Evidence for common pharmacological properties of [3H]5-hydroxytryptamine binding sites,presynaptic 5-hydroxytryptamine autoreceptors in CNS and inhibitory presynaptic 5-hydroxytryptamine receptors on sympathetic nerves

The affinities of 16 5-hydroxytryptamine (5-HT) receptor agonists (indole derivatives) and 7 5-HT receptor antagonists for [3H]5-hydroxytryptamine [( 3H]5-HT) binding sites in rat cerebral cortex membranes were determined. In addition, the potencies of the agonists for inhibiting the electrically induced tritium overflow from rat brain cortex slices preincubated with [3H]5-HT and from canine saphenous veins preincubated with [3H]noradrenaline were measured. Furthermore, the potencies of the indole derivatives for inducing contractile responses of canine saphenous veins were recorded. In addition, the interaction of the antagonists with unlabelled 5-HT at the 5-HT autoreceptor was studied in rat brain cortex slices. There was a good correlation between the binding affinities of the indole derivatives for the [3H]5-HT sites of rat brain cortex membranes and their potencies for inhibiting the evoked tritium overflow from both rat brain cortex slices and strips of canine saphenous vein. Comparison of the inhibition constants derived from the overflow experiments in both tissues again revealed a high correlation coefficient while there was only weak correlation between the binding affinities in rat brain cortex and the contractile potencies of the drugs in canine saphenous vein strips. When 5-HT receptor antagonists were investigated, metitepin and metergoline showed moderate affinities for the 5-HT autoreceptors in rat brain cortex slices, whereas quipazine had only weak affinity, and ketanserin, metoclopramide, cinanserin and cyproheptadine exhibited no antagonistic property. In binding experiments, the competition curves of most 5-HT receptor antagonists were biphasic, suggesting that the [3H]5-HT binding sites are heterogeneous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

Summary The agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [³H]5-hydroxytryptamine ([³H]5-HT) binding sites and the affinities of the antagonists for [¹²⁵Iodo]LSD ([¹²⁵I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the K _D values published for displacement of [³H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT₂ binding sites labelled by both [¹²⁵I]LSD and [³H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT₁ binding sites labelled by [³H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT₂-like receptors.     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

5-HT1A-receptors mediate stimulation of adenylate cyclase in rat hippocampus

Serotonin (5-HT) stimulated adenylate cyclase activity in homogenates of rat hippocampus. This effect was pharmacologically characterised with a series of agonists and antagonists of various structural classes. These compounds where also tested in radioligand binding studies using selective ligands for the various subtypes of 5-HT1 and 5-HT2 receptors. 5-HT1A, 5-HT1B and 5-HT1C recognition sites were labelled with [3H]8-OH-DPAT([3H]8-hydroxy-2-(di-n-propylamino)-tetralin) in pig cortex membranes, [125I]CYP([125I]iodocyanopindolol) in rat cortex and [3H]mesulergine in pig choroid plexus membranes, respectively. The rank order of potency of 13 agonists stimulating adenylate cyclase activity in homogenates of rat hippocampus was in good agreement with the rank order of affinity of these agonists for the 5-HT1A binding site: N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT) greater than 5-carboxamidotryptamine (5-CT) greater than 8-OH-DPAT greater than 5-HT greater than 5-methoxytryptamine (5-OCH3T) greater than d-LSD greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) greater than alpha-methylserotonin (alpha-CH3-5-HT) greater than dopamine greater than 2-methylserotonin (2-CH3-5-HT). The correlation between the respective potencies and affinities of these agonists was r = 0.934, P less than 0.001. There was no correlation between stimulation of adenylate cyclase activity by these agonists and their affinity for 5-HT1B, 5-HT1C or 5-HT2 binding sites. r = 0.381-0.108, P less than 0.20-0.73.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

The pharmacological properties of the presynaptic serotonin autoreceptor in the pig brain cortex conform to the 5-HT1D receptor subtype

Summary The effects of serotonin receptor agonists and antagonists on the electrically (3 Hz) evoked ³H overflow were determined on pig brain cortex slices preincubated with ³H-serotonin and superfused with physiological salt solution containing indalpine (an inhibitor of serotonin uptake) plus phentolamine. The potencies of the serotonin receptor agonists and antagonists were compared with their affinities for 5-HT_1A, 5-HT_1B, 5-HT_1c, and 5-HT_1D binding sites in pig or rat tissue membranes; in addition, the potencies of the agonists were compared to their potencies in inhibiting adenylate cyclase activity in membranes of calf substantia nigra. In the superfusion experiments on pig brain cortex slices the following rank orders of potencies were obtained: agonists, serotonin > 5-methoxytryptamine = 5-carboxamidotryptamine >R U 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) > SDZ 21009 (4(3-terbutylamino- 2-hydroxypropoxy)indol- 2-carbonic-acid-isopropylester) yohimbine cyanopindolol > 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) CGS 12066 B (7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrrolo[1,2-a]quinoxaline); ipsapirone and urapidil were ineffective; antagonists (antagonism determined against 5methoxytryptamine as an agonist), metitepine > metergoline > mianserin. Propranolol, spiperone or mesulergine did not produce a shift of the concentration-response curve for 5-methoxytryptamine. The potencies of the serotonin receptor agonists in pig brain cortex slices were significantly correlated with their affinities for 5-HT_1c and 5-HT_1D binding sites in membranes of the pig choroid plexus and caudate nucleus, respectively, but not with their affinities for 5-HT_1A and 5-HT_1B sites in membranes of the cerebral cortex of pig and rat, respectively. The agonist potencies in decreasing ³H overflow were also significantly correlated with their potencies in inhibiting adenylate cylase activity in calf substantia nigra (i.e., a 5-HT_1D receptor-mediated effect). In conclusion, the pig brain cortical 5-HT autoreceptor probably belongs to the 5-HT_1D subtype. The involvement of 5-HT_1c recognition sites was excluded by the low potency of mianserin as an antagonist and, in particular, by the ineffectiveness of the 5-HT_1c receptor antagonist mesulergine.E. S. and M. G. were supported by grants of the Deutsche ForschungsgemeinschaftSend offprint requests to M. Göthert at the above address     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selectivity of some ergot derivatives for 5-HT1 and 5-HT2 receptors of rat cerebral cortex

The affinities of several ergot derivatives for 5-HT1 and 5-HT2 receptors were evaluated using [3H]5-HT and [3H]mianserin, and membranes prepared from rat frontal cortex. CQ 32-084, which had a Ki value against the 5-HT2 component of [3H]mianserin binding of 9 nM, had little effect on [3H]5-HT binding and was selective for the 5-HT2 receptor. Lisuride and LY-158A also displayed preferential affinity for the 5-HT2 receptor. Dihydroergocryptine, CM 29-712, lergotrile and LY-062 were more selective for the 5-HT1 receptor. Bromocriptine showed little selectivity for either subtype. Overall, the ergot derivatives displayed markedly different affinities and selectivities for central 5-HT receptors suggesting that their serotonergic actions should be considered when evaluating their pharmacological spectrum of activity.     

[3H]Ketanserin labels 5-HT2 receptors and α1-adrenoceptors in human and pig brain membranes

The binding characteristics of [3H]ketanserin (a reported selective radioligand for serotonin 5-HT2 receptors) and [125I]BE 2254 (which labels selectively alpha 1-adrenoceptors) were characterized in brain frontal cortex membranes of pig and man. Saturation experiments indicated that both radioligands label apparently a homogeneous class of binding sites in human and pig fontal cortex membranes. Competition experiments with [125I]BE 2254 using 17 agonists and antagonists showed monophasic and steep curves in human and pig frontal cortex membranes. The pharmacological profile of these sites is typical of alpha 1-adrenoceptors. In competition experiments with [3H]ketanserin, most of the tested compounds displayed shallow or biphasic curves. In particular, alpha 1-adrenoceptor-selective antagonists (prazosin, WB 4101, BE 2254...) displaced with nanomolar affinity about 15 and 40% of the specific [3H]ketanserin binding in human and pig frontal cortex membranes, respectively. The minor component of [3H]ketanserin binding correlated highly significantly with [125I]BE 2254 binding in both membrane preparations. The major component of [3H]ketanserin binding to pig and human frontal cortex membranes correlated significantly with [3H]ketanserin binding in rat brain cortex membranes (which is essentially to 5-HT2 receptors). The present data demonstrate that [3H]ketanserin in nanomolar concentrations binds significantly to alpha 1-adrenoceptors in human and pig frontal cortex membranes; this suggests a rather limited degree of selectivity of ketanserin for 5-HT2 receptors in pig and human tissues.     

3H]rauwolscine behaves as an agonist for the 5-HT1A receptors in human frontal cortex membranes.

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential sensitivity of 5-HT1B auto and heteroreceptors

The effect of the native and rodent-selective 5-HT1B receptor agonists (5-hydroxytryptamine (5-HT) and CP93,129) on the K+-evoked overflows of [3H]5-HT, [3H]dopamine (DA) and [3H]acetylcholine (ACh) was studied in synaptosome preparations obtained from rat brain striatum or hippocampus loaded with radiolabeled neurotransmitter. The aim of the study was to compare the different potencies of the specific 5-HT1B receptor agonists to stimulate the auto and heteroreceptors and to modulate the different neurotransmitter release. Results show that under the same experimental conditions, 5-HT and CP93,129 exhibited significantly higher potencies in inhibiting the K+-evoked overflow of [3H]5-HT from synaptosomes of rat striatum (IC50=2.0+/-1.8 nM and 20.5+/-3.1 nM, respectively) than in inhibiting the K+-evoked overflow of [3H]DA from synaptosomes of the same cerebral region (IC50= 0.8+/-0.2 microM and 1.8+/-0.4 microM, respectively), or [3H]ACh from synaptosomes of hippocampus (IC50=1.7+/-0.8 microM for CP93,129). The inhibitory effects of the 5-HT1B receptor agonists on [3H] K+-overflows were antagonized by the selective 5-HT1B receptor antagonist (SB224289), further indicating that the observed effects were 5-HT1B receptor specific. Sumatriptan, a selective r5-HT1D receptor agonist, did not show any significant effect on the K+-overflow of [3H]5-HT in the range of concentrations (10(-10) to 10(-6) M), and did not affect the K+ overflow of [3H]DA or [3H]ACh at concentrations (10(-9) to 10(-4) M), which exclude the involvement of 5-HT1D receptors. These inhibitory effects of the 5-HT1B receptor agonists were highly attenuated by pertussis toxin in the three systems studied, suggesting the involvement of Gi/Go-proteins in the transduction mechanism pathway of the receptor generated signal. In conclusion, these results suggest that 5-HT1B heteroreceptors located on dopaminergic and cholinergic terminals exhibit a lower sensitivity to 5-HT1B receptor agonist and antagonist than do 5-HT1B autoreceptors. The observed difference in functional sensitivities of 5-HT1B auto- and heteroreceptors may represent important consequences in the physiological control of the release of serotonin versus that of other neurotransmitters.     

3H]-SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues.

The selective 5-HT7 receptor antagonist radioligand, [3H]-SB-269970, has been reported to radiolabel the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex (thomas et al, 2000). Saturation analysis of [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes was consistent with labelling a homogenous population of binding sites in each tissue. K(D) values for [3H]-SB-269970 binding in these tissues ranged from 0.9 to 2.3 nM, being similar to those reported for the human cloned and guinea pig cortex 5-HT7 receptors (1.3 and 1.7 nM, respectively). Bmax values for [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain membranes were 20, 30, 31, 14 and 68 fmoles x mg x protein(-1), respectively. For each species the profile of inhibition of [3H]-SB-269970 binding, using a number of 5-HT7 receptor agonists and antagonists, correlated well with that reported for the human cloned 5-HT7(a) receptor (correlation coefficients were 0.95, 0.94, 0.92, 0.95, 0.97 versus the mouse, rat, pig, marmoset and human tissues, respectively). In conclusion, [3H]-SB-269970 has been shown to radiolabel 5-HT7 receptors in rodent, pig and primate brain and represents a valuable tool with which to further characterise the distribution and function of 5-HT7 receptors in native tissues and elucidate their potential role in disease states.     

Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal M1, atrial M2 and ileal M3 receptors.

1. Affinity constants for 15 non-selective or putatively selective muscarinic antagonists were determined at muscarinic autoreceptors and postsynaptic receptors (linked to phosphatidylinositol (PI) hydrolysis) in rat hippocampal slices, at muscarinic receptors mediating contractility in guinea-pig atria or ileal smooth muscle and at binding sites in rat cerebral cortical membranes labelled with [3H]-1-quinuclidinyl benzilate or [3H]-pirenzepine. 2. Comparison of the affinities of these antagonists at central M1 receptors (inositol-monophosphate formation in rat hippocampal slices) with their affinities at peripheral M1 receptors (inhibition by McN-A-343 of electrically stimulated twitches in rabbit vas deferens) provides support for the suggestion that these receptors may differ pharmacologically. 3. Comparison of affinity constants obtained by displacement of specifically bound [3H]-pirenzepine from rat cerebral cortical membranes with those obtained in functional tests showed poor correlations between affinities for binding sites and for functional atrial receptors or for hippocampal autoreceptors. A significant correlation was found between affinities for [3H]-pirenzepine binding and those determined at muscarinic receptors linked to PI turnover in rat hippocampus. A significant correlation was also obtained between the affinities for specific [3H]-pirenzepine binding sites in cortical membranes and the affinities at ileal receptors. 4. Comparison of the affinity values for muscarinic autoreceptors in rat hippocampus with affinity values obtained from in vitro models of muscarinic receptor subtypes showed no significant correlations between these autoreceptors and either M1 or M3 receptors. A significant correlation was found between antagonist affinities for hippocampal autoreceptors and muscarinic receptors in the heart. Therefore, muscarinic autoreceptors in rat hippocampus are pharmacologically similar to the M2 (cardiac) muscarinic receptor subtype.     

Eltoprazine, a drug which reduces aggressive behaviour, binds selectively to 5-HT1 receptor sites in the rat brain: an autoradiographic study

Eltoprazine, a phenylpiperazine derivative, selectively reduces offensive aggression in animal models. The present study was designed to localize and characterize the binding sites of [3H]eltoprazine in the rat brain and to compare the distribution of these sites with the distribution of [3H]5-HT binding sites. The binding of [3H]eltoprazine to whole tissue sections was saturable and revealed an apparent dissociation constant (Kd) of 11 nM. Autoradiographic studies demonstrated a widespread distribution of [3H]eltoprazine binding sites throughout the brain. Specific [3H]eltoprazine binding was completely displaced by 5-HT; conversely, unlabelled eltoprazine reduced [3H]5-HT binding to the levels of non-specific binding. The overall distribution of [3H]eltoprazine binding sites showed a strong resemblance to the location of 5-HT1 binding sites labelled with [3H]5-HT. Yet, regions enriched in 5-HT1A and 5-HT1C sites (e.g. dentate gyrus and choroid plexus, respectively) revealed relatively more [3H]5-HT binding as compared to [3H]eltoprazine binding, whereas [3H]eltoprazine binding was more pronounced in 5-HT1B receptor dense areas such as the dorsal subiculum, substantia nigra, ventral pallidum and globus pallidus. Displacement of [3H]eltoprazine with various selective serotonergic drugs demonstrated binding of [3H]eltoprazine to 5-HT1 receptor subtypes. The pharmacological and anatomical data indicate that eltoprazine binds to 5-HT1A, 5-HT1B and to a lesser extent to 5-HT1C binding sites in the rat brain. These results emphasize the important role of serotonin in the regulation of offensive aggression and suggest that eltoprazine may serve as a new tool to study the involvement of central 5-HT1 receptors in the expression of this behaviour.     

Molecular pharmacology of 5-HT1D recognition sites: Radioligand binding studies in human,pig and calf brain membranes

1) The binding characteristics of [3H]5-HT (5-hydroxytryptamine, serotonin) were investigated in membrane preparations of several regions from calf, pig and human brain in the presence of 100 nmol/l 8-OH-DPAT (8-hydroxy-2[di-n-dipropylamino]tetralin) and 100 nmol/l mesulergine in order to mask 5-HT1A and 5-HT1C sites. 2) [3H]5-HT bound rapidly, reversibly and stereo-selectively to a population of high affinity recognition sites in membranes from pig caudate, calf caudate and human cortex, caudate and substantia nigra. 3) Saturation experiments carried out with [3H]5-HT in the presence of 100 nmol/l 8-OH-DPAT and 100 nmol/l mesulergine revealed that non-5-HT1A non-5-HT1C sites represented from 50 to more than 90% of the total 5-HT1 sites (determined with [3H]5-HT in the absence of 8-OH-DPAT and mesulergine), depending on the tissue source. 4) The pharmacological profile of these sites was characterized in competition experiments performed with a variety of ligands in membranes of calf, pig and human caudate membranes. Under these conditions, [3H]5-HT labelled a population of "5-HT1-like" sites which display nanomolar affinity for tryptamines (5-carboxamidotryptamine greater than 5-HT greater than or equal to 5-methoxytryptamine greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the rat brain 5-HT1A and 5-HT2 receptors after chronic administration of levoprotiline, (+)-oxaprotiline and other antidepressant drugs.

The effects of levoprotiline (LEV), a (-)-enantiomer of oxaprotiline (OXA) and a clinically effective antidepressant, on the binding parameters of hippocampal 5-HT1A and cortical 5-HT2 receptors of rats were compared with those of (+)-enantiomer of OXA ((+)-OXA), imipramine and mianserin. Both LEV and (+)-OXA displayed in vitro some affinity for 5-HT1A receptors labelled with [3H]-8-OH-DPAT, and for 5-HT2 receptors labelled with [3H]-ketanserin. Repeated administration of LEV, for 14 days led to a marked increase in the number of 5-HT1A binding sites in the rat hippocampus, with no change in the KD values. (+)-OXA, imipramine and mianserin produced similar effects on 5-HT1A binding parameters. The number of 5-HT2 receptors was increased after two weeks of LEV administration, not altered after (+)-OXA, and decreased after imipramine or mianserin. The number of [3H]-ketanserin binding sites was decreased after four weeks of (+)-OXA administration, but not altered after LEV. The specific binding of [3H]-ketanserin in the rat cerebral cortex was decreased after repeated treatment with LEV and (+)-OXA (ex vivo). In competition studies the affinity of serotonin for [3H]-ketanserin binding sites was decreased in LEV- and increased in (+)-OXA-treated rats. The results suggest that LEV similarly to other antidepressants increases the number of 5-HT1A receptors, however without common alteration in 5-HT2 receptor number and function.     

(3)H]-8-OH-DPAT binding in the rat brain raphe area: involvement of 5-HT(1A) and non-5-HT(1A) receptors

1. The 5-HT(1A) agonist 8-OH-DPAT has been shown to have additional 5-HT uptake inhibiting properties. The present work was undertaken to examine further the binding of [(3)H]-8-OH-DPAT in the raphe area of the rat brain, a region rich in 5-HT(1A) receptors and 5-HT uptake sites. 2. 5-HT inhibited [(3)H]-8-OH-DPAT binding in a biphasic manner (pK(i1): 8.82+/-0.01, pK(i2): 6.07+/-0.05, n=4) with the low affinity site representing 36+/-4% of the total population. A biphasic inhibition curve was found also with the 5-HT(1A) antagonist, WAY 100635 (pK(i1): 8.65+/-0.17, pK(i2): 4.26+/-0.38, n=3). In the presence of 1 microM WAY 100635 to mask 5-HT(1A) receptors, 5-HT inhibited [(3)H]-8-OH-DPAT binding in a monophasic manner (pK(i): 6.04+/-0.07, n=3). 3. The affinities of various compounds for sites labelled by [(3)H]-8-OH-DPAT in the presence of 1 microM WAY 100635 and for sites labelled by [(3)H]-citalopram (a selective 5-HT uptake inhibitor) were determined. There was a significant correlation between pK(i) values at 5-HT uptake sites and at non-5HT(1A) sites labelled by [(3)H]-8-OH-DPAT (r=0.80, P<0. 001, n=17), suggesting these latter sites to be 5-HT uptake sites. 4. Whereas the affinities of R(+) and S(-) enantiomers of 8-OH-DPAT for the 5-HT uptake site are similar, R(+)8-OH-DPAT has 10 times higher affinity for the non-5-HT(1A) site than S(-)8-OH-DPAT and was considered as an outlier in the correlation. It is suggested that [(3)H]-8-OH-DPAT labels other, as yet unknown binding sites in the raphe.     

Selective 5-HT1B agonists identify the 5-HT autoreceptor in lumbar spinal cord of rat

The purpose of the present study was to characterize the 5-HT autoreceptor in the lumbar spinal cord of the rat. The effect of selective 5-HT1A and 5-HT1B agonists on K+-evoked release of [3H]5-HT and the binding of [3H]5-HT were examined. The 5-HT1B compounds, mCPP and quipazine were more potent than exogenous 5-HT at decreasing K+-evoked release of [3H]5-HT in slices of spinal cord. The pEC40 values of 5-HT agonists tested, determined from release assays, significantly correlated with the relative affinities (pKD's) of these compounds for the binding of [3H]5-HT to the 5-HT1B receptor subtype in the presence of 2 microM 8-OHDPAT, as determined from radioligand binding studies (r = 0.98, P = 0.003). Conversely, the potencies of the 5-HT1A agonists 5-MeODMT and 8-OHDPAT, at the 5-HT autoreceptor, were negatively correlated (r = -0.77, P less than 0.10) with their potencies at displacing [3H]5-HT from the 5-HT1A subsite (binding of [3H]5-HT in the presence of 1 microM mCPP). Thus, the 5-HT autoreceptor in spinal cord appears to bear a significant pharmacological similarity to the 5-HT1B binding site. Further testing of the present results requires the development of new 5-HT1 agonists which are selective (1000-fold difference) for the 5-HT1A and 5-HT1B subsites.     

Species variations in RU 24969 interactions with non-5-HT1A binding sites

The interaction of RU 24969 with [3H]5-hydroxytryptamine ([3H]5-HT) binding to non-5-HT1A binding sites was analyzed in nine species. Non-5-HT1A binding was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. Computer-assisted iterative curve fitting of RU 24969 competition studies indicate that RU 24969 distinguishes two distinct components of non-5-HT1A sites in rat and mouse brain. These two binding components display apparent Ki values of 0.33-0.39 and 66-130 nM for RU 24969. By contrast, RU 24969 competition for non-5-HT1A binding in guinea-pig, cow, human, dog, chicken, turtle and frog brain membranes is consistent with a homogeneous population of [3H]5-HT binding sites. This population of sites displays apparent Ki values of 23-130 nM for RU 24969 and appear to be analogous to the low affinity component of non-5-HT1A sites identified in rat and mouse.     

Effects of chronic treatment with fluoxetine and citalopram on 5-HT uptake, 5-HT1B autoreceptors, 5-HT3 and 5-HT4 receptors in rats

The effect in rats of chronic treatment with two specific 5-HT reuptake inhibitors (SSRI) with antidepressant properties, citalopram (10 mg/kg, i.p. twice a day for 14 days, one day washout) and fluoxetine (15 mg/kg, p.o. twice a day for 21 days, 7 days washout), was evaluated on some mechanisms involved in central 5-HT neurotransmission. No adaptive modifications of brain 5-HT uptake (sites) were found by measuring functional [³H]5-HT uptake and [³H]citalopram binding in cortical and hippocampal synaptosomes, and by [³H]citalopram binding autoradiography in the raphe nuclei (5-HT cell bodies) and the ventral tegmental area (5-HT axonal pathway). Chronic treatments had no effect on presynaptic 5-HT_1B autoreceptors, functionally evaluated by measuring 5-HT_1B-mediated inhibition of depolarization-induced [³H]5-HT release from cortical and hippocampal synaptosomes. Chronic citalopram or fluoxetine did not significantly affect the binding of [³H]BRL-43694 to 5-HT₃ receptors in the rat brain cortex. Citalopram had no effect on [¹²⁵I]SB-207710 binding to 5-HT₄ receptors, measured by autoradiography in the substantia nigra. Negative results, such as those reported in the present study, could be due to a number of variables including the animal species, the treatment schedule or the brain areas considered, thus explaining the differences from some previous reports of significant effects of SSRI. However, our negative data are in agreement with many other published studies, suggesting that adaptive modifications of brain 5-HT transporters, terminal 5-HT_1B receptors, 5-HT₃ and 5-HT₄ receptors may not be a general effect induced by all SSRI. Received: 9 December 1996 / Accepted: 1 March 1997