-426. [Abstract]
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Watts SW, Webb RC. Mechanism of
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8. Vane JR. A sensitive method for the assay of 5-hydroxytryptamine. Br J Pharmacol. 1957;12:344-349.
9. Johnson TD, Clarke DE. In: Stone TW, ed. Quinolinic Acid and the Kynuramines. Boca Raton, Fla: CRC Press; 1989:213-226.
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Miwa Y, Hirata K-I, Matsuda Y, Suematsu M,
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Mecca TE, Webb RC. Vascular responses
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