Introduction

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Serotonin, also known as 5-hydroxytryptamine (5-HT), is a hormone with complicated and interesting cardiovascular actions. In some but not all species, 5-HT causes a contraction of the vasculature. The contraction that results from 5-HT, and the changes in this contraction associated with certain disease states (McGregor and Smirk, 1970; Turla and Webb, 1990; Watts, 1998) make this a particularly interesting compound in cardiovascular research. Most of the research involving 5-HT done to date has been performed using the rat and the serotonergic pharmacology of the vasculature of the rat has been well characterized. A 5-HT_2A receptor, as defined by activation by the agonist -methyl-5-hydroxytryptamine maleate (-methyl-5-HT) and inhibition by ketanserin or MDL100907, mediates contraction in the vasculature of the normotensive rat (Cohen et al., 1981; Nakaki et al., 1985; Roth et al., 1986; Watts et al., 1996). Currently, the cardiovascular researcher is beginning to use the mouse for models of both genetic (Schlager, 1994; Schlager and Sides, 1997) and experimental hypertension. However, little is known about the serotonergic pharmacology of the vasculature of the mouse.

The grandparent strain to many but not all genetically altered mice is the C57BL/6J mouse (Jackson Laboratories, Bar Harbor, ME). To date, the aortic smooth muscle receptor stimulated by 5-HT in any mouse strain has not been characterized. Using classical pharmacology techniques, we presently test the hypothesis that a 5-HT_2A receptor, as is found in the normotensive rat (Cohen et al., 1981; Nakaki et al., 1985; Roth et al., 1986; Watts et al., 1996; Florian and Watts, 1997), mediates 5-HT-induced contraction in the mouse aorta and is coupled to L-type calcium channels, phospholipase C (PLC), and tyrosine kinases. Using a series of 5-HT receptor agonists and antagonists, as well as signaling pathway inhibitors to modulate aortic contraction from the C57BL/6J mouse, we determined which receptor(s) and pathways mediate contraction in the mouse aortic smooth muscle.

	Experimental Procedures

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All animal procedures followed were in accordance with institutional guidelines of Michigan State University.

Isolated Tissue Bath Protocol. C57BL/6J mice were euthanized with carbon dioxide to effect, and the thoracic aortae were removed. Tissues were placed in cold physiological salt solution and kept at 4°C until the following day. Physiological salt solution contained 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂0PO₄, 1.17 mM MgSO₄·7H₂O, 1.6 mM CaCl₂·H₂O, 14.9 mM NaHCO₃, 5.5 mM dextrose, and 0.03 mM CaNa₂EDTA. The aortae were dissected into helical strips (0.15 × 1 cm). The endothelial cell layer was removed by rubbing the luminal side of the vessel with a moistened cotton swab. Tissues were placed in physiological salt solution for measurement of isometric contractile force. One end of the helical strip was attached to a glass rod, and the other was attached to a force transducer (FT03; Grass Instruments, Quincy, MA), and placed under optimal tension (250 mg, determined previously). Tissue baths were filled with physiological salt solution, warmed to 37°C, and aerated with 95% O₂, 5% CO₂. Changes in isometric contractile force were recorded on a Grass polygraph (Grass Instruments). After a 1-h equilibrium, the aortae were challenged with a maximal concentration of the ₁-adrenergic receptor agonist phenylephrine (PE, 1 × 10⁵ M). The tissues were washed and the removal of the endothelium was confirmed by observing a lack of aortic relaxation to the endothelium-dependent agonist acetylcholine (1 × 10⁶ M) in tissues after first contracting the tissue with a half-maximal concentration of phenylephrine (1 × 10⁸ M). Tissues were washed multiple times and then one of the following protocols was performed.

Test of Agonist-Induced Contraction. Cumulative concentration-response curves to two of 16 agonists were generated in a random manner. Tissues were exposed to cumulative additions of one of the following: 5-HT, 1-(3-chlorophenyl)piperazine (m-CPP), 5-carboxamidotryptamine maleate (5-CT), CGS-12066A maleate, N,N-dipropyl-5-carboxamidotryptamine maleate (dipropyl-5CT), PAPP, ±-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OH-DPAT), sumatriptan, BRL 54443 maleate, -methyl-5-HT, ±-DOI-hydrochloride (DOI), 5-methoxytryptamine, BW 723C86, 2-methyl-5-hydroxytryptamine maleate (2-Me-5-HT), 1-(m-chlorophenyl)-biguanide hydrochloride (m-CPBG), or BIMU8 (1 × 10⁹-3 × 10⁵ M). Tissues that were exposed to an agonist had a 30-min washout period before generation of a second cumulative concentration response and tissues were exposed to a maximum of two agonists in a randomized order. However, curves to BW 723C86 were always performed last because contraction to BW 723C86 was difficult to wash out. We will address the selectivity of the above-mentioned agonists later, as well as how their selectivity influences the interpretation of the results from these experiments.

Test of Antagonist, L-Type Calcium Channel, PLC, Tyrosine Kinase, Monoamine Oxidase, or 5-HT Reuptake Inhibition against 5-HT or BRL 54443. Antagonists, L-type calcium channel inhibitors, PLC inhibitors, tyrosine kinase inhibitors, a monoamine oxide (MAO) inhibitor, or a 5-HT reuptake inhibitor was administered using similar procedures as was used for agonists, but in separate experiments. The following were administered separately to tissues for a 1-h incubation: vehicle [0.1-0.5% dimethyl sulfoxide (DMSO), 0.1-0.5% deionized H₂O, or 0.1-0.5% ethanol], 1-(1-naphthyl)piperazine hydrochloride (1-NP, 5 × 10⁸ M), LY215840 (1 × 10⁸ or 3 × 10⁸ M), LY53857 (0.5 × 10⁸ or 1 × 10⁸ M), ketanserin tartrate (1 × 10⁸ M), LY272015 (5 × 10⁸ M), spiperone (1 × 10⁸ M), 3-tropanyl-indole-3-carboxylate hydrochloride (ICS 205-930, 1 × 10⁵ M), clozapine (1 × 10⁷ M), L-type calcium channel blocker nifedipine (5 × 10⁸ or 1 × 10⁶ M), L-type calcium channel blocker nitrendipine (1 × 10⁷ M), PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC, 1 × 10⁴ M), general tyrosine kinase inhibitor genistein (5 × 10⁶ M), inactive isomer of genistein, daidzein (5 × 10⁶ M), mitogen-activated protein kinase kinase (MAPKK) inhibitor PD 098,059 (1 × 10⁵ M), MAO inhibitor pargyline hydrochloride (1 × 10⁵ or 1 × 10⁶ M), or 5-HT reuptake inhibitor fluoxetine (1 × 10⁶ M). After this hour, either 5-HT or BRL 54443 was added to the bath, depending on the experiment, in a cumulative manner (1 × 10⁹-3 × 10⁴ M).

Determination of Effective Nifedipine Concentration. The concentration of nifedipine to use in investigation of 5-HT receptor signaling was validated by administering one concentration (5 × 10⁸ or 1 × 10⁶ M; 1-h incubation in the dark) of nifedipine to the tissues and then performing a cumulative concentration-response curve to KCl (6-100 mM). KCl was used as an indirect stimulus for L-type calcium channel activation, and only one concentration of nifedipine was examined for each tissue.

Determination of Effective NCDC Concentration. The concentration of NCDC used (100 µM) was determined based on its ability to abolish 5-HT-stimulated PLC activation in the rat aorta (Turla and Webb, 1990).

Data Analysis. Contractile data are presented as a mean ± S.E.M. for the number of animals indicated by N and are reported as a percentage of the initial PE (1 × 10⁵ M) contraction. The initial contraction to PE was not significantly different between compared experimental groups and thus this value has been used to normalize contractile data. Effective concentration values (EC₅₀, defined as the agonist concentration necessary to produce a half-maximal response) were calculated using a nonlinear regression analysis, using the following algorithm:

in the graphics package GraphPad Prism (San Diego, CA). Where a maximum response was not obtained but contraction was clearly observed, EC₅₀ values were estimated as greater or equal to that derived from the equation above. The apparent dissociation constant for antagonists was calculated using the following equation:
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 and [B] is the concentration of the antagonist tested.

Materials. All solutions were made daily in deionized water with the exceptions of clozapine, daidzein, genistein, ketanserin, LY215840, nitrendipine, and PD 098,059, which were made soluble in DMSO; NCDC, nifedipine, spiperone, and pargyline, which were made in ethanol; and PAPP, which was made in dilute aqueous acid. All chemicals, with the exceptions of BIMU8 (gift from Boehringer Ingleheim, Monza, Italy), BW 723C86 (Tocris, Ballwin, MO), genistein (Biomol, Plymouth Meeting, PA), LY272015 (Eli Lilly and Company, Indianapolis, IN), LY215840 (gift from Eli Lilly and Company), 5-methoxytryptamine (Sigma, St. Louis, MO), NCDC (Sigma), pargyline (Sigma), and sumatriptan (gift from Glaxo Research, Stevenage, Hertfordshire, UK) were purchased from Research Biochemicals International (Natick, MA).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of Monoamine Oxidase Inhibition and Desensitization to 5-HT. Pargyline, used to determine the extent of tissue enzymatic degradation of 5-HT by MAO, did not alter the potency of 5-HT in the mouse aorta, nor was the efficacy of 5-HT altered (Fig. 1). The serotonin reuptake inhibitor fluoxetine (1 × 10⁶ M; N = 4) did not shift contraction leftward but shifted contraction to 5-HT rightward by 8-fold (data not shown). This is consistent with fluoxetine's known interaction with 5-HT receptors, particularly 5-HT_2A and 5-HT_2C receptors (Bonhaus et al., 1997). Given these findings, neither pargyline nor fluoxetine were included in the following experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of monoamine oxidase inhibitor pargyline (1 × 106 M, 1 × 105 M) on 5-HT-induced contraction in the C57BL/6J aortae. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Vehicle (ethanol, 0.1%, N = 8, ), pargyline (1 × 106 M, ), and pargyline (1 × 105 M, N = 3, ). Data are reported as a percentage of initial contraction to a maximal concentration of PE (1 × 105 M).

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Effect of Serotonergic Agonists. Figure 2 displays the effects of serotonergic receptor agonists that caused the most potent and efficacious contraction (top), and the agonists that caused contraction with lesser potency and efficacy (Fig. 2, bottom), with 5-HT as a reference. For clarity's sake, the response to some agonists are described here in the text, and included in Table 1. The agonists removed from Fig. 1 include the nonselective receptor agonist m-CPP (log EC₅₀ [M] = 6.75, maximal contraction = 2.8 ± 1.9% PE), the 5-HT_1A agonist CGS-12066A (EC₅₀ value [M]= not calculated, maximal contraction = 0% PE), the 5-HT₃ agonist m-CPBG (EC₅₀ value [M]= not calculated, maximal contraction = 0% PE), and the 5-HT₄ agonist BIMU8 (log EC₅₀ [M] = 5.93, maximal force of contraction = 1.6 ± 1.6% PE). Agonists such as 5-HT, 5-HT₂ receptor partial agonist DOI, and 5-HT₂ receptor agonist -methyl-5-HT caused the most potent contraction in the mouse aorta. It should be noted that -methyl-5-HT also has significant affinity for the 5-HT_1E and 5-HT_1F receptor (Zgombick et al., 1992; Adham et al., 1993). BRL 54443, an agonist developed for having high affinity for the 5-HT_1E and 5-HT_1F receptor but which has measurable affinity for the 5-HT_2A receptor (Brown et al., 1998), was a partial agonist in the mouse aorta. Finally, the nonselective agonists 5-methoxytryptamine and 5-CT contracted the mouse aorta with significant potency and efficacy.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Top, comparison of contraction elicited by 5-HT, 5-CT, -methyl-5-HT, 5-HT2 receptor agonist DOI, BRL 54443, and nonselective 5-HT receptor agonist 5-methoxytryptamine. Bottom, comparison of contraction elicited by 5-HT, 2-methyl-5-hydroxytryptamine maleate, sumatriptan, PAPP, dipropyl-5CT, BW 723C86, and 8-OH-DPAT. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Data are reported as a percentage of initial contraction to a maximal concentration of PE (1 × 105 M).

View this table: [in this window] [in a new window]   TABLE 1 Negative log EC50 values [M], E values, and slope generated from the cumulative response curves to serotonergic receptor agonists in the C57BL/6J mouse aortae N = 4-10; log EC50 values are reported as means ± S.E.M.

Effect of Ketanserin on BRL 54443-Induced Contraction. Because a response to BRL 54443 and -methyl-5-HT could indicate either 5-HT_2A or 5-HT_1F receptor activation, we performed additional experiments to characterize the contraction to BRL 54443 contraction. Ketanserin, an antagonist of 5-HT_2A and 5-HT_2C receptors, was incubated with tissues for 1 h, and a cumulative concentration-response curve to BRL 54443 was generated. Figure 3 displays results of experiments in which contraction caused by BRL 54443 in the presence of ketanserin was rightward shifted with an apparent antagonist dissociation constant (pK_B) of 9.4 ± 0.15. Thus, these data indicate that contraction caused by BRL 54443 is likely mediated through stimulation of 5-HT_2A receptors and not 5-HT_1F receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of 5-HT2A receptor antagonist ketanserin on BRL 54443-induced contraction in the C57BL/6J aortae. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Vehicle (DMSO, 0.1%, N = 4, ) and ketanserin (1 × 108 M, N = 4, ). Data are reported as a percentage of initial contraction to a maximal concentration of PE (1 × 105 M). *Statistically significant (P < 0.05) between responses of tissues incubated with ketanserin and vehicle.

Effect of Serotonergic Antagonists. We next examined the ability of serotonergic antagonists to shift 5-HT-induced contraction in the mouse aorta. Antagonists that caused a large shift in contraction stimulated by 5-HT are displayed in Fig. 4, top, as well as antagonists that caused a less significant shift of contraction (Fig. 4, bottom). The experiments run with LY53857, LY215840, and spiperone had control responses that tended to be lower than that generated in other experiments, so we display in Fig. 4 two controls curves. The reason for this difference is unknown. However, in every experiment, arteries incubated with these antagonists were able to achieve a maximum contraction to 5-HT that was statistically similar to that of tissues incubated with vehicle, and thus we have treated these compounds as competitive antagonists. As was expected, ketanserin, 1-NP, LY53857, LY215840, clozapine, and spiperone, all of which have significant affinity at the 5-HT_2A receptor, caused a significant shift in 5-HT-induced contraction, whereas LY272015 and ICS205930, a 5-HT_2B receptor antagonist and a 5-HT_3/4 receptor antagonist, respectively, did not greatly shift contraction (Table 2).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Top, effect of ketanserin, 1-NP, LY53857, clozapine, spiperone, and LY215480 on 5-HT-induced contraction in the C57BL/6J aortae. Bottom, effect of LY272015 and ICS 205-930 on 5-HT-induced contraction in the C57BL/6J aortae. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Data are reported as a percentage of initial contraction to a maximal concentration of PE (1 × 105 M).

Correlations of Functional Parameters with Literature Values. We combined the pK_B and the log EC₅₀ values, and tested whether there was a correlation between these values with compound binding affinities reported in literature for the rat 5-HT receptors. This approach with combined functional data was taken for 14 different 5-HT receptors. Binding at the 5-HT_2A receptor had by far the highest correlation (r = 0.927) with functional parameters of serotonergic compounds in the mouse aorta (Fig. 5). We then separated out the log EC₅₀ values and pK_B values and performed separate correlations between each of these functional parameters and available binding data. For the 5-HT_2A receptor, the correlation value between binding data and log EC₅₀ values was 0.750 and was 0.923 between pK_B values and binding data. Similar segregated correlations with all other 5-HT receptors resulted in r values that were negative or less than 0.4. Thus, it is likely that it is the 5-HT_2A receptor that is the main contractile receptor in the mouse aorta.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Correlation of functional parameters of agonists and antagonists with literature-derived binding data for the 5-HT2A receptor.

Effects of Signaling Pathway Inhibitors. We next examined signaling mechanisms of 5-HT-induced contraction. The effect of L-type calcium channel blockers against 5-HT is shown in Figs. 6, bottom, and 7, top. Two concentrations of nifedipine were examined for their ability to inhibit KCl-induced contraction (Fig. 6, top). KCl-induced contraction was inhibited by nifedipine (5 × 10⁸ M) and a greater concentration of nifedipine (1 µM) caused no further reduction in 5-HT-induced contraction (Fig. 6, bottom). The fold shift and percentage of maximum response of L-type calcium channel blockers are listed in Table 3. L-type calcium channel blockers caused a small but significant shift and reduction in the percentage of maximal response for nitrendipine and nifedipine. Similarly, the PLC inhibitor NCDC also reduced the percentage of maximal response (47.9% of contraction, see Table 3 and Fig. 7, center) but did not significantly alter the potency of 5-HT.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Top, effect of L-type calcium channel blocker nifedipine on KCl-induced contraction in the C57BL/6J aortae. Bottom, effect of L-type calcium channel blocker nifedipine on 5-HT-induced contraction in the C57BL/6J aortae. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Data are reported as a percentage of initial contraction to a maximal concentration of PE (1 × 105 M). *Statistically significant (P < 0.05) between responses of tissue incubated with nifedipine and vehicle.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Top, effect of L-type calcium channel blocker nitrendipine on 5-HT-induced contraction in the C57BL/6J aortae. Center, effect of phospholipase C inhibitor NCDC on 5-HT-induced contraction in the C57BL/6J aortae. Bottom, effect of the general tyrosine kinase inhibitor genistein, MAPKK-specific inhibitor PD 098,095, and inactive isomer of genistein, daidzein, on 5-HT-induced contraction in the C57BL/6J aortae. Points represent the means ± S.E.M. for N, which indicates the number of C57BL/6J aortae tested. Data are reported at a percentage of initial contraction to a maximal concentration of PE (1 × 105 M). *Statistically significant (P < 0.05) between responses of tissue incubated with signaling pathway inhibitors and vehicle.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The mouse is an important model in research. Making use of the mouse as a model for research involving 5-HT, especially with the exciting advances in knockout mice and spontaneously hypertensive mice that are now available, requires us to first have an understanding and proficiency in the use of mouse vasculature and of the receptors that mediate contraction to 5-HT. Using classic pharmacological techniques, we have addressed this issue in the normal C57BL/6J male mouse.

Pargyline did not cause a shift or a change in the magnitude of contraction to 5-HT, a change we would have expected if 5-HT were rapidly metabolized through MAO. The inability of pargyline and fluoxetine to potentiate the arterial response to 5-HT suggests that 5-HT is not rapidly metabolized or taken up in the mouse aorta, and therefore, we did not add it to our buffer for these experiments.

Our experiments have generated several findings that support the hypothesis that the primary 5-HT receptor mediating contraction in the mouse aorta is likely a 5-HT_2A receptor. These experiments did not address the possibility of 5-HT-induced relaxation or the role of endothelium in modulating 5-HT-induced contraction because all experiments were performed in the absence of endothelium. Our first finding is the significant potency and efficacy of agonists with significant affinity for the 5-HT_2A receptor, such as -methyl-5-HT, DOI, and BRL 54443, to cause aortic contraction and the lower potency and efficacy of drugs that have a lower affinity for the 5-HT_2A receptor. DOI, as has been found in other preparations, acted as a partial agonist. 5-CT has low affinity for the 5-HT_2A receptor (Hoyer, 1989) and 5-methoxytryptamine has higher affinity for the 5-HT_2A receptor than 5-CT (Bonhaus et al., 1997), so the rank order of agonist potencies is consistent with interaction with a 5-HT_2A receptor.

It is important to note here that truly selective agonists for the 5-ht_5A, 5-ht_5B, 5-HT₆, and 5-HT₇ receptor were not available and therefore could not be tested, but that several of the agonists examined have measurable affinity for these newer receptors. For example, 2-methyl-5-HT has significant affinity for the 5-HT₆ receptors; 5-CT for the 5-HT₅, 5-HT₆ (Erlander et al., 1993; Boess et al., 1996), and 5-HT₇ receptors (Ruat et al., 1993); and 8-OH-DPAT and dipropyl-5-CT at 5-HT₇ receptors (Shen et al., 1993; To et al., 1995, respectively). The lack of significant potency and efficacy of these compounds, with the exception of 5-CT, would argue that it is unlikely that 5-HT₆ and 5-HT₇ receptors play a role in mediating 5-HT-induced contraction in the mouse thoracic aorta. One can speculate that this would be the case because one manner by which these two receptor families mediate their intracellular signaling is through activation of Gs and adenylate cyclase, a process that would result in arterial relaxation. From these standpoints, the fact that some of the 5-HT receptor compounds tested had affinity for these receptors, in addition to the receptor for which we were using the compound, was not a concern. Moreover, we are unaware of reports that have localized the 5-HT₆ receptor to the vasculature; the 5-HT₇ receptor has been found and demonstrated to be functionally active in arterial tissue (Terron, 1996). The lack of presence of a receptor or clear dissociation of activation of the receptor from modulating arterial contractile function allows us to make a conclusion from the experiments with 5-HT receptor agonists with some confidence.

It was interesting that BRL 54443, a 5-HT_1E/1F receptor agonist, caused contraction. As current research indicates, it is unlikely that 5-HT_1F receptor mediates 5-HT-induced contraction of the vasculature (Cohen and Schenck, 1999) and our findings agree with this. This is based on the finding that BRL 54443-induced contraction could be blocked by ketanserin and ketanserin does not have significant affinity for the 5-HT_1F receptor (human; Adham et al., 1993). Moreover, other agonists that also possess significant affinity for the 5-HT_1F receptor such as sumatriptan and 2-methyl-5-HT do not cause a significant contraction in the mouse aorta.

The second finding supporting the hypothesis is the shift of 5-HT-induced contraction caused by antagonists with a high affinity for the 5-HT₂ receptor. Ketanserin, LY53857, spiperone, LY215840, clozapine, and 1-NP caused a significant shift in response to 5-HT, as was expected for blockers with significant affinity for 5-HT₂ receptor. Of these blockers, spiperone is selective for the 5-HT_2A receptor over the 5-HT_2C receptor (Hoyer, 1989) and the ability of spiperone to shift 5-HT-induced contraction, in combination with the shifts caused by other antagonists, suggests that it is the 5-HT_2A receptor mediating contraction in the mouse aorta.

The correlations generated using the log EC₅₀ values and pK_B values we obtained from our own experiments versus the binding data we derived from literature based on the rat model were used to determine what receptor was primary in mediating contraction. The correlation of functional parameters, individually or in combination, with binding data for rat showed a strong correlation at the 5-HT_2A receptor. These were by far the strongest correlations compared with the value obtained for other 5-HT receptors. The only correlation that came close to the correlation at the 5-HT_2A receptor was the correlation at the 5-HT_2C receptor of 0.512 (agonists and antagonists included in the correlation); this receptor has been shown to have a very similar structure to the 5-HT_2A receptor (Hoyer et al., 1994). There remains the possibility of the presence of another small population of receptors, such as the 5-HT_2C receptor, although the 5-HT_2C receptor protein has never been found peripherally. Such correlations suggest that the most likely receptor mediating contraction to 5-HT in the mouse aorta is a 5-HT_2A receptor, as it is in the rat (Fig. 5).

Our experiments also generated evidence that 5-HT couples to L-type calcium channels, phospholipase C, and tyrosine kinase-dependent signaling pathways for contraction, as is seen in the rat (Florian and Watts, 1997). The greatest shift of contraction, which also partially reduced the maximal force of contraction, was to genistein, a general tyrosine kinase inhibitor, and this suggests the 5-HT contractile receptor in the mouse aorta is dependent on tyrosine kinase as a signaling pathway. PD 098,095 caused a small shift, suggesting that MAPKK is one but not the only tyrosine kinase involved. The rightward shift in the presence of daidzein (2.5-fold) likely indicates that although it is the inactive isomer of genistein, it is not completely devoid of its inhibitory properties. Similar to that found for 5-HT-induced contraction in rat aorta, L-type calcium channel inhibitors nifedipine and nitrendipine caused a significant reduction in the maximum contraction to 5-HT with a small rightward fold shift. This indicates that L-type calcium channel activation is necessary for 5-HT to contract mouse aorta. The same appears true of tissues in the presence of phospholipase C inhibitor NCDC. There was not a great rightward shift in contraction as seen with genistein, but there was a significant reduction in the ability of 5-HT to contract back to maximal response in the presence of NCDC. Because contraction was not able to be completely abolished in the presence of any one of the signaling pathway inhibitors, it is likely a combination of these signaling pathways is responsible for this contraction.

In summary, the correlation for functional parameters of serotonergic compound values generated in our own experiments versus the values found in published literature for the rat strongly support the receptor being a 5-HT_2A receptor. These pharmacological data support a 5-HT_2A receptor mediating contraction in the isolated mouse aorta, and that this contraction is coupled to L-type calcium channels, phospholipase C, and tyrosine kinase signaling pathways.