Abstract
The purpose of this study was to characterize pharmacologically the 5-HT receptor(s) mediating contraction in the mouse aorta and the pathways these receptors are coupled with to mediate contraction. We hypothesized that a 5-HT2A receptor, as in the rat, mediates contraction by activating L-type calcium channels, phospholipase C (PLC), and tyrosine kinase(s). Endothelium-denuded aortic strips were placed in a tissue bath for measurement of isometric contractile force. 5-HT, the 5-HT2A receptor agonist α-methyl-5-HT, and partial 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (±-DOI) caused the most potent and efficacious contraction. The 5-HT1E/1F receptor agonist BRL 54443 also induced contraction (−log EC50 = 6.52); however, the 5-HT2A receptor antagonist ketanserin antagonized this contraction. Our hypothesis was further supported by the finding that antagonists with affinity for the 5-HT2A receptor, ketanserin, 1-(1-naphthyl)piperazine, spiperone, and LY53857, reduced 5-HT-induced contraction. A correlation of 0.927 was found between literature-derived compound binding affinities for the agonists and antagonists at the 5-HT2A receptor of the rat and the data generated in our experiments (−log EC50 and pKB values). The L-type calcium channel blockers nifedipine and nitrendipine, PLC inhibitor 2-nitro-4-carboxyphenylN,N-diphenylcarbamate, and tyrosine kinase inhibitors genistein and PD 098,059 all shifted and/or reduced maximum contraction to 5-HT. We conclude that contraction to 5-HT in the mouse aorta is mediated primarily by a 5-HT2A receptor and is coupled to L-type calcium channels, PLC, and tyrosine kinases.
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-HT2A receptor, as defined by activation by the agonist α-methyl-5-hydroxytryptamine maleate (α-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-HT2Areceptor, 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
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 α1-adrenergic receptor agonist phenylephrine (PE, 1 × 10−5 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 × 10−6 M) in tissues after first contracting the tissue with a half-maximal concentration of phenylephrine (1 × 10−8 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, α-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 × 10−9–3 × 10−5 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 × 10−8 M), LY215840 (1 × 10−8 or 3 × 10−8M), LY53857 (0.5 × 10−8 or 1 × 10−8 M), ketanserin tartrate (1 × 10−8 M), LY272015 (5 × 10−8 M), spiperone (1 × 10−8 M), 3-tropanyl-indole-3-carboxylate hydrochloride (ICS 205-930, 1 × 10−5 M), clozapine (1 × 10−7 M), L-type calcium channel blocker nifedipine (5 × 10−8 or 1 × 10−6 M), L-type calcium channel blocker nitrendipine (1 × 10−7 M), PLC inhibitor 2-nitro-4-carboxyphenylN,N-diphenylcarbamate (NCDC, 1 × 10−4 M), general tyrosine kinase inhibitor genistein (5 × 10−6 M), inactive isomer of genistein, daidzein (5 × 10−6 M), mitogen-activated protein kinase kinase (MAPKK) inhibitor PD 098,059 (1 × 10−5 M), MAO inhibitor pargyline hydrochloride (1 × 10−5 or 1 × 10−6 M), or 5-HT reuptake inhibitor fluoxetine (1 × 10−6 M). After this hour, either 5-HT or BRL 54443 was added to the bath, depending on the experiment, in a cumulative manner (1 × 10−9–3 × 10−4 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 × 10−8 or 1 × 10−6 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 × 10−5 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:
Efficacy (E) value was determined using the formula agonists average maximal contraction/5-HT's average maximal contraction. Slope was determined using the graphics package GraphPad Prism by using a nonlinear regression algorithm. Correlations were generated using binding data found in literature for 5-HT receptors in the rat (Fozard, 1989; Hoyer, 1989; Wainscott et al., 1993; Cushing et al., 1996;Bonhaus et al., 1997; Colas et al., 1997; Brown et al., 1998) versus the EC50 and/or pKB values generated from our own data using a linear regression analysis within GraphPad Prism.
Fold shift from vehicle was calculated using the following formula:
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
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 × 10−6 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-HT2A and 5-HT2C receptors (Bonhaus et al., 1997). Given these findings, neither pargyline nor fluoxetine were included in the following experiments.
To justify testing two serotonergic agonists consecutively, experiments investigating the reproducibility of 5-HT-induced contraction were performed. 5-HT (1 × 10−6 M) was added to the tissue bath and contraction was allowed to plateau. Tissues were then washed every 5 min for a total of 30 min. Tissues were again challenged with 5-HT (1 × 10−6 M). The contraction to this second administration of 5-HT was 126.25 ± 13.29% of the first contraction and this was not statistically significantly different from the first response (P > 0.05). Thus, serotonergic responsiveness is maintained throughout the experiment.
Effect of Serotonergic Agonists.
Figure2 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 agonistm-CPP (−log EC50 [M] = 6.75, maximal contraction = 2.8 ± 1.9% PE), the 5-HT1A agonist CGS-12066A (EC50 value [M]= not calculated, maximal contraction = 0% PE), the 5-HT3 agonistm-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 α-methyl-5-HT caused the most potent contraction in the mouse aorta. It should be noted that α-methyl-5-HT also has significant affinity for the 5-HT1E and 5-HT1F receptor (Zgombick et al., 1992; Adham et al., 1993). BRL 54443, an agonist developed for having high affinity for the 5-HT1E and 5-HT1F receptor but which has measurable affinity for the 5-HT2Areceptor (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.
Of the agonists listed in the bottom of Fig. 2, only BW 723C86, an agonist of the 5-HT2B receptor, caused a concentration-dependent and observably efficacious contraction. This contraction, however, was far removed in magnitude from that observed with 5-HT itself. PAPP, dipropyl-5-CT, and 8-OH-DPAT were used as agonists with significant affinity for the 5-HT1Areceptor; sumatriptan as an activator of 5-HT1B, 5-HT1D, and 5-HT1Freceptors (Adham et al., 1993); and 2-methyl 5-HT as an agonist of 5-HT3 receptors, as well as possessing affinity for 5-HT2B and 5-HT6receptors (Boess et al., 1996; Wainscott et al., 1996). Potency values [−log EC50 values] and E values for each agonist were generated and they are listed in Table 1. Collectively, the data displayed in Fig. 2 and Table 1 demonstrate that agonists that possess measurable and significant affinity for the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2B, 5-HT3, and 5-HT4 receptors caused a contraction weak in potency and efficacy. The highest E values were found for 5-HT2 receptor agonist α-methyl-5-HT and nonselective receptor agonist 5-methoxytryptamine. In addition to those agonists, 5-CT, BRL 54443, and the 5-HT2 receptor agonist DOI had E values above 0.5.
Effect of Ketanserin on BRL 54443-Induced Contraction.
Because a response to BRL 54443 and α-methyl-5-HT could indicate either 5-HT2A or 5-HT1F receptor activation, we performed additional experiments to characterize the contraction to BRL 54443 contraction. Ketanserin, an antagonist of 5-HT2A and 5-HT2Creceptors, was incubated with tissues for 1 h, and a cumulative concentration-response curve to BRL 54443 was generated. Figure3 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 (pKB) of 9.4 ± 0.15. Thus, these data indicate that contraction caused by BRL 54443 is likely mediated through stimulation of 5-HT2A receptors and not 5-HT1F receptors.
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-HT2A receptor, caused a significant shift in 5-HT-induced contraction, whereas LY272015 and ICS205930, a 5-HT2B receptor antagonist and a 5-HT3/4 receptor antagonist, respectively, did not greatly shift contraction (Table 2).
Correlations of Functional Parameters with Literature Values.
We combined the pKB 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-HT2A 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 pKB values and performed separate correlations between each of these functional parameters and available binding data. For the 5-HT2A receptor, the correlation value between binding data and −log EC50 values was 0.750 and was 0.923 between pKB values and binding data. Similar segregated correlations with all other 5-HT receptors resulted inr values that were negative or less than 0.4. Thus, it is likely that it is the 5-HT2A receptor that is the main contractile receptor in the mouse aorta.
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 × 10−8 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.
The effects of tyrosine kinase inhibitors on 5-HT-induced contraction in the mouse aorta are displayed in Fig. 7 (bottom). The shifts of contraction to the tyrosine kinase inhibitors are listed in Table 3. There was a rightward shift in the presence of daidzein (2.5-fold), but also an increase in 5-HT percentage of maximum response at 117.1% that was not statistically significant. The general tyrosine kinase inhibitor genistein had the greatest shift (5.85) and also reduced the maximal contraction to 5-HT. The MAPKK-specific inhibitor PD 098,095 caused a 1.05-fold shift in comparison to its vehicle.
Discussion
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-HT2A 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-HT2Areceptor, such as α-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-HT2A receptor. DOI, as has been found in other preparations, acted as a partial agonist. 5-CT has low affinity for the 5-HT2A receptor (Hoyer, 1989) and 5-methoxytryptamine has higher affinity for the 5-HT2A receptor than 5-CT (Bonhaus et al., 1997), so the rank order of agonist potencies is consistent with interaction with a 5-HT2A receptor.
It is important to note here that truly selective agonists for the 5-ht5A, 5-ht5B, 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-HT7receptors (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-HT1E/1Freceptor agonist, caused contraction. As current research indicates, it is unlikely that 5-HT1F 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-HT1Freceptor (human; Adham et al., 1993). Moreover, other agonists that also possess significant affinity for the 5-HT1Freceptor 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-HT2A receptor over the 5-HT2C 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-HT2A receptor mediating contraction in the mouse aorta.
The correlations generated using the −log EC50values and pKB 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-HT2A 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-HT2A receptor was the correlation at the 5-HT2C 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-HT2A receptor (Hoyer et al., 1994). There remains the possibility of the presence of another small population of receptors, such as the 5-HT2C receptor, although the 5-HT2C 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-HT2A 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-HT2A receptor. These pharmacological data support a 5-HT2A 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.
Footnotes
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Send reprint requests to: Carolyn McKune/Dr. Stephanie W. Watts, Department of Pharmacology and Toxicology, B445 Life Sciences Bldg., Michigan State University, East Lansing, MI 48824-1317. E-mail:wattss{at}msu.edu
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The American Heart Association (Michigan Affiliate), as well as the College of Veterinary Medicine at Michigan State University, and National Institutes of Health HL 58489 generously funded part of this project.
- Abbreviations:
- 5-HT
- 5-hydroxytryptamine
- α-methyl-5-HT
- α-methyl serotonin maleate
- PLC
- phospholipase C
- PE
- phenylephrine
- m-CPP
- 1-(3-chlorophenyl)piperazine
- 5-CT
- 5-carboxamidotryptamine maleate
- dipropyl-5-CT
- N,N-dipropyl-5-carboxamidotryptamine maleate
- PAPP
- p-aminophenylethyl-m-trifluoromethylphenyl piperazine
- 8-OH-DPAT
- (±)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide
- DOI
- (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride
- 2-Me-5-HT
- 2-methyl-5-hydroxytryptamine maleate
- m-CPBG
- 1-(m-chlorophenyl)-biguanide hydrochloride
- MAO
- monoamine oxidase
- DMSO
- dimethyl sulfoxide
- 1-NP
- 1-(1-naphthyl)piperazine hydrochloride
- ICS 205-930
- 3-tropanyl-indole-3-carboxylate hydrochloride
- NCDC
- 2-nitro-4-carboxyphenylN,N-diphenylcarbamate
- MAPKK
- mitogen-activated protein kinase kinase
- E
- efficacy
- Received July 21, 2000.
- Accepted December 7, 2000.
- The American Society for Pharmacology and Experimental Therapeutics