Presynaptic modulation of 5-HT release in the rat septal region.

5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [(3)H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S(1)), S(2), S(3); 360 pulses, 3 Hz, 2 ms, 26-28 mA). In all septal regions the Ca(2+)-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [(3)H] was inhibited by the 5-HT(1B) agonist CP-93,129 and the alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the mu- and kappa-opioid receptor agonists (d-Ala(2), N-Me-Phe(4), glycinol(5))-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the delta-opioid receptor agonist (d-Pen(2), d-Pen(5))-enkephalin (DPDPE), the dopamine D(2) receptor agonist quinpirole and the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine were all ineffective; the GABA(B) receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S(1). It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT(1B) autoreceptors, as well as by alpha(2)-, mu- and kappa-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.

Presynaptic serotonergic modulation of 5-HT and acetylcholine release in the hippocampus and the cortex of 5-HT1B-receptor knockout mice.

Lesioning of serotonergic afferents increases hippocampal ACh release and attenuates memory deficits produced by cholinergic lesions. Improved memory performance described in 5-HT1B-knockout (KO) mice might thus be due to a weaker 5-HT1B-mediated inhibitory influence of 5-HT on hippocampal ACh release. The selective delay-dependent impairment of working memory observed in these KO mice suggests, however, that cortical regions also participate in task performance, possibly via indirect influences of 5-HT on ACh release. To provide neuropharmacological support for these hypotheses we measured evoked ACh and 5-HT release in hippocampal and cortical slices of wild-type (WT) and 5-HT1B KO mice. Superfused slices (preincubated with [3H]choline or [3H]5-HT) were electrically stimulated in the absence or presence of 5-HT1B receptor ligands. In hippocampus and cortex, 5-HT1B agonists decreased and antagonists increased 5-HT release in WT, but not in 5-HT1B KO mice. In 5-HT1B KO mice, 5-HT release was enhanced in both structures, while ACh release (in nCi) was reduced. ACh release was inhibited by 5-HT1B agonists in hippocampal (not cortical) slices of WT but not of 5-HT1B KO mice. Our data (i) confirm the absence of autoinhibition of 5-HT release in 5-HT1B-KO mice, (ii) demonstrate a reduced release of ACh, and the absence of 5-HT1B-receptor-mediated inhibition of ACh release, in the hippocampus and cortex of 5-HT1B-KO mice, and (iii) are compatible with an indirect role of cortical ACh in the working memory impairment observed in these KO mice.

Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin.

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5-HT) release and 5-HT reuptake activity, hippocampal extracellular 5-HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5-HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5-HT levels. Adenosine and A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), decreased extracellular 5-HT levels, whereas non-selective antagonist, caffeine, and A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased them. When 5-HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5-HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and A3 receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not affect 5-HT levels; however, when A1 receptor was blocked by CPT, 5-HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5-HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5-HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5-HT reuptake activity might be modulated by hippocampal A3 receptor.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

Iodine-123-5-iodo-6-nitroquipazine: SPECT radiotracer to image the serotonin transporter.

UNLABELLED: Because serotonergic function has been implicated in the pathophysiology of a number of diseases of the nervous system, efforts to image this system in vivo have received considerable recent attention. Promising preliminary results with the tracer 5-iodo-6-nitroquipazine (INQUIP) have prompted us to perform further studies designed to validate the use of the tracer as an in vivo ligand for the serotonin transporter. METHODS: We studied six adult macaca mulatta in eight experiments which involved SPECT imaging at 17 to 24 hr post-tracer injection, including three experiments with coinjection of the 123I-and 125I-radiolabeled tracer for direct comparison of autoradiography and SPECT, and three experiments in which animals were lesioned with the serotonergic neurotoxin (+/-)3,4-methyl-enedioxymethamphetamine (MDMA). In addition, we evaluated the metabolism of the tracer in the brain and periphery. RESULTS: SPECT images obtained at 17 and 24 hr reflected the known pattern of distribution of serotonin transporters and also showed close correspondence to the autoradiograms. Ratios of binding in the brain-stem to binding in the cerebellum were close to 3 at 17 hr. autoradiograms from an MDMA-treated animal showed up to 95% reductions of binding, while the SPECT data showed smaller reductions. Virtually all of the tracer in the brain stem was in the form of unmetabolized parent compound, but plasma showed rapid peripheral metabolism of the tracer. CONCLUSION: These results demonstrate that INQUIP SPECT images are sensitive measures of in vivo binding to the serotonin transporter, and support the further development of the tracer as a method for the in vivo study of serotonergic neurons in humans.

Growth-inhibitory effects of serotonin uptake inhibitors on human prostate carcinoma cell lines.

Growth stimulation of a variety of cell types by the neurotransmitter serotonin has been reported. We have examined the effects of three serotonin-uptake inhibitors, 6-nitroquipazine, zimelidine and fluoxetine (Prozac, Eli Lilly Co., Indianapolis, Indiana) on human prostate carcinoma cell lines. In vitro, all 3 of these compounds inhibited the proliferation of PC-3, DU-145 and LNCaP cells in a dose-dependent manner. Also, all 3 compounds blocked the uptake of a radiolabeled analog of serotonin by the prostate carcinoma cell lines. The order of potency for inhibition of growth as well as for serotonin uptake was fluoxetine > zimelidine > 6-nitroquipazine. The growth of subcutaneous, PC-3 xenografts in athymic nude mice was significantly inhibited by fluoxetine. These results implicate biogenic amines such as serotonin in the growth of prostate carcinoma cells and indicate the potential use of serotonin-uptake inhibitors for the treatment of prostate cancer.

In vivo imaging of the 5-hydroxytryptamine reuptake site in primate brain using single photon emission computed tomography and [123I]5-iodo-6-nitroquipazine.

Previous experiments have demonstrated that 5-iodo-6-nitro-2-piperazinylquinoline (5-I-6-NQP) is a potent and selective ligand for studying brain 5-hydroxytryptamine (5-HT) reuptake sites. We performed in vivo imaging in non-human primates using single photon emission computed tomography (SPECT) and the 123I-labeled compound [123I]5-I-6-NQP. These studies showed rapid brain uptake, with slow egress of the tracer from the brainstem, a region rich in 5-HT reuptake sites. Loss of the tracer from regions with a lower density of these sites, such as cerebellum, was relatively more rapid. Pretreatment of animals with paroxetine increased the washout of tracer from the brainstem to rates similar to that seen in cerebellum. Brainstem to cerebellar ratios of tracer accumulation were > 2 by 8 h after injection, and in paroxetine pretreated animals remained close to 1. These results indicate that the radiotracer has characteristics suitable for use as a SPECT imaging agent of serotonin reuptake sites.

[125I]5-iodo-6-nitroquipazine: a potent and selective ligand for the 5-hydroxytryptamine uptake complex. I. In vitro studies.

In search of a potent and selective radioiodinated ligand for the 5-hydroxytryptamine (serotonin or 5-HT) uptake complex, we synthesized and evaluated the in vitro properties of [125I]5-iodo-6-nitroquipazine. The binding properties and pharmacological profile of this radioligand were studied in rat brain homogenates, and it was found to display high affinity and selectivity for the serotonin uptake complex. Scatchard analysis of the binding data indicated a single population of sites with a Kd of 23 +/- 6 pM and a Bmax of 430 +/- 50 fmol/mg protein (mean +/- S.E.M., n = 7). Inhibitors of serotonin uptake were the most efficient competitors for [125I]5-iodo-6-nitroquipazine binding with Ki values similar in rank order and magnitude to those obtained in studies of other established serotonin uptake blockers. Inhibitors of dopamine and norepinephrine uptake as well as a wide variety of postsynaptic receptor agents were relatively ineffective in inhibiting [125I]5-iodo-6-nitroquipazine binding to rat brain membranes. Serotonin was the only monoaminergic neurotransmitter capable of effectively competing for [125I]5-iodo-6-nitroquipazine binding sites and gave a Ki value of 2.8 +/- 0.6 microM. Lesions of the serotonergic system with p-chloroamphetamine resulted in a dramatic loss (> 90%) of [125I]5-iodo-6-nitroquipazine binding to rat cortical membranes. Non-radiolabeled 5-iodo-6-nitroquipazine potently inhibited the binding of [3H]paroxetine to serotonin reuptake sites in rat cortical membranes with a Ki of 0.17 +/- 0.06 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

[125I]5-iodo-6-nitroquipazine: a potent and selective ligand for the 5-hydroxytryptamine uptake complex. II. In vivo studies in rats.

The in vivo regional distribution and pharmacological profile of [125I]5-iodo-6-nitroquipazine in the rat brain were studied to evaluate this compound as a potential in vivo imaging agent of the 5-hydroxytryptamine (serotonin or 5-HT) uptake complex. This radioligand penetrated the blood-brain barrier quickly and efficiently, with 1.9% of injected dose found in the whole brain at 5 min post i.v. injection. The regional brain distribution of radioactivity at time points later than 2 h was highly correlated with the known distribution of serotonin uptake sites and terminals. Coadministration of 2 mg/kg paroxetine inhibited > 90% of the total in vivo binding of [125I]5-iodo-6-nitroquipazine. Other serotonin uptake inhibitors, such as fluoxetine and sertraline, were also effective inhibitors of [125I]5-iodo-6-nitroquipazine brain binding in vivo. Non-serotonergic uptake blockers (desipramine, nomifensine, and GBR-12909) and the postsynaptic serotonin receptor agent LSD had no effect on [125I]5-iodo-6-nitroquipazine binding in vivo even at high doses. Lesioning of the serotonergic system by p-chloramphetamine produced approximately 90% decrease in specific in vivo binding. Extraction and analysis of brain radioactivity indicated that approximately 95% of the extractable radioactivity was unmetabolized [125I]5-iodo-6-nitroquipazine. These results indicate that [125I]5-iodo-6-nitroquipazine is a specific, useful radioligand for studying serotonergic uptake sites and terminals in animals, and an 123I-radiolabeled form of the drug would be an excellent candidate for non-invasive single photon emission computed tomography (SPECT) imaging of these sites in the living human brain.

New hybrids of quipazine and trazodone as selective inhibitors of uptake of 5-hydroxytryptamine.

Two new congeners, 4-(chloropropyl)-1-(2-quinolyl)piperazine- and 2-[3-[4-[2-(quinolyl)]-1-piperazinyl]propyl]-1,2,4-triazolo] 4,3-a]pyridin-3(2H)-one, of trazodone were synthesized and found to be potent and selective inhibitors of synaptosomal uptake of 5-hydroxytryptamine [5-HT, serotonin; IC50 = norepinephrine greater than 5 microM, 5-HT = 210-890 nM], with minimal effects in antagonizing (-)-apomorphine-induced climbing behavior and suppression of spontaneous locomotor activity in mice (ED50 greater than 50 mg/kg). The two compounds behaved like atypical antidepressants, since they weakly antagonized reserpine-induced hypothermia. The acute toxicity studies have shown that these compounds were less lethal when compared with imipramine or quipazine. Furthermore, chronic treatments (20 mg/kg, daily for 10 and 21 days) significantly decreased the isoprenaline-induced increase in cyclic AMP in the rat brain cortex, suggesting desensitization of beta-adrenoceptors. These findings point to the effects of these compounds as potential antidepressants dealing with specific serotonergic mechanisms.

Antinociceptive and anti-inflammatory activity of a novel quipazine derivative (AAL-13): a selective inhibitor of 5-hydroxytryptamine reuptake.

The anti-inflammatory and antinociceptive activities of a novel quipazine derivative 2(4-(3-chloropropyl)piperazinyl) quinoline (AAL-13), a selective inhibitor of 5-hydroxytryptamine (5-HT) reuptake, has been examined. Anti-inflammatory activity was assessed by mesuring the inhibition of a cotton pellet granuloma and of carrageenan-induced paw oedema in rats, and of cantharidin-induced topical inflammation in the mouse ear. Antinociceptive activity was studied by using the modified Randall-Selitto method. Indomethacin was used as a reference. AAL-13 slightly inhibited granuloma formation (13%, P less than 0.02) at 100 mg kg-1 day-1 for 7 days, whereas half that dose had no significant effect. There was significant inhibition of carrageenan-induced rat paw oedema (35%, P less than 0.05 and 103%, P less than 0.001) 3 h after single doses of AAL-13 (50 and 100 mg kg-1 p.o., respectively). Three hours after i.p. injection, the oedema inhibition was 58% (P less than 0.05) and 86% (P less than 0.001) for doses of 25 and 50 mg kg-1, respectively. In comparison, indomethacin (3, 6 and 12 mg kg-1 p.o.) inhibited oedema by 59% (P less than 0.02), 65% (P less than 0.01) and 63% (P less than 0.02), respectively. Intraperitoneally, only the 12 mg kg-1 dose produced significant inhibition (82%, 3 h after carrageenan injection, P less than 0.05). AAL-13 (1.5 mg/ear) had a significant anti-inflammatory effect on the mouse ear (52%, inhibition, P less than 0.05), while indomethacin (3 mg/ear) gave 43% inhibition (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of melanization in human melanoma cells by a serotonin uptake inhibitor.

Significant levels of intracellular catecholamines were found in a human melanoma cell line and were enhanced by increasing the extracellular tyrosine concentration. Intracellular dopa, 5-cysteinyldopa, tyrosinase, and melanin also rose under these conditions. 5-HT (serotonin) was synthesized by the melanoma cells but further study was hindered by the high level of 5-HT in fetal calf serum. A 5-HT uptake antagonist, DU 24565 (6-nitroquipazine), was employed as an alternative method for studying 5-HT action. This compound, which in contrast to tunicamycin had no inhibitory effects on cell proliferation or tyrosinase activity, strongly inhibited melanization and decreased the levels of dopa, 5-cysteinyldopa, dopamine, noradrenaline, adrenaline, and 3,4-dihydroxyphenylacetic acid. DU 24565 had little effect on 5-HT or tyrosine accumulation in these cells but suppressed the uptake of extracellular dopa. The results show that human melanoma cells synthesize a wide range of biogenic amines in culture and suggest a new approach to regulating intracellular levels of dopa and of a variety of dopa products.

A serotonin agonist-antagonist reversible effect on Na+-K+-ATPase activity in the developing rat brain.

In brain of adult and developing rats the Na+-K+-adenosine triphosphatase (Na+-K+-ATPase) system seems to react to serotonin (5-HT) changes induced pharmacologically. A 5-HT agonist (quipazine) elicits a response of the enzyme activity in the cerebral cortex in vivo, which is neutralized with a 5-HT antagonist (methysergide). This effect was observed from day 21 to adulthood. Also in a state of 5-HT receptor hypersensitivity (rats treated early with 5,6-dihydroxytryptamine), the response of Na+-K+-ATPase to the 5-HT agonist was higher than without neurotoxic lesion of 5-HT paths. These data suggest an involvement of the Na+-K+-ATPase system in 5-HT receptor sensitivity in the rat brain.