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We previously reported a significant mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small-cell lung carcinoma cells (SCLC, GLC-8), mediated by both 5-HT1D and 5-HT1A receptors. Here we investigate possible interactions between the two receptor subtypes. Dose-effect curves obtained by simultaneously applying equipotent concentrations of the selective 5-HT1A agonist 8-OH-DPAT and the selective 5-HT1D receptor agonist sumatriptan are shifted to the right, although maximal effects are additive. The nonselective 5-HT antagonist metergoline displays higher potency when both receptor subtypes are activated. The 5-HT1D receptor antagonist GR127935 is markedly more potent against sumatriptan than against the sensitive portion of 5-HT effect. Indeed, both GR127935 and the 5-HT1A antagonist spiperone shift the EC50 for the residual effect of 5-HT from approximately 300 to 120-150 nM, suggesting that blocking one receptor subtype may facilitate activation of the other. Preincubation with either 8-OH-DPAT or sumatriptan suppresses the mitogenic response to the other specific receptor agonist; suppression is complete within 10 min at 37 degrees C, and is not observed when the preincubation is done at 4 degrees C. Measurements of adenylate cyclase activity do not help in interpreting the results. Conversely, measurements of MAP kinase activity reveals biphasic activation with a delayed activation at 1 h, and reproduce the suppression of the effect of the second drug by 15 min preincubation. These findings constitute the first evidence of a reciprocal negative interference between human 5-HT1A and 5-HT1D receptors, and indicate that SCLC GLC-8 cells simultaneously express both receptor subtypes. Mere reciprocal antagonism of the drugs employed cannot account for these data. We suggest that in this cell system cross-talk occurs in the transduction pathways of the two receptor subtypes.

Small cell lung cancer (SCLC) cells express cholinergic nicotinic receptors on their membranes, and the stimulatory effect on cell growth of nicotine has been described in cell cultures. We investigated three SCLC cell lines, all showing high levels of fluorescence intensity when labeled with FITC alpha-bungarotoxin in most cells. From one of these cell lines, NCI-N592 a tumor line in athymic nude mice was established. Nude mice were subcutaneously grafted on the same day with tumor fragments and with Alzet osmotic minipumps (200 microliters, 14 days of infusion) infused with serotonin or nicotine to investigate their effects on tumor growth. Two dose levels of each compound were used, namely 20 and 200 micrograms/day. In mice treated with 200 micrograms serotonin, tumors took a shorter time than those of untreated controls to reach 50 mg in volume, meaning that the first steps of tumor growth were faster. In contrast, a delay in tumor appearance was observed in mice treated with low-dose serotonin. No differences were found in tumor growth in the groups of mice treated with nicotine. When the treatment was delivered to already established and vascularized tumors (around 100 mm3), no effect on tumor growth was achieved by serotonin or nicotine. Therefore, in the experimental conditions used in the study, the stimulatory effect of nicotine on an SCLC tumor was not demonstrated.

We have recently shown that the mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small lung carcinoma (SCLC) cells is at least partly due to stimulation of a 5-HT1D receptor type. We now report that the 5-HT1A receptor agonist R(+)-8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) is also capable of stimulating [3H]thymidine incorporation into SCLC GLC-8 cells, although with lower efficacy than 5-HT. The simultaneous administration of maximal doses of 8-OH-DPAT and the 5-HT1D receptor agonist sumatriptan reproduced the maximal [3H]thymidine incorporation observed with 5-HT alone. The 5-HT1A receptor antagonists spiperone and SDZ 216-525 completely abolished the effect of 8-OH-DPAT (IC50 30 nM for both drugs) behaving as pure antagonists. Accordingly, the two drugs partially inhibited the mitogenic effect of 5-HT. These data indicate that the mitogenic effect of 5-HT in SCLC cells involves both 5-HT1A and 5-HT1D receptor types.

Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter and vasoactive agent, is contained in two small cell lung carcinoma cell lines GLC8 and NCI-N-592 and is released in the culture medium. It also stimulates DNA synthesis in the same cell lines. In GLC8 cells this mitogenic effect is not counteracted by ketanserin, ICS 205-930 and GR 113-808 which are antagonists of the 5-HT2, 5-HT3 and 5-HT4 receptors, respectively. On the contrary, the antagonists metergoline, methysergide, SDZ 21-009 and methiothepin inhibit the 5-HT-stimulated incorporation of [3H]thymidine in GLC8 cells. The 5-HT1D agonist sumatriptan is capable of mimicking 5-HT action on cell proliferation. Both sumatriptan and 5-HT inhibit adenylate cyclase activity at doses which correlate with the mitogenic effect. We conclude that a 5-HT1D receptor type contributes to the mitogenic effect of 5-HT in GLC8 cells. This is the first demonstration of an involvement of the 5-HT1D receptor type in human cell proliferation. The design of specific antagonists for this type of receptor might be useful for the growth control of this very aggressive tumor.

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small-cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose-dependent manner; this effect is antagonized by alpha-bungarotoxin (alpha Bgtx) and alpha-conotoxin MI (alpha Ctx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both alpha Bgtx and alpha Ctx. By PCR analysis, we demonstrate the presence in SCLC of both the alpha 7 and the beta 2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that alpha Bgtx-sensitive receptors of the alpha 7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

Small-cell lung carcinoma cells express different plasma membrane nicotinic acetylcholine receptor subtypes. We have now found that interacting with these receptors (-)-nicotine induces a dose-dependent and stereoselective release of [3H]serotonin which is dependent on external calcium and blocked by the specific ganglionic nicotinic antagonist mecamylamine. With the same potency (-)-nicotine stimulates tumor cell proliferation, an effect also blocked by mecamylamine. Serotonin itself stimulates cell proliferation in a dose-dependent manner, an effect blocked by the selective serotonergic receptor antagonists methiotepine and metergoline. These data suggest that nicotine might affect proliferation of small-cell lung carcinoma cells by inducing the release of hormones (such as serotonin) with autocrine capabilities and place both the nicotinic and the serotonergic receptors at key positions in the biological and, possibly, pharmacological approach to this human lung cancer.

Systemic administration of parachlorophenylalanine (PCPA, 100 mg/kg sc on alternate days X two times), a blocker of serotonin (5-HT) synthesis, considerably decreased brain 5-HT and plasma prolactin (PRL) levels in young male rats. Intraventricular (IVT) administration of 5,7-dihydroxytryptamine (5,7-DHT, 200 mug/20 mul), a neurotoxic drug which destroys 5-HT nerve terminals, induced, 3, 12, and 30 days after treatment, a marked depletion of brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and considerably reduced plasma PRL levels at each time interval. Feeding of rat for up to 4 days with a tryptophan (TP)-deficient diet, caused a depletion of brain 5-HT and 5-HIAA contents and did not modify plasma PRL levels. Addition of TP (2 g/kg of diet) to the TP-deficient diet resulted in increased brain 5-HT and 5-HIAA contents and significantly increased PRL levels. These data provide evidence for the role of the 5-HT system in the maintenance of tonic PRL secretion.