Cortical 5-HT(1A) receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Identification of a novel 5-HT1 binding site in rat spinal cord

High affinity, specific [³H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT_1A selective agonist demonstrated high affinity competition (K_{i = 1.3 nM}) for 24.6 ± 0.7% of the total [³H]5-HT binding sites. Competition studies employing the 5-HT_1B selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K_{i = 1.1 nM}) competitive inhibition for 26.2 ± 1.4% of all [³H]5-HT binding sites. Neither 5-HT_1C, 5-HT_1D, 5-HT₂ nor 5-HT₃ selective compounds demonstrated any high affinity competition for the residual 49% of specific [³H]5-HT binding. Therefore, three major classes of [³H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT_1A, 5-HT_1B and a novel [³H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [³H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT₁ receptor subtype present in spinal cord.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT_1A receptor. Both antisera allowed the immunoprecipitation of 5-HT_1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT_1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT_1A sites labelled by selective radioligands such as [³H]8-OH-DPAT and [¹²⁵I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT_1A receptor binding subunits with G proteins from one brain area to another.     

Phenylpiperazine derivatives with strong affinity for 5HT1A, D2A and D3 receptors

Four 7-[3-(4-phenyl-1-piperazinyl)propoxy]coumarins were synthesized. The affinities of these compounds for DA (D_2A, D₃) and 5HT_1A receptors were evaluated for their ability to displace [³H]-raclopride and [³H]-8-OH-DPAT respectively from their specific binding sites. The affinities of the target compounds were all in the nanomolar range and followed the order 5-HT_1A > D₂ > D₃.     

Synthesis and SAR of highly potent dual 5-HT1A and 5-HT1B antagonists as potential antidepressant drugs

Novel 5-HT₁ autoreceptor ligands based on the N-4-aryl-piperazinyl-N′-ethyl-5,6,7,8-tetrahydropyrido[4′, 3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one core are described. Aiming at antidepressants with a novel mode of action our objective was to identify potent antagonists showing balanced affinities and high selectivity for the 5-HT_1A and 5-HT_1B receptors. Strategies for the development of dual 5-HT_1A and 5-HT_1B antagonists based on 1 and 2 as leads and the corresponding results are discussed. Isoquinoline analogue 33 displayed high affinity and an antagonistic mode of action for the 5-HT_1A and the 5-HT_1B receptors and was characterized further with respect to selectivity, electrically stimulated [³H]5-HT release and in vivo efficacy.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [H]GR65630 to rat cortical membranes

In the absence of detergent, specific binding of [³H]GR65630, a 5-hydroxytryptamine₃ (5-HT₃) antagonist, determined in the presence of 5-HT₃ receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [³H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X-100) decreased nonspecific binding, increasing the proportion of specific binding to 70% of total binding. In the presence of 0.1% Triton X-100, binding of [³H]GR65630 was rapid, reversible and saturable at 25°C. The rank order of 5-HT₃ receptor active drugs in inhibiting [³H]GR65630 binding was quipazine > ICS205-930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [³H]GR65630 efficiently labeled 5-HT₃ receptors in the presence of Triton X-100. Triton X-100 improved 5-HT₃ receptor binding with rat brain membranes.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Differential Regulation of Somatodendritic Serotonin 5-HT1A Receptors by 2-Week Treatments with the Selective Agonists Alnespirone (S-20499) and 8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract : Single treatment with the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) and alnespirone (S-20499) reduces the extracellular 5-HT concentration (5-HT_ext) in the rat midbrain and forebrain. Given the therapeutic potential of selective 5-HT_1A agonists in the treatment of affective disorders, we have examined the changes in 5-HT_1A receptors induced by 2-week minipump administration of alnespirone (0.3 and 3 mg/kg/day) and 8-OH-DPAT (0.1 and 0.3 mg/kg/day). The treatment with alnespirone did not modify baseline 5-HT_ext but significantly attenuated the ability of 0.3 mg/kg s.c. alnespirone to reduce 5-HT_ext in the dorsal raphe nucleus (DRN) and frontal cortex. In contrast, the ability of 8-OH-DPAT (0.025 and 0.1 mg/kg s.c.) to reduce 5-HT_ext in both areas was unchanged by 8-OH-DPAT pretreatment. Autoradiographic analysis revealed a significant reduction of [³H]8-OH-DPAT and [³H]WAY-100635 {³H-labeled N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexanecarboxamide · 3HCl} binding to somatodendritic 5-HT_1A receptors (but not to postsynaptic 5-HT_1A receptors) of rats pretreated with alnespirone but not with 8-OH-DPAT. In situ hybridization analysis revealed no change of the density of the mRNA encoding the 5-HT_1A receptors in the DRN after either treatment. These data indicate that continuous treatment for 2 weeks with alnespirone, but not with 8-OH-DPAT, causes a functional desensitization of somatodendritic 5-HT_1A receptors controlling 5-HT release in the DRN and frontal cortex.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

Serotonin-1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT_1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT_1A receptors to activate G proteins was a general mechanism by which 5-HT_1A receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT_1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT_1A autoreceptor function was not accompanied by a decrease in 5-HT_1A receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT_1A receptor-stimulated [³⁵S]GTPγS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT_1A somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT_1A receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT_1A autoreceptors is regulated.     

The pharmacological characterization of 5-HT3 receptor binding sites in rabbit ileum: Comparison with those in rat ileum and rat brain

We have further characterized the 5-HT₃ receptors in rat and rabbit tissues by evaluating the binding of the 5-HT₃ receptor ligand, [³H]GR67330 to homogenates of rabbit ileum, rat ileum and rat brain (entorhinal cortex). In each tissue specific [³H]GR67330 binding represented a single saturable, high affinity site (K_d = 0.14, 0.18, 0.076 nM in rabbit ileum, rat ileum and rat brain respectively). The densities of sites present in rabbit and rat ileum were similar to that present in rat brain (B_max = 63, 47, 72 fmol/mg protein in rabbit ileum, rat ileum and rat brain respectively).

In each tissue, 5-HT₃ receptor agonists and antagonists potently competed for [³H]GR67330 binding. Derived inhibition constants were similar in rat ileum and brain. However marked differences in IC₅₀s were apparent for rabbit ileum compared with rat brain or ileum. These were most apparent with agonists. Thus, mCPBG [1-(meta-chlorophenylbiguanide)], phenylbiguanide, 5-HT and 2-methyl 5-HT were at least 5 times less potent to inhibit [³H]GR67330 binding in rabbit ileum than rat brain. The most pronounced differences were evident with phenylbiguanide and mCPBG which were 70 and 300 times less potent in the rabbit ileum respectively compared with the rat tissues. These differences were unlikely to be due to depletion effects because tissue combination experiments (rabbit ileum and rat brain) yielded biphasic inhibition curves for phenylbiguanide with affinities for each component similar to those in the individual tissues. Antagonist affinities also varied between the rabbit and rat tissues, although less markedly. Amongst the antagonists, the most marked differences were apparent with SDZ 206–830 and quipazine each being 10 times less potent to inhibit binding to rabbit than rat tissue.

Hill coefficients for inhibition of binding varied with tissue. In rat brain, as previously described for [³H]GR67330, Hill coefficients for agonist (and quipazine) inhibition of binding were greater than unity. This was less marked in rat and rabbit ileum tissues.

The present studies provide further evidence for species variation in 5-HT₃ receptors.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

The GABAA receptor complex in the developing chick optic tectum: characterization of benzodiazepine and ionophore-linked convulsant/barbiturate recognition sites

By use of membrane preparations and incubation conditions optimized for each binding site, we have characterized the benzodiazepine and ionophore-linked-convulsant/barbiturate modulatory sites within the chick tectal GABA_A receptor complex. Using [³H]flunitrazepam (FNZ) and [³⁵S]t-butylbicyclophosphorothionate (TBPS), respectively, as specific radioligand probes for the two sites, we have found in each case one single population of high-affinity, saturable, specific binding sites. The apparent dissociation constants (K_d) show no change during tectal development (9 nM for [³H]FNZ, and 25–28 nM for [³⁵S]TBPS) while the respective densities of binding sites at saturation (B_max) experience in both cases a twofold increase between embryonic day 16 and postnatal day 10. Ligand-specific pharmacological profiles and allosteric interactions between the transmitter and modulatory sites appear to be well preserved in the chick tectal membrane preparations employed in this study.     

Acetaminophen-induced antinociception via central 5-HT(2A) receptors.

Acetaminophen is one of the most widely used analgesic drugs. Although the mechanism of analgesic action of acetaminophen is still not known, the involvement of the central serotonin (5-hydroxytryptamine: 5-HT) system is one possibility. In the present study, we examined the antinociceptive effect of acute and chronic intraperitoneally (i.p.) administered acetaminophen by tail flick latency measurements in the rat. A significantly increased tail flick latency was observed in acute and 15-day acetaminophen-treated rats, but not in 30-day acetaminophen-treated rats, at a dose of 400 mg/kg/day. To investigate the plasticity of receptors at postsynaptic membrane, we conducted a series of experiments by radioligand binding method on frontal cortex and brainstem membrane. The technique involved radioligand binding with [phenyl-4-³H]spiperone and ketanserin for studying 5-HT_2A receptor characteristics. A significant decrease in the maximum number of 5-HT_2A binding sites (B_max) was demonstrated in all treatment groups with acetaminophen 300 and 400 mg/kg on frontal cortex membrane, whereas the value of the dissociation equilibrium constant (K_d) remained unchanged. The down-regulation of 5-HT_2A binding sites in frontal cortex was of a lesser magnitude after 30 days of treatment and the tail flick latency was as in the control animals. These results suggest that down-regulation of 5-HT_2A receptor in response to 5-HT release is a major step in the mechanism underlying analgesia produced by this agent. On the contrary, chronic use of acetaminophen may result in 5-HT depletion, which in turn produces re-adaptation of postsynaptic 5-HT_2A receptors. These data provide further evidence for a central 5-HT-dependent antinociceptive effect of acetaminophen.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

Activation of paraventricular nucleus of hypothalamus 5-HT1A receptor on sodium intake

Hypothalamic paraventricular nucleus (PVN) has an important role in the regulation of water and sodium intake. Several researches described the presence of 5-HT₁ receptors in the central nervous system. 5-HT_1A was one of the prime receptors identified and it is found in the somatodendritic and post-synaptic forms. Therefore, the aim of this study was to investigate the participation of serotonergic 5-HT_1A receptors in the PVN on the sodium intake induced by sodium depletion followed by 24 h of deprivation (injection of the diuretic furosemide plus 24  h of sodium-deficient diet). Rats (280–320 g) were submitted to the implant of cannulas bilaterally in the PVN. 5-HT injections (10 and 20 μg/0.2 μl) in the PVN reduced NaCl 1.8% intake. 8-OH-DPAT injections (2.5 and 5.0 μg/0.2 μl) in the PVN also reduced NaCl 1.8% intake. pMPPF bilateral injections (5-HT_1A antagonist) previously to 8-OH-DPAT injections have completely blocked the inhibitory effect over NaCl 1.8% intake. 5-HT_1A antagonists partially reduced the inhibitory effect of 5-HT on NaCl 1.8% intake induced by sodium depletion. In contrast, the intake of palatable solution (2% sucrose) under body fluid-replete conditions was not changed after bilateral PVN 8-OH-DPTA injections. The results show that 5-HT_1A serotonergic mechanisms in the PVN modulate sodium intake induced by sodium loss. The finding that sucrose intake was not affected by PVN 5-HT_1A activation suggests that the effects of the 5-HT_1A treatments on the intake of NaCl are not due to mechanisms producing a nonspecific decrease of all ingestive behaviors.     

Expression of serotonin receptor mRNAs in blood vessels

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1 _β, 5-HT_2A, 5-HT_2B, 5-HT₄, and 5-Ht₇ receptor mRNA to different extents. New splice variants of the porcine 5-HT₄ receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT_{1 β}, 5-HT_2b, and 5-HT₄ receptor mRNA, whereas in smooth muscle cells 5-HT_{1 β}, 5-HT_2A, 5-HT₇, and in some experiments 5-HT_2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.