A number of masking drugs were used in the present study in order to inhibit the binding of [3H]-mesulergine to other receptors for which it has affinity. Mesulergine has been reported to have affinity for 5-HT2A and 5-HT2C receptors as well as some affinity for dopamine D2 and α1/α2-adrenoceptors (Closse, 1983; Rinne, 1983; Hoyer et al., 1994), (see Methods). The masking drugs, some of which also have affinity for 5-HT7 receptors, were chosen at concentrations which would theoretically occupy at least 90% of their targetted receptor populations (Kalkman et al., 1986; Hoyer et al., 1994; Sleight et al., 1995a; Bonhaus et al., 1997), without significantly affecting the binding of the radioligand to the 5-HT7 receptor.
Saturation studies performed in rat brain and guinea-pig ileum provided pKD values of 8.0±0.04 and 7.9±0.11 respectively for [3H]-mesulergine, which were in accordance with a previously reported pKB value of 7.8 (Carter et al., 1995) and a pKi value of 8.15 (Hoyer et al., 1994; To et al., 1995) at the 5-HT7 receptor. However, no evidence of specific binding to a 5-HT7 site was apparent in the rat jejunum.
Displacement of [3H]-mesulergine binding in rat brain and guinea-pig ileum was observed using the agonists 5-CT (pKi=11.4, 12.1), 5-HT (9.9, 10.0), 5-MeOT (9.2, 9.6) as well as sumatriptan (7.6, 7.0) and the antagonists risperidone (8.3, 9.4), ritanserin (7.6, 8.3), metergoline (8.0, 8.8), LSD (8.1, 9.4) and pindolol (4.5, 6.3), respectively. The pKi values of these compounds correlated well with those from other studies of the 5-HT7 receptor in both order and magnitude (Kalkman et al., 1986; Bard et al., 1993; Plassat et al., 1993; Ruat et al., 1993; Shen et al., 1993; Roth et al., 1994; To et al., 1995).
5-CT, 5-HT, 5-MeOT and sumatriptan displayed two-site displacement binding in the rat brain and guinea-pig ileum, which is not unusual for agonists, since they can bind to both a high and low affinity state of the receptor (Kenakin, 1984). However, the two-site binding displayed by ritanserin in the rat brain is more difficult to explain, and was not detected in the guinea-pig ileum nor reported in guinea-pig brain using [3H]-5-CT (To et al., 1995).
The possibility of [3H]-mesulergine binding to non-serotonergic receptors, such as dopamine receptors, were ruled out by the addition of masking drugs such as raclopride (Rinne, 1983) and by the use of the agonists 5-CT, 5-HT and 5-MeOT which are specific for serotonin receptors. In addition, a preliminary experiment performed with haloperidol (0.01 nM to 1 μM), a potent D2 receptor antagonist (pKi=9.3), with little affinity for 5-HT7 receptors (pKi=6.6; Roth et al., 1994), failed to displace mesulergine binding in rat brain.
The binding site identified in both rat brain and guinea-pig ileum is unlikely to be of the 5-HT1A or 5-HT1B (rat brain) subtype, since pindolol was found to have a low (micromolar) affinity in this study, consistent with 5-HT7 but not 5-HT1 receptors, where it has nanomolar affinity (Lovenberg et al., 1993; Ruat et al., 1993; Hoyer et al., 1994; Carter et al., 1995; McLean & Coupar, 1996). The other 5-HT1 receptor subtypes; 5-HT1D (guinea-pig ileum), 5-ht1E, and 5-ht1F were ruled out by the high affinity of 5-CT in both the rat brain (pKi=11.4) and guinea-pig ileum (pKi=12.1, Hoyer et al., 1994).
The receptor is also unlikely to be the 5-HT2A or 5-HT2C subtype, firstly because these receptors were excluded with cinanserin and RS 102221, respectively, and more importantly the antagonist order of affinity of risperidone (pKi=8.7, 9.4), metergoline (8.0, 8.8) and ritanserin (7.7, 8.3) in rat brain and guinea-pig ileum, respectively did not correlate with either 5-HT2A (risperidone>ritanserin>metergoline) or 5-HT2C (metergoline>ritanserin>risperidone) receptors (Hoyer et al., 1994, Sleight et al., 1995a). In addition, 5-CT was found to have nanomolar affinity in this study whilst it has micromolar affinity or lower at both 5-HT2A and 5-HT2C receptor sites (Bard et al., 1993; Hoyer et al., 1994; Roth et al., 1994). Furthermore, sumatriptan was found to have micromolar affinity in this study whilst it has only millimolar affinity at 5-HT2A and 5-HT2C receptors (Hoyer et al., 1994).
The involvement of 5-HT4, 5-ht5 and 5-ht6 receptors is excluded due to the use of a nanomolar concentration of [3H]-mesulergine in displacement studies. Mesulergine has only micromolar affinity at 5-ht5A, 5-ht5B and 5-ht6 receptors (Plassat et al., 1993; Erlander et al., 1993; Monsma et al., 1993) and is inactive at 5-HT4 receptors (Bard et al., 1993). Further evidence against 5-ht5B and 5-ht6 receptor involvement is the low affinity of 5-CT (pKi=7.4 and 6.6 respectively) for these receptors (Hoyer et al., 1994).
While the 5-HT orphan receptor reported by Castro et al. (1997) has a high affinity for 5-CT, it cannot be the receptor in question since mesulergine has only a micromolar affinity at the orphan receptor (Castro et al., 1997). In addition the 5-HT orphan receptor has a similar affinity for both 5-CT and 5-HT (Castro et al., 1997), a characteristic not displayed by the receptor in the present study.
This suggestion is further supported by the high affinity of atypical antipsychotics such as clozapine and risperidone for the 5-HT7 receptor (Roth et al., 1994). Moreover, the 5-HT7 receptor has also been implicated in other affective disorders such as depression, where it was found that a down-regulation of the receptor occurs after chronic antidepressant treatment (Sleight et al., 1995a). In addition 5-HT7 receptors may have a role in the regulation of mammalian circadian rhythms (Lovenberg et al., 1993).
The high affinity binding of LSD in rat brain in the present study adds further support to a 5-HT7 site where a pKi value similar to that reported in guinea-pig brain by To et al. (1995) was obtained (pKi 8.2 compared with 7.8). This raises the possibility that the hallucinogenic action of LSD may be mediated in part via the 5-HT7 receptor.
Peripheral tissue studies
The results of the binding studies in guinea-pig ileum lend further support to the functional findings of Feniuk et al. (1984), Kalkman et al. (1986) and Carter et al. (1995) where a postjunctional 5-HT site was seen to induce relaxation of pre-contracted guinea-pig ileum. The binding studies performed in this investigation are concordant with those of Kalkman et al. (1986), where [125I]-LSD was used to characterize a postjunctional 5-HT site in guinea-pig ileum. In the study by Kalkman et al. (1986), cinanserin (300 nM) was used to mask 5-HT2 receptors, a concentration which the authors claim theoretically inhibits 98% of 5-HT2 receptors. Unfortunately, this concentration of cinanserin was also found to occupy close to 50% of the 5-HT7-like site in a displacement study in the same investigation. This may have led to an underestimation of the KD value of [125I]-LSD at the 5-HT7 site. Nevertheless, the antagonist order of affinity reported (iodo-LSD>metergoline = mesulergine>spiperone> >haloperidol>propranolol) correlates well with a 5-HT7 receptor.
Despite the lack of binding of [3H]-mesulergine in the rat jejunum, the functional experiments performed in the present study suggest the presence of a 5-HT7-like receptor in this tissue, in agreement with the findings of McLean & Coupar (1996). The low affinity of pindolol and lack of antagonist affinity of yohimbine, cinanserin, RS 102221 and GR 113808 rule out a 5-HT1A, α2, 5-HT2A, 5-HT2C and 5-HT4 receptor site, in contrast to the findings of Javid & Naylor (1997), who reported the involvement of 5-HT2 and 5-HT4 receptors in mediating the contractile response of 5-HT in the proximal region of the rat small intestine. The use of ondansetron (1 μM) and the relatively high potency of 5-CT rules out 5-HT3 receptors. The presence of 5-ht5 and 5-ht6 receptors can also be excluded by the potency of 5-CT at this site. The relatively low potency of 5-CT compared to its high affinity in binding studies is characteristic of the 5-HT7 receptor and was also reported by Carter et al. (1995), and Martin & Wilson (1995). The correlation between the pKB and apparent pKB values obtained in the rat jejunum and recombinant 5-HT7 receptors from rat or mouse expressed in transfected cells (Plassat et al., 1993; Ruat et al., 1993; Shen et al., 1993; Roth et al., 1994) was very high (see Results).
It is interesting to note that the affinity values of mesulergine (7.3) and ritanserin (7.3) were lower in the present study with the rat isolated jejunum than reported in a similar study by McLean & Coupar (1996), where mesulergine and ritanserin were reported to have pA2 values of 8.1 and 8.0 respectively. Affinity values for LSD, mesulergine and ritanserin reported in the guinea-pig ileum, receptors expressed from 5-HT7 receptor genes (Ruat et al., 1993; Carter et al., 1995) and binding studies in the rat brain and guinea-pig ileum in this investigation were also slightly higher than those obtained in the rat jejunum. However, binding studies performed in the guinea-pig brain (To et al., 1994), have shown a pKD value of 7.8 for LSD, which agrees with the pKB value obtained in the present study. In addition, risperidone, ritanserin and LSD resulted in a non-competitive antagonism of the serotonin response in the presence of ondansetron (1 μM). At higher concentrations, risperidone (10–100 nM) and ritanserin (100 nM–1 μM) caused a depression of the maximum response to 5-HT without further shifting the control curve. A comparison of the functional characteristics of LSD and risperidone at the 5-HT7 receptor can not be made, as the effect of these compounds has not been investigated in other studies. However, at a concentration range of 10–100 nM, ritanserin was reported to act as a competitive inhibitor of the 5-HT7-like response in the rat jejunum, (McLean & Coupar, 1996). The observation that an increased concentration of ritanserin and risperidone did not further shift the mean curve to 5-HT may indicate that the interaction of the antagonists with the 5-HT7 receptor is of a non-competitive nature such as that seen with allosteric modulation. Negative allosteric modulators are known to produce parallel shifts of agonist concentration-response curves up to a limiting value (Ehlert, 1988; Lanzafame et al., 1996). Often, as seen with ritanserin, they may appear to be competitive at lower concentration ranges. Further studies are required to fully define the mechanism by which ritanserin and risperidone interact with the 5-HT7 site.
The lack of [3H]-mesulergine binding in the rat jejunum in this study may be explained by a low 5-HT7 receptor density. This is supported by the observation that a response to 5-HT in the rat jejunum after blockade of 5-HT3 receptors with ondansetron was not always present; (see Results). The response observed in the rat jejunum differs from other smooth muscles where activation of the 5-HT7 receptor results in relaxation or various preparations in different species, such as porcine vena cava and myometrium (Sumner et al., 1989; Kitazawa et al., 1998), canine coronary artery (Cushing & Cohen, 1992), marmoset aorta (Dyer et al., 1994) guinea-pig ileum (Feniuk et al., 1984; Kalkman et al., 1986; Carter et al., 1995) and cat saphenous vein (Hoyer et al., 1994). In addition, the contractile response is not consistent with the coupling mechanism of the 5-HT7 receptor, which to date has been shown to only involve activation of adenylate cyclase, (Shenker et al., 1987; Eglen et al., 1997).
5-HT7 receptor subtypes?
The possibility of 5-HT7 receptor subtypes cannot be excluded; Ruat et al. (1993) isolated a 448 amino acid cDNA in the rat (5-HT7(a)), while Lovenberg et al. (1993) isolated a 435 amino acid cDNA (5-HT7(b)). The difference between the two cDNAs was only in the carboxy-terminal of the seven transmembrane structure of the expressed receptor and was said to result from alternative splicing (Boess & Martin, 1994). The generation of two receptor variants differing in the carboxy regions, can result in differential coupling to G-proteins (Lucas & Hen, 1995) and possibly a different physiological response as has been reported with the prostaglandin EP3 (Namba et al., 1993) and somatostatin SST2 receptors (Vanetti et al., 1993). In fact, the neuronal 5-HT7(a) isoform has been reported to increase intracellular calcium resulting in activation of calmodulin-stimulated, adenylate cyclase isoforms AC1 and AC8, independent of phosphoinositide and protein kinase C (Baker et al., 1998). This unique mechanism of coupling for any of the serotonin receptors was reported to be Gs-independent in studies with whole cells (see Wayman et al., 1994; Sunahara et al., 1996). It is possible some similar mechanism may exist in the rat jejunum, where a contraction instead of a relaxation to a 5-HT7-like receptor was observed.
Furthermore, an additional rat 5-HT7 isoform, a 470 amino acid cDNA, (5-HT7(c)) resulting from a retained exon cassette, has also been reported (Heidmann et al., 1997). However, unlike the other two isoforms, the rat 5-HT7(c) was reported not to be present in human tissue, which suggests a species difference for the presence of various 5-HT7 isoforms (Heidmann et al., 1997). This again raises the possibility of a difference in the physiological response produced by 5-HT7 receptors and may explain why an ‘atypical’ response was obtained in the rat jejunum.
In conclusion binding studies performed with [3H]-mesulergine were able to detect 5-HT7 sites in rat brain and guinea-pig ileum, but not rat jejunum, where a functional 5-HT7-like site was present.