Inactivation of 5-HT(1A) receptors in hippocampal and cortical homogenates

5-HT(1A) receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [35S]GTPgammaS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT]-stimulated [35S]GTPgammaS binding and [3H]8-OH-DPAT binding assays were used to assess 5-HT(1A) receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E(max) but not the EC(50) of R(+)-8-OH-DPAT to stimulate [35S]GTPgammaS binding was decreased by EEDQ in both preparations. Additionally, the IC(50) for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [35S]GTPgammaS and [3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [35S]GTPgammaS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl- cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT(1A) receptor coupling to G(alpha) in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT(1A) receptors to inactivation by EEDQ in vitro is the same.     

Irreversible blockade of central 5-HT1A receptor binding sites by the photoaffinity probe 8-methoxy-3'-NAP-amino-PAT

A new photoaffinity ligand derived from the potent 5-HT agonist, 8-OH-DPAT, has been synthesized. In the dark, this compound, 8-methoxy-2-(N-n-propyl,N-3-(2-nitro-4-azidophenyl)aminopropyl) aminotetralin or 8-methoxy-3'-NAP-amino-PAT, displaced [3H]8-OH-DPAT and [3H]5-HT bound to 5-HT1A and 5-HT1 sites in hippocampal membranes with IC50 values of 6.6 and 18.1 nM respectively. The apparent affinity of 8-methoxy-3'-NAP-amino-PAT for the 5-HT1A binding sites was at least 20 times higher than for the other 5-HT receptor sites (5-HT2 and 5-HT3) or the dopamine-related [3H]spiperone and [3H]7-OH-DPAT binding sites. Under UV irradiation (lambda = 366 nm), 8-methoxy-3'-NAP-amino-PAT produced an irreversible blockade of 5-HT1A sites which could be prevented by prior site occupancy by a saturating concentration (10 microM) of reversible 5-HT ligands such as 5-HT itself, 8-OH-DPAT or LSD. The blockade of 5-HT1A binding sites was concentration-dependent, and two successive irradiations of rat brain membranes in the presence of 30 nM 8-methoxy-3'-NAP-amino-PAT were found to be more efficient that a single exposure to 100 nM of the photosensitive ligand. Thus, a 55-60% irreversible blockade of 5-HT1A binding sites was achieved following 2 cumulative irradiations of hippocampal membranes with 30 nM 8-methoxy-3'-NAP-amino-PAT. Under such conditions, cortical 5-HT2 receptor binding sites as well as striatal 5-HT3 and dopamine-related binding sites remained unaltered.     

Differential sensitivity of 3H-agonist binding to pre- and postsynaptic 5-HT1A receptors in bovine brain.

1. The full and weak partial 5-HT1A agonist ligands [3H]-8-OH-DPAT and [3H]-BMY-7378 were used to characterize the binding parameters of pre- and postsynaptic 5-HT1A binding sites in bovine dorsal raphe and hippocampal membranes, respectively. The Kd and Bmax values for the individual radioligands were indistinguisable across the regions tested, as were the Ki values generated by a series of agents acting at 5-hydroxytryptamine (5-HT) receptors. 2. The concentration-dependent allosteric attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding produced by the nonhydrolyzable guanyl nucleotide, Gpp(NH)p, generated similar IC50 values within a particular region; however, these were significantly different between regions. While the maximal attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding was similar in dorsal raphe, Gpp(NH)p produced a significantly greater attenuation of [3H]-8-OH-DPAT binding in hippocampal membranes when compared to [3H]-BMY-7378. The maximal attenuation of [3H]-8-OH-DPAT binding by Gpp(NHp) in hippocampus was also significantly greater than that seen with either radioligand in dorsal raphe. 3. Although exposure to Gpp(NH)p had no effect on the affinity constants of either radioligand in either region, it produced a concentration-dependent reduction in the maximal number of binding sites for both radioligands in both regions. While the percentage reduction in Bmax values were similar for both radioligands in the dorsal raphe, Gpp(NH)p reduced the Bmax of [3H]-8-OH-DPAT in hippocampus significantly more than that of [3H]-BMY-7378. 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]8-OH-DPAT labels the 5-hydroxytryptamine uptake recognition site and the 5-HT1A binding site in the rat striatum

The binding of [3H]8-hydroxy-2-(di-N-propylamino)-tetralin ([ 3H]8-OH-DPAT) to rat hippocampal and striatal membranes has been compared. In the hippocampus, low concentrations of [3H]8-OH-DPAT bound to a single, high affinity site which was sensitive to inhibition by spiperone, buspirone and ergotamine but not by mianserin, quipazine or (-)-propranolol. This is consistent with a selective labeling of the 5-HT1A receptor. In the striatum, [3H]8-OH-DPAT bound to two sites with high and low affinity (KD's 1.18 and 109 nM). The high affinity component was blocked by low concentrations of buspirone, spiperone and ergotamine. The low affinity component was blocked only by high concentrations of buspirone and spiperone, and was not displaced by ergotamine at concentrations up to 1 microM. The ergotamine-resistant component of striatal [3H]8-OH-DPAT binding was blocked by low concentrations of the 5-HT uptake inhibitors fluvoxamine and paroxetine, and by relatively low concentrations of 5-HT itself. Thus [3H]8-OH-DPAT labels the 5-HT transporter in the rat striatum. Unlike [3H]imipramine binding, the binding of [3H]8-OH-DPAT to the 5-HT transporter was independent of external sodium ions. It is therefore suggested that 8-OH-DPAT acts as substrate for the 5-HT transporter and labels the 5-HT recognition site of the transporter complex.     

Fentanyl and sufentanil inhibit agonist binding to 5-HT1A receptors in membranes from the rat brain.

The influence of several opioid narcotics and related drugs, on the binding of [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin. ([3H]8-OH-DPAT), a serotonergic agonist, to 5-HT1A receptors was determined in membranes from the brain of the rat. Sufentanil and fentanyl inhibited binding of [3H]8-OH-DPAT to hippocampal membranes, with IC50 values of 5.5 and 3.4 microM, respectively. In contrast, IC50 values for meperidine, alfentanil and naloxone exceeded 100 microM. The inhibition of binding by sufentanil appeared to be competitive insofar as 10 microM sufentanil increased the apparent KD from 1.0 +/- 0.1 to 3.9 +/- 0.3 nM, without affecting the number of binding sites and the inhibition was easily reversed. The binding of [3H]8-OH-DPAT to hippocampal membranes was inhibited by 5'-guanylylimidodiphosphate, a stable analogue of GTP, in a concentration-dependent manner. None of the opioid drugs examined altered the sensitivity of binding of [3H]8-OH-DPAT to guanine nucleotides. These results suggest that certain opioid narcotics, disrupt serotonergic neurotransmission as a result of direct interactions with 5-HT1A receptors. No effects of opioid narcotics on 5-HT1A receptor-G protein coupling were noted.     

Prenatal stress influences 8-OH-DPAT modulated startle responding and [3H]-8-OH-DPAT binding in rats

The present study was to investigate some aspects of the 5-HT1A receptor system in adult-aged rats (50-60 days) that were either exposed to prenatal stress (PS) or not exposed to prenatal stress (CON). In the first series of experiments, rats were pretreated with vehicle, the 5-HT1A agonist 8-OH-DPAT or the 5-HT1A antagonist, WAY-100635 and exposed to 120 acoustic startle stimuli (95 dB) using a 30 s inter-trial interval. 8-OH-DPAT produced a dose-dependent increase in acoustic startle responding in CON and PS rats, with the PS rats exhibiting greater responding than CON rats. WAY-100635 depressed startle amplitudes only in the CON group. Finally, radioligand binding studies using [3H]-8-OH-DPAT indicated a significant decrease in receptor density in hippocampal homogenates from PS rats but no difference in [3H]-8-OH-DPAT binding from homogenates of the amygdala. Our results are consistent with earlier reports indicating that prenatal stress alters the serotonergic system. Specifically, our results indicate that gestational exposure to chronic mild stress enhances startle amplitudes following 8-OH-DPAT administration, prevents the depression in startle amplitudes following WAY-100635 administration and reduces [3H]-8-OH-DPAT binding in hippocampal preparations.     

Chloroamphetamine did not prevent the effects of chronic antidepressants on 5-hydroxytryptamine inhibition of forskolin-stimulated adenylate cyclase in rat hippocampus.

Administration of p-chloroamphetamine (PCA, 10 mg/kg i.p. on two occasions) to rats resulted in a severe depletion of [3H]paroxetine binding sites, a measure of presynaptic serotonergic terminals, in both cortex and hippocampus, but did not affect [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT) binding or 5-hydroxytryptamine (5-HT)-induced inhibition of forskolin-stimulated adenylate cyclase in hippocampal membranes. Administration of either imipramine (15 mg/kg i.p. for 2 weeks) or lithium (0.2% for 2 weeks) to PCA-treated rats did not affect [3H]8-OH-DPAT binding but reduced the degree of inhibition of forskolin-stimulated adenylate cyclase by 5-HT in hippocampal membranes. It is concluded that the effects of imipramine and Li+ on 5-HT1A receptor-mediated responses in the hippocampus are exerted postsynaptically, possibly at a level distal to the receptor.     

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.     

(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.     

Serotonin-2 receptor-mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat

5-Hydroxytryptamine (5-HT) inhibited the K+-induced release of [3H]acetylcholine [( 3H]ACh) from slices of the hippocampus of the rat, dose-dependently. Minaprine (3-(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine, Fig. 1) had no effect on the release of [3H]ACh. However, it inhibited the (formula; see text) Fig. 1. Chemical structure of minaprine dihydrochloride. attenuation of the release of [3H]ACh by 5-HT dose-dependently. The 5-HT2 receptor antagonists, mianserine, methysergide and spiperone, prevented the inhibitory effect of the 5-HT, as well as did minaprine. The attenuating effect of 5-HT was not mimicked by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and was not prevented by a 5-HT1A and 5-HT1B mixed receptor antagonist, propranolol, or by the 5-HT3 receptor antagonists, cocaine and metoclopramide. Minaprine inhibited the bindings of [3H]5-HT, [3H]8-OH-DPAT and [3H]ketanserin in the hippocampus. The inhibitory effect of minaprine on the binding of [3H]ketanserin was more marked than on the binding of [3H]5-HT and [3H]8-OH-DPAT, and was non-competitive. The Ki value of minaprine for the binding of [3H]ketanserin was 2.9 microM. The inhibitory effect of 5-HT on the release of [3H]ACh was observed in the presence of tetrodotoxin. By electrolytic lesioning of the medial septum, the K+-induced release of [3H]ACh from the slices of hippocampus was significantly reduced and the release was no longer inhibited by 5-HT. The lesioning significantly decreased the binding of [3H]ketanserin in the hippocampus, with hardly any reduction in the binding of [3H]5-HT and [3H]8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen modulates 5-HT(1A) agonist inhibition of lordosis behavior but not binding of [(3)H]-8-OH-DPAT

Previous studies showed that repeated estrogen treatment reduces the ability of the 5-HT(1A) receptor agonist, 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT), to inhibit lordosis behavior of female rats. The present study evaluated the effects of repeated estrogen treatment on lordosis behavior and 5-HT(1A) receptor binding and coupling to G protein in the hypothalamus-preoptic area using the agonist ligand [3H]-8-OH-DPAT, which binds selectively to G-protein-coupled 5-HT(1A) receptors. Rats were injected twice with 25 or 50 microg of estradiol benzoate (EB) 7 days apart followed by 500 microg of progesterone (P) 48 h after the second EB injection. Controls received a single injection of 25 or 50 microg EB followed 48 h later by 500 microg of P. Four hours after P, 0.15 mg/kg 8-OH-DPAT was injected, and lordosis behavior examined for 30 min. Rats treated twice with EB showed significantly less 8-OH-DPAT inhibition of lordosis behavior than rats receiving a single EB injection. For receptor binding, rats received EB without P treatment. None of the estrogen treatments reduced [3H]-8-OH-DPAT binding density or affinity in the hypothalamus-preoptic area or hippocampus. These studies suggest that estrogen modulates 5-HT(1A) agonist potency without a measurable change in 5-HT(1A) receptor density or coupling to G protein.     

Regional CNS densities of serotonin 1A and dopamine D2 receptors in periadolescent alcohol-preferring P and alcohol-nonpreferring NP rat pups

The objective of the present study was to use quantitative autoradiography to determine binding densities of serotonin(1A) (5-HT(1A)) and dopamine (DA) D(2) receptors in alcohol-naive periadolescent P and NP rat pups. P (n=8) and NP (n=7) rat pups, 25 days of age, from different litters were used. Coronal brain sections were incubated with 2 nM [3H]8-OH-DPAT or 20 nM [3H]sulpiride for 5-HT(1A) or D(2) binding, respectively. Approximately 15-40% higher densities of [3H]8-OH-DPAT binding were observed in the anterior cortical regions of the periadolescent P rat compared with NP rat pups. Similar differences were also observed in posterior cortical regions with P rats having 25-40% higher [3H]8-OH-DPAT binding than NP rats. [3H]8-OH-DPAT binding was approximately 10-20% higher in posterior hippocampal regions of the P rat pups compared with the NP line. [3H]sulpiride binding was significantly different only in the ventral tegmental area (VTA), where binding was approximately 20% lower in the periadolescent P rats compared with the NP rat pups. Overall, these results are very similar to findings observed in adult alcohol-naive P and NP rats, and suggest that the innate differences in the neural systems implicated in high alcohol drinking behaviors may already be established in the periadolescent animal.     

S 14506: novel receptor coupling at 5-HT(1A) receptors

S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.     

5-HT1A receptor agonists inhibit carbachol-induced stimulation of phosphoinositide turnover in the rat hippocampus

The selective 5-HT1A agonists, 8-hydroxy-2-(di-n-dipropylamino)tetralin (8-OH-DPAT) and ipsapirone, and the 5-HT1A/5-HT1B agonist, 1-(m-trifluoromethylphenyl)piperazine, partially inhibited the carbachol-stimulated [3H]inositol phosphate formation in rat hippocampal slices. The effect of 8-OH-DPAT was antagonized by cyanopindolol. Selective 5-HT1B, 5-HT2 and 5-HT3 agonists were inactive. 8-OH-DPAT failed to affect the phosphoinositide turnover stimulated by KCl, quisqualate or noradrenaline in hippocampal slices and by carbachol in striatal or cortical slices. These results suggest that 5-HT1A receptors are negatively coupled to phosphoinositide phosphodiesterase in the hippocampus.     

Regulation of the avidity of ternary complexes containing the human 5-HT(1A) receptor by mutation of a receptor contact site on the interacting G protein alpha subunit

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein.     

The naturally occurring Arg219Leu variant of the human 5-HT1A receptor: impairment of signal transduction

The present study in transfected HEK293 cells aimed to investigate whether the pharmacological and/or transductional properties of the naturally occurring Arg219Leu variant (VAR) in the third intracellular loop of the h5-HT1A receptor differ from those of the wild-type receptor. Binding of [3H]8-hydroxy-2-(di-n-propylamino)tetraline ([H]8-OH-DPAT) and of [35S]GTPgammaS to membranes, as well as inhibition of forskolin-stimulated [3H]cAMP formation by 5-HT receptor agonists in whole cells, were estimated. The VAR and wild-type h5-HT1A receptors were found to be expressed at virtually identical densities. The VAR and wild-type receptors did also not differ with respect to the potencies of 5-HT receptor agonists and antagonists in inhibiting [3H]8-OH-DPAT binding. The ability of 5-HT to stimulate [35S]GTPgammaS binding (a measure of G protein coupling) to the VAR receptor and of the agonists 5-HT, buspirone and urapidil to inhibit forskolin-stimulated cAMP accumulation in HEK293 cells expressing the VAR receptor was decreased by 60-90%. In conclusion, the Arg219Leu variation of the human 5-HT1A receptor does not change the binding properties, but is associated with a drastic impairment of signal transduction. In patients carrying this variation, disturbances of 5-HT1A receptor-mediated functions and diminished responses to drugs acting via this receptor may occur.     

Allosteric modulation of the human 5-HT(7A) receptor by lipidic amphipathic compounds.

Human 5-HT7A receptors positively modulated adenylyl cyclases via Gs subtypes of G proteins in human embryonic kidney 293 cells, and bound 5-hydroxytryptamine (HT) with high and low affinity (K(I) values of 1.5 +/- 0.3 and 93 +/- 4 nM). More than 60% of 5-HT7A receptors, however, displayed the high-affinity 5-HT binding with no sensitivity to 5'-guanylylimidodiphosphate. In this study, we found that select amphipathic agents affected the high-affinity 5-HT binding to 5-HT7A. Oleic acid at low concentrations (<15 microM), but not palmitic, stearic, and arachidonic acids, increased maximal [3H]5-HT binding without affecting its K(D) value and [3H]mesulergine (antagonist) binding. Fatty acid-free bovine serum albumin (FF-BSA), a scavenger of fatty acids and lipid metabolites, substantially reduced maximal [3H]5-HT binding (no change in K(D) value and antagonist binding) but lost its action upon treatment with inactive stearic acid. FF-BSA and oleic acid produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors 5-HT1D and 5-HT2C. Among various lysophospholipids, lysophosphatidyl choline (50 microM) decreased maximal [3H]5-HT binding, and a similar zwitterion, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; 0.1%), increased it (no change in K(D)). Functionally, 5-HT-induced guanosine-5'-O-(3-[35S]thio)triphosphate (GTPgamma35S) binding was enhanced by oleic acid and CHAPS, but reduced by FF-BSA and lysophosphatidyl choline; the amphipathic agents and FF-BSA did not affect dopamine-induced GTPgamma35S binding at D1, a prototypic Gs-coupled receptor. At 5-HT7A, oleic acid, FF-BSA, CHAPS, and lysophosphatidyl choline also brought about corresponding changes in the half-maximal 5-HT concentration for cAMP production, without affecting the maximal and basal levels. We propose that endogenous, amphipathic lipid metabolites may modulate 5-HT7A receptors allosterically to promote high-affinity 5-HT binding and to enable receptors to couple more efficiently to Gs subtypes of G proteins.     

Central 5-HT1A receptors and the mechanism of the central hypotensive effect of (+)8-OH-DPAT, DP-5-CT, R28935, and urapidil

This study investigated the central hypotensive effects of drugs that possess a high affinity for central 5-hydroxytryptamine (5-HT1A) binding sites; (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N,N-dipropylcarboxamidotryptamine (DP-5-CT), erythro-1-(1-[2(1,4-benzodioxan-2-yl)-2-hydroxyethyl]- 4-piperidyl)-2-benzimidazolinone (R28935) and urapidil proved to possess high affinity and selectivity for central 5-HT1A binding sites, labeled by [3H]8-OH-DPAT. (+)8-OH-DPAT (0.1-10 micrograms/kg) given through the left vertebral artery of chloralose-anesthetized cats, lowered blood pressure by a biphasic dose-response curve. When given systemically, 10- to 100-fold higher doses of (+)8-OH-DPAT were necessary to obtain the same hypotensive effect when compared with central administration. Besides 8-OH-DPAT, R28935, DP-5-CT and urapidil also lowered blood pressure by a central mechanism in doses that were ineffective when given systemically. The central hypotensive effect of 0.3 micrograms/kg (+)8-OH-DPAT, 3 micrograms/kg DP-5 CT, and 3 micrograms/kg R28935 could be blocked by 100 micrograms/kg (-)pindolol, indicating that central 5-HT1A receptors are involved. High doses of (+)8-OH-DPAT (3-10 micrograms/kg) can also lower blood pressure by activating central alpha 2-adrenoceptors. The hypotensive effect of 300 micrograms/kg urapidil given through the vertebral artery could not be blocked by (-)pindolol. These results indicate the involvement of central 5-HT1A receptors in the mechanism of the central hypotensive activity of (+)8-OH-DPAT, DP-5-CT, and R28935.     

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.     

Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain

The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.