Serotonin induces a wide spectrum of effects in the cardiovascular system, mediated through a variety of receptor subtypes (1,2). In many vascular beds, serotonin elicits a vasoconstrictor effect and does so generally by activating 5-HT2A receptors. For example, 5-HT2A receptors mediate the serotonin-induced contraction in the rabbit (3-5) and rat (6) aorta, dog femoral artery (7), and many other vessels from several species (1,8). The receptor subtype responsible for contraction in selected vessels such as canine saphenous vein (9,10), canine (11), rabbit (12), and human (13) basilar arteries, and bovine pial arteries (14) is the 5-HT1-like receptor subtype. In addition, 5-HT also activates α-adrenoceptors in rabbit ear (3,15-17), iliac (18) and femoral arteries (19), and in rabbit aorta under special circumstances (20). Serotonin inhibits stimulation-induced neurotransmitter release from sympathetic nerve terminals (9), and it also acts on endothelium to release endothelium-derived relaxing factor (21).
An important property of serotonin is its capacity to interact synergistically with other vasoconstrictor agents. de la Lande et al. (22) were first to show a synergistic interaction between serotonin and norepinephrine. Many other arteries also exhibit such synergism, including rabbit thoracic aorta (23), femoral artery (24), and rat caudal artery (25). The rabbit femoral artery is of particular interest because of the wide variety of synergistic interactions observed in this vessel. For example, Van Nueten et al. (24) used the rabbit femoral artery in a study of the synergism between serotonin and several other vasoconstrictor agents. They showed that serotonin amplifies the contractile responses to angiotensin II, histamine, and prostaglandin F2α (PGF2α) by 500-2,000%.
Under certain experimental conditions in selected blood vessels, serotonergic receptors that are normally inactive or "silent" become enabled if the tissue is precontracted with a threshold concentration of either a nonserotonergic agonist or a depolarizing concentration of K+(26). These newly enabled receptors can then mediate the contractile responses to serotonergic agonists. Such inactive or silent receptors of the 5-HT1-like subtype have been found in guinea pig iliac artery (27) and rabbit ear (28) and renal (29) arteries, precontracted with PGF2α, methoxamine, and histamine, respectively. Pre-contraction of rabbit ear (28) and iliac (30) arteries with an α-adrenergic agonist, phenylephrine (PHE), also activates previously silent 5-HT1-like receptors.
In rabbit femoral artery, it has been reported that activation of thromboxane A2 receptors by U46619 can convert 5-HT-induced contraction from one mediated predominantly by 5-HT2A receptors to one mediated predominantly by 5-HT1-like receptors (31). To our knowledge, no study has identified the receptor(s) mediating the amplified response to serotonin in rabbit femoral artery precontracted with an α-adrenergic agonist.
In this study, focus on the femoral artery arose, in part, from a comparison of the magnitude of synergism between serotonin and α-adrenergic agonists in this vessel with that in the rabbit ear artery and thoracic aorta (32). In 1986, Stupecky et al. (23) showed that the sensitivity of the rabbit thoracic aorta to serotonin increased by ∼10-fold in the presence of a threshold concentration of an α-agonist. The rabbit ear artery exhibited a 1,000-fold increase in sensitivity, whereas the rabbit femoral artery was intermediate with 100-fold increase (32). The purpose of our study was to characterize the magnitude of the synergism between α-adrenergic agonist and serotonin in rabbit femoral artery and to identify the receptor(s) mediating the amplified response.
The term, 5-HT1-like, is used in this study. There is a consensus building in the literature that the vascular 5-HT1-like receptor mediating contraction is the 5-HT1B(26). However, that the mRNA for the 5-HT1D receptor subtype is present in some blood vessels raises the possibility that this receptor subtype also could be involved (33).
Femoral arteries from specific-pathogen-free New Zealand White rabbits (2-3 kg; Myrtle's Rabbitry, Thompson Station, TN, U.S.A.) were used in these experiments. The rabbits were killed by exposure to 100% CO2 to produce deep anesthesia (34), followed by rapid decapitation. The femoral arteries were isolated and placed in Krebs' solution at room temperature, cleaned, and cut into 3-mm rings. In a previous study, we found that endothelial removal had no effect on the response of control compared with PHE-precontracted blood vessel rings to serotonin (28). Thus no effort was made to remove the endothelium. Vessel rings were mounted for the measurement of isometric contraction (35) in tissue baths containing 30 ml of 95% O2/5% CO2 gassed Krebs' bicarbonate solution at 37°C. The composition of the Krebs' solution in millimoles per liter was NaCl, 119.2; KCl, 4.9; CaCl2, 1.3; MgSO4, 1.2; NaHCO3, 25; glucose, 11.1; ascorbic acid, 0.114; and tetrasodium ethylenediamine tetraacetate, 0.03. The rings were placed under 1.5-g resting force, a value found in preliminary experiments to provide optimal active force development (i.e., the largest repeatable contraction to the standard stimulus of 68 mM KCl). After 60 min under resting force, baths were drained and refilled with Krebs' solution containing 68 mM KCl prepared by equimolar reduction of Na+. This concentration of KCl was found in preliminary experiments to cause the maximal contraction of rabbit femoral artery. When tissues achieved steady-state contraction, baths were drained and refilled 3 times over 5 min with normal Krebs' solution, and tissues were allowed to relax to baseline. Resting force was adjusted to 1.5 g as needed. Thirty minutes later, this potassium exposure was repeated, and the resulting contraction was expressed as 100% and used to normalize subsequent contractile responses. After recovery from potassium exposure, some tissues were precontracted with PHE, to produce 0.5- to 1.0-g contraction. This represented between 4 and 8% of the maximal response of femoral artery to 68 mM K+. If antagonists were used, they were added 30 min before the PHE precontraction. Immediately after the PHE precontraction, agonist concentration response curves (CRCs) were obtained by cumulative addition in 0.5-log increments (Fig. 1). The PHE precontraction was subtracted from all subsequent contractile responses to either serotonin (Fig. 2) or sumatriptan (Fig. 3) in the preparation of the CRCs, reported in the Results section. Magnitudes of leftward CRC shift caused by precontraction (Fig. 2A) or rightward CRC shift caused by antagonists (Fig. 2C) were determined at specified levels of contraction (see Results section) by interpolation from linear regression between the nearest two data points on the respective CRCs. Isometric contractions were recorded using Grass FT03C strain gauges (Grass Co., Quincy, MA, U.S.A.) connected to a Maclab Electronic Data Acquisition System (Castle Hill, Australia). The following drugs were used: 5-hydroxytryptamine (serotonin), phenylephrine HCl, and prazosin (Sigma Chemical Co., St. Louis, MO, U.S.A.); ritanserin (RBI, Natik, MA, U.S.A.); sumatriptan and GR127935T (gifts from the Glaxo Pharmaceutical Co., Stevenage, U.K.).
CRCs in Figs. 2, 3, and 6 were based on three artery rings per treatment, per experiment. Statistical analysis was based on the number (n) of experiments (animals) used. CRCs were compared by repeated measures, two-way analysis of variance using SuperANOVA statistical software (Abacus Concepts Inc., Berkeley, CA, U.S.A.). Contractile responses at a given serotonin concentration on different CRCs were considered significantly different when p < 0.05 using Scheffé's post hoc test.
In this study, experiments were performed to assess the sensitivity of the femoral artery to serotonin in the presence of the α-agonist, PHE. In quiescent arteries (in the absence of precontraction with PHE), serotonin did not induce any contraction at low concentrations (1 × 10−9 to 1 × 10−6M), but it induced concentration-dependent contractions at higher concentrations (3 × 10−6 to 3 × 10−4M). In the presence of PHE, the concentration-response curve to serotonin was shifted to the left and became biphasic (Figs. 1 and 2). PHE-induced precontraction increased this sensitivity to serotonin by ∼1,326-fold (determined at the 30% level of contraction, the lowest level at which the PHE-amplified and control curves were parallel; Fig. 2A). The change in sensitivity occurred at the lower concentrations (1 × 10−9 to 1 × 10−6M) and not at the higher concentrations (3 × 10−6 to 3 × 10−4M), over which the PHE-amplified and control CRCs were not significantly different.
The 5-HT2A-receptor antagonist, ritanserin (0.01 μM) and the 5-HT1B-receptor antagonist, GR127935T (0.01 μM) (36) were used to determine the possible involvement of 5-HT2A and 5-HT1-like receptors in the PHE-amplified response to serotonin. Ritanserin had little or no effect on the PHE-amplified serotonin CRC (Fig. 2A). In contrast, GR127935T inhibited the enhanced response to serotonin, causing an ∼200-fold rightward shift of the CRC (Fig. 2A). The combination of ritanserin plus GR127935T had no greater effect than that of GR127935T alone.
Careful inspection of the data used to prepare Fig. 2A revealed that they were made up of two fundamentally different sets of results concerning the combination of ritanserin plus GR127935T. In one set of three experiments, the combination had no further effect on the serotonin CRC than did GR 127935T alone (Fig. 2B). In the other set, GR127935T plus ritanserin shifted the serotonin CRC an additional 52-fold to the right of the CRC in the presence of GR127935T alone (determined at the 20% level, approximately the highest level of contraction achieved at the highest serotonin concentration used in the presence of GR127935T plus ritanserin; Fig. 2C).
Experiments also were performed using the 5-HT1-receptor agonist, sumatriptan, and the results are shown in Fig. 3. Sumatriptan did not cause contraction in the control tissues. However, in the presence of PHE, a concentration-dependent response of ∼20% of the maximal contraction to serotonin was observed.
The experiments shown in Figs. 1 through 3 were conducted between January and April 1995. Throughout this time period, the serotonin CRCs in the absence of PHE were monophasic, as also shown in Fig. 4A. Moreover, these CRCs were sensitive to α-adrenergic receptor blockade with prazosin (Fig. 4A). In experiments conducted during the 2 months subsequent to April 1995, control CRCs in the absence of PHE became variable and biphasic, and the first phase fell to the left (i.e., was obtained at lower concentrations than those observed in the earlier, monophasic CRCs). Examples are shown in Fig. 4B-D.
As a first approach to analyzing the mechanisms underlying the variable, biphasic control CRCs, a series of experiments were run in which serotonin CRCs were obtained in the presence and absence of 0.1 μM prazosin. Three individual experiments are shown in Fig. 5. These were chosen to illustrate the range of variability of the control CRCs. In addition, these experiments revealed that the nature of blockade by prazosin varied with the position of the control curve on the X axis. Prazosin caused a monophasic rightward shift of the serotonin CRC when the control CRC had a threshold of 3 μM. In contrast, as the control CRC was shifted to the left and became biphasic, the CRC in the presence of prazosin became increasingly biphasic, and prazosin lost the ability to block the lower part of the serotonin CRC.
Further to characterize this phenomenon, four experiments were pooled in which the control CRCs were biphasic and had a serotonin threshold of 0.1 μM. In addition, 0.1 μM prazosin, 0.01 μM ritanserin, and the combination of these antagonists were used in these experiments. As shown in Fig. 6, prazosin had no significant effect on the serotonin CRC until serotonin concentrations of 10 μM were reached. Thereafter, prazosin caused a significant rightward shift of the serotonin CRC. In contrast, ritanserin significantly shifted the serotonin curve to the right from threshold to near maximum. The greatest magnitude of rightward shift by ritanserin occurred between 0.1 and 10 μM serotonin, and ritanserin blockade declined at serotonin concentrations >30 μM. The combination of prazosin plus ritanserin nearly abolished the contraction to serotonin.
It is well documented that the predominant receptor subtype mediating the contractile response of the rabbit femoral artery to serotonin is the 5-HT2A receptor (24,37). We have confirmed this (19) and found that, in addition, serotonin acts on α-adrenoceptors at very high concentrations, those >3 μM. This latter observation is not surprising because we reported a similar finding in the rabbit aorta (20), a vessel in which serotonin also acts predominantly on 5-HT2A receptors (3-5). There also is great interest in the mechanism(s) by which precontraction of certain blood vessels with a nonserotonergic agonist markedly enhances the sensitivity of these vessels to serotonin. In many cases, the enhanced response is mediated by a newly enabled receptor, usually the 5-HT1-like receptor subtype (26). MacLennan and Martin (31) have reported that precontraction of the rabbit femoral artery with the thromboxane A2-mimetic, U46619, increases the sensitivity of this vessel to serotonin. Moreover, the contraction in the absence and presence of U46619 is mediated by 5-HT2A and 5-HT1-like receptors, respectively (31). In a preliminary study, we found that precontraction of the femoral artery with an α-adrenergic agonist also enhanced the sensitivity to serotonin (32). The purpose of our study was to characterize further the effect of precontraction of the femoral artery with an α-adrenergic agonist, using PHE.
Two unexpected results were observed in this study. First, the untreated femoral artery was ≥100-fold less sensitive to serotonin than reported by several other laboratories (5,24,37) or by us in an earlier study (19). Second, during the course of the study, the untreated femoral artery exhibited a change in responsiveness to serotonin in which the vessel became both more variable and more sensitive to serotonin, and the serotonin CRC became biphasic. In the discussion later, the effect of PHE on the sensitivity of the femoral artery to serotonin is addressed first, followed by an exploration of the unexpected changes in the behavior of the untreated femoral artery rings.
Precontraction of the rabbit femoral artery with PHE increased the sensitivity of this vessel to serotonin dramatically. The serotonin CRC was shifted to the left 1,326-fold (Fig. 2A). Ritanserin had little or no effect on the PHE-enhanced response to serotonin, whereas GR127935T exhibited a marked blockade, shifting the CRC >200-fold to the right. The combination of ritanserin plus GR127935T had no greater effect than that by GR127935T alone (Fig. 2A). These results strongly suggest that the PHE-enhanced response to serotonin was mediated by 5-HT1-like receptors, and that the 5-HT2A receptor was not involved.
It was found that the six experiments pooled to generate Fig. 2A could be divided equally into two subgroups, based on different effects of ritanserin plus GR127935T. In both subgroups, ritanserin alone had little or no effect, and GR127935T alone caused a marked rightward shift of the PHE-enhanced CRC. Ritanserin plus GR127935T had no further effect beyond that of GR127935T alone in one subgroup (Fig. 2B), but caused a greater inhibition than GR127935T alone in the other (Fig. 2C). There is no obvious explanation for the differential effects of the combined antagonists. In the case in which the combination exhibited greater inhibition than GR127935T alone, the following conditions could have been operative. It is possible that both 5-HT2A and α-adrenergic receptors contributed to the response of the untreated femoral artery to serotonin in this subgroup, whereas the 5-HT1-like receptor was the predominant receptor mediating the PHE-enhanced response. In this case, the GR127935T-mediated blockade would have shifted the CRC to the right, close to the concentration range in which the 5-HT2A receptor could contribute to the serotonin-induced contraction. In turn, this would allow the addition of ritanserin to produce a further rightward shift. Because ritanserin alone had no effect (Fig. 2C), it is unlikely that PHE precontraction enabled both 5-HT1-like and 5-HT2A receptors, allowing both to mediate the enhanced response.
Several laboratories (24,37), including ours (19), have reported that 5-HT2A receptors are the predominant receptors mediating the response of untreated rabbit femoral arteries to serotonin. Moreover, all these investigators have found that the serotonin CRC falls approximately in the same concentration range, 0.01-1 μM. Thus it was surprising in this study to obtain monophasic serotonin CRCs that were consistently in the range of 3-300 μM. In an earlier study, we reported that serotonin produces a biphasic CRC in the rabbit femoral artery (19). We demonstrated that the second phase occurred over the concentration range of 3-300 μM and was mediated by α-adrenoceptors. The monophasic serotonin CRCs obtained in the present study also were mediated by α-adrenoceptors, as these were sensitive to blockade by prazosin (see Figs. 4-6). Thus the femoral arteries used in the present study exhibited what must be considered anomalous behavior. Throughout most of the study, it appears that these vessels failed to express 5-HT2A-receptor activity, based on functional measurements. However, late in the study, the serotonin CRCs became biphasic, with the first phase residing in a lower concentration range more characteristic of mediation by 5-HT2A receptors (see Fig. 6). The use of ritanserin and prazosin confirmed that the first and second phases were mediated by 5-HT2A and α-adrenergic receptors, respectively (see Fig. 5). We are unaware of any reports in the literature in which the functional expression of 5-HT2A receptors exhibits such variability. That these receptors were absent from January through April 1995, followed by a variable return during the subsequent 2 months, suggests that seasonal factors could have played a role. However, the fact that 5-HT2A-receptor functional activity was absent at all is surprising. A partial explanation also could be related to the sources and characteristics of the rabbits. Rabbits (not specific pathogen free) obtained from Simunek (Vista, CA, U.S.A.) were used in our earlier study in which the femoral arteries exhibited normal 5-HT2A-receptor activity (19). In contrast, specific-pathogen-free rabbits obtained from Myrtle's Rabbitry, Inc., were used in this study.
In conclusion, these results demonstrate that PHE precontraction can markedly enhance the sensitivity of the rabbit femoral artery to serotonin. In addition, the use of selective receptor antagonists suggested that the enhanced contractile response was mediated by 5-HT1-like receptors.
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