Spinal astrocytes stimulated by tumor necrosis factor-α and/or interferon-γ attenuate connexin 43-gap junction via c-jun terminal kinase activity.

Spinal astrocytes have important mechanistic contributions to the initiation and maintenance of neurodegenerative diseases and chronic pain. Under inflammatory conditions, spinal astrocytes are exposed to cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and these cytokines could alter astrocytic function by modulating connexin (Cx43), subunits that form channels that modulate intercellular communication in astrocytes. The current study investigated the alteration of Cx43-gap junction in rat primary cultured spinal astrocytes stimulated with cytokines by real-time PCR and Western blotting. The transcriptional and translational levels of Cx43 were significantly but partially reduced 24 and 48 hr treatment with either TNF-α (10 ng/ml) or IFN-γ (5 ng/ml). A mixture of TNF-α and IFN-γ led to a robust decrease of Cx43 expression and, moreover, a moderate reduction of gap junction intercellular communication (GJIC), which was evaluated by a scrap loading/dye transfer assay. Both the decrease of Cx43 expression and the reduction in GJIC induced by the mixture of TNF-α and IFN-γ were prevented by blocking c-jun terminal kinase (JNK) but not by blocking extracellular signaling molecules ERK and p38 kinase, indicating a specific role of astrocytic JNK in the response to cytokines. In addition, treatment with cytokines potently induced the phosphorylation of JNK and c-jun in a time-dependent manner. These results indicate that intercellular communication of astrocytes is significantly disrupted in the inflammatory state and that stimulation of spinal astrocytes with inflammatory cytokines leads to significant inhibition of Cx43-GJIC through activation of the JNK signaling pathway.

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice.

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.

Activation of transient receptor potential ankyrin 1 evokes nociception through substance P release from primary sensory neurons.

To examine mechanisms underlying substance P (SP) release from primary sensory neurons in response to activation of the non-selective cation channel transient receptor potential ankyrin 1 (TRPA1), SP release from cultured rat dorsal root ganglion neurons was measured, using radioimmunoassay, by stimulating TRPA1 with allyl isothiocyanate (AITC), a TRPA1 agonist. AITC-evoked SP release occurred in a concentration- and time-dependent manner. Interestingly, p38 mitogen-activated protein kinase (p38) inhibitor SB203580 significantly attenuated AITC-evoked SP release. The in vivo effect of AITC-evoked SP release from primary sensory neurons in mice was evaluated. Hind paw intraplantar injection of AITC induced nociceptive behaviors and inflammation (edema, thermal hyperalgesia). AITC-induced thermal hyperalgesia and edema were inhibited by intraplantar pre-treatment with either SB203580 or neurokinin-1 receptor antagonist CP96345. Moreover, intrathecal pre-treatment with either CP96345 or SB203580 inhibited AITC-induced nociceptive behaviors and thermal hyperalgesia. Immunohistochemical studies demonstrated that intraplantar AITC injection induced the phosphorylation of p38 in mouse dorsal root ganglion neurons containing SP. These findings suggest that activation of TRPA1 evokes SP release from the primary sensory neurons through phosphorylation of p38, subsequent nociceptive behaviors and inflammatory responses. Furthermore, the data also indicate that blocking the effects of TRPA1 activation at the periphery leads to significant antinociception.

Asymmetric alternation of the hemodynamic response at the prefrontal cortex in patients with schizophrenia during electroconvulsive therapy: a near-infrared spectroscopy study.

OBJECTIVE: Although electroconvulsive therapy (ECT) is a well-established treatment for psychiatric disorders, its mechanism of action remains unclear. To investigate the cerebral hemodynamic response during ECT, we measured the changes in the regional cerebral blood flow (rCBF) at the bilateral prefrontal cortex (PFC) using near-infrared spectroscopy (NIRS). Method. The participants included eleven patients with schizophrenia and ten patients with mood disorders. The normalized tissue hemoglobin index (nTHI) was used as a sensitive parameter of rCBF by the SRS method and was measured during bilateral ECT using a two-channel NIRS. Results. 1. All patients responded to ECT treatment. 2. The levels of bilateral nTHI indicated a transient decrease during electrical stimulation and immediately were increased at both ictal and post-ictal phases by approximately 20% above baseline. 3. Patients with schizophrenia, but not mood disorders, showed significant asymmetric alteration of nTHI levels (left>right) during both the ictal and post-ictal phases. 4. The asymmetry index of nTHI, which indicates the difference between the left and right sides of the nTHI, was negatively correlated with the period of illness for schizophrenia, although the asymmetry index was not significantly correlated with any other clinical data, such as the effect of ECT treatment. Conclusion. Preliminary data demonstrated that bilateral ECT caused hemodynamic changes in bilateral PFC, and asymmetric alteration was found for schizophrenia, but not for mood disorders. Although further studies are necessary, the asymmetric hemodynamic response by ECT may be associated with the pathophysiology of schizophrenia, especially in the early stages.

Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production.

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.

Riluzole-induced glial cell line-derived neurotrophic factor production is regulated through fibroblast growth factor receptor signaling in rat C6 glioma cells.

Riluzole is approved for the treatment of amyotrophic lateral sclerosis (ALS); however, recent accumulating evidence suggests that riluzole is also effective for the treatment of psychiatric disorders, such as mood disorders. Plastic change in the brain induced by neurotrophic factors/growth factors is thought to be involved in the mechanism of antidepressants. This study investigated the mechanism of riluzole-induced glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells), a model of astrocytes. The study investigated the phosphorylation of cAMP response element binding protein (CREB), an important transcriptional factor of the gdnf gene, and found that riluzole increased CREB phosphorylation in a time-dependent manner, peaking at 40min after treatment. The riluzole-induced CREB phosphorylation was completely blocked by a mitogen-activated protein kinase kinase (MEK) inhibitor (U0126). Riluzole increased extracellular signal-regulated kinase (ERK) activation prior to CREB phosphorylation. These results suggest that riluzole rapidly activates the MEK/ERK/CREB pathway. Furthermore, two types of fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors (SU5402 and PD173074) completely blocked riluzole-induced CREB phosphorylation. In addition, riluzole rapidly phosphorylated FGFR substrate 2α (FRS2α), a major adaptor protein of FGFR. These findings suggest that riluzole induces CREB phosphorylation through FGFR. In addition, PD173074 inhibited riluzole-induced GDNF production. In contrast, l-glutamate and a glutamate transporter inhibitor (t-PDC) did not yield any effects in either CREB phosphorylation or GDNF production. These findings suggest that riluzole rapidly activates a MEK/ERK/CREB pathway through FGFR in a glutamate transporter-independent manner, followed by GDNF expression in C6 cells.

[The role of transactivation as a signal transduction mechanism in the central nervous system].

Transactivation represents the process whereby G-protein coupled receptors (GPCRs) activate receptor tyrosine kinases (RTKs), which are receptors for several neurotrophic factors and growth factors, with a subsequent downstream signaling such as mitogen-activated protein kinase (MAPK). Transactivation has been shown to be involved in the pathophysiology of cardiac hypertrophy and chronic kidney diseases, and it is targeted to develop novel therapeutic agents for these diseases. Recent accumulating evidence indicates that monoamines as well as neurotrophic factors and growth factors are thought to be involved in the therapeutic effects for psychiatric disorders. We have reported that serotonin (5-HT), which is important for the effect of antidepressant, increases glial cell line-derived neurotrophic factor (GDNF) via the 5-HT2R-mediated fibroblast growth factor receptor 2 transactivation pathway in glial cells (Tsuchioka et al, 2008). Transactivation in the CNS may play a role in the crosstalk between receptors for neurotransmitters, such as monoamines, and RTKs. Thus, transactivation will be an important basis for producing novel treatment strategies for psychiatric disorders. In this review, we outline a role of transacitvation in the CNS by explaining the concept and the mechanism of transactivation and summarizing recent reports about it in the CNS with our results.

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells.

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT(2) receptor (5-HT(2)R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT(2)R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Galpha(q/11) and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Galpha(q/11), 5-HT(2)R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT(2)R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT(2)R-mediated FGFR2 transactivation in C6 cells.

Antidepressants induce acute CREB phosphorylation and CRE-mediated gene expression in glial cells: a possible contribution to GDNF production.

Recently, the changes of neuronal and glial plasticity related gene expression following the increase of monoamine are suggested to be important for the therapeutic effect of antidepressants. We previously showed that antidepressants increased glial cell line-derived neurotrophic factor (GDNF) expression, which was dependent on acute activation of protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK) in rat C6 glioma cells (C6 cells) and normal human astrocytes (NHA). Transcription of many genes including GDNF is directed by the cAMP responsive element (CRE) and its cognate transcription factor CRE binding protein (CREB). In this study, we showed that amitriptyline, a tricyclic antidepressant, acutely increased phosphorylation of CREB, without altering the level of total CREB in C6 cells as well as in NHA. In contrast, acute amitriptyline treatment did not affect phosphorylation of CREB in SH-SY5Y cells, a human neuroblastoma cell line. Different classes of antidepressants as well as amitriptyline acutely increased phosphorylation of CREB, but haloperidol and diazepam did not. The amitriptyline-induced phosphorylation of CREB was completely blocked by U0126 [a mitogen-activated protein (MAP) kinase kinase 1 inhibitor] and genistein (a PTK inhibitor), but not by inhibitors of protein kinase A, p38 MAP kinase, or Ca(2+)/calmodulin-dependent kinase. Amitriptyline treatment also increased the expression of luciferase reporter gene regulated by CRE elements. The amitriptyline-induced luciferase activity was completely inhibited by U0126 in the same as phosphorylation of CREB. These results suggest that antidepressants acutely increase CREB activity in PTK and ERK-dependent manners, which might contribute to gene expression including GDNF in glial cells.

Prior neonatal isolation reduces induction of NGF mRNA and decreases GDNF mRNA in the hippocampus of juvenile and adult rodents subjected to immobilization stress.

Numerous studies have demonstrated that early adverse experiences are associated with the development of susceptibility to stress later in life. Although it is known that early experience of adversity, such as neonatal isolation, maternal separation, and low maternal care, enhances the activity of the hypothalamo-pituitary-adrenalaxis in rodents, the detailed mechanism underlying stress susceptibility induced by early adversity remains to be elucidated. Since neurotrophins have been shown to have a neuroprotective effect, we examined the influence of repeated neonatal isolation on expression of nerve growth factor (NGF), glia cell-derived neurotrophic factor (GDNF), and neurotrophin-3 mRNA in the hippocampus of juvenile and adult rats subsequently exposed immobilization stress, using real-time quantitative PCR and in situ hybridization. Neonatal isolation did not affect the basal hippocampal expression of these neurotrophin mRNAs in either juvenile or adult rats not subsequently exposed to immobilization. Similarly, there was a significant interaction between neonatal isolation and immobilization that affected the expression of NGF and GDNF mRNAs. Neonatal isolation attenuated the induction of NGF mRNA in both groups of rats and decreased GDNF mRNA in juvenile rats in response to immobilization. The decreased induction of NGF mRNA and reduced GDNF mRNA in response to immobilization was found in the CA3 pyramidal cell layer and dentate gyrus granular cell layer in the hippocampus of adult rats that had been subjected to neonatal isolation. These findings suggest that susceptibility to stress arising from prior neonatal isolation might be a result of decreased neuroprotective support through NGF and GDNF.

[Glia as targets for antidepressants: an involvement in glial cell line-derived neurotrophic factor].

Recently, clinical and animal studies have shown that neuronal and glial plasticity are important for the therapeutic action of antidepressants. Thus, it has been suggested that neurotrophic factors or growth factors, which are potent regulators for neuronal and glial plasticity, might be involved in the effect of antidepressants. Post-mortem studies provide evidence for glial reduction in different brain areas in mood disorders. Therefore, we focused on glial cell line-derived neurotrophic factor (GDNF) in mood disorders, because GDNF plays an important role in neurogenesis and high-ordered brain function, such as learning and memory. GDNF family ligands have shown promise of efficacy for neurodegenerative disorders such as Parkinson's disease, suggesting that GDNF family ligands exist in the closest position to clinical development for treatment of diseases of the central nervous system. We reported that total GDNF levels in whole blood in patients with mood disorders were significantly lower than those in healthy control subjects (Takebayashi et al, 2006), and antidepressants increased GDNF production through monoamine-independent activation of protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK) in glial cells (Hisaoka et al, 2007). Clarifying the monoamine-independent novel target of antidepressants in glia might contribute to the development of more efficient therapeutics for depression.

Antidepressants increase glial cell line-derived neurotrophic factor production through monoamine-independent activation of protein tyrosine kinase and extracellular signal-regulated kinase in glial cells.

Recent studies show that neuronal and glial plasticity are important for therapeutic action of antidepressants. We previously reported that antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells). Here, we found that amitriptyline, a tricyclic antidepressant, increased both GDNF mRNA expression and release, which were selectively and completely inhibited by mitogen-activated protein kinase kinase inhibitors. Indeed, treatment of amitriptyline rapidly increased extracellular signal-regulated kinase (ERK) activity, as well as p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase activities. Furthermore, different classes of antidepressants also rapidly increased ERK activity. The extent of acute ERK activation and GDNF release were significantly correlated to each other in individual antidepressants, suggesting an important role of acute ERK activation in GDNF production. Furthermore, antidepressants increased the acute ERK activation and GDNF mRNA expression in normal human astrocytes as well as C6 cells. Although 5-hydroxytryptamine (serotonin) (5-HT), but not noradrenaline or dopamine, increased ERK activation and GDNF release via 5-HT2A receptors, ketanserin, a 5-HT2A receptor antagonist, did not have any effect on the amitriptyline-induced ERK activation. Thus, GDNF production by amitriptyline was independent of monoamine. Both of the amitriptyline-induced ERK activation and GDNF mRNA expression were blocked by genistein, a general protein tyrosine kinase (PTK) inhibitor. Actually, we found that amitriptyline acutely increased phosphorylation levels of several phosphotyrosine-containing proteins. Taken together, these findings indicate that ERK activation through PTK regulates antidepressant-induced GDNF production and that the GDNF production in glial cells may be a novel action of the antidepressant, which is independent of monoamine.

Decreased levels of whole blood glial cell line-derived neurotrophic factor (GDNF) in remitted patients with mood disorders.

Recent post-mortem and imaging studies provide evidence for a glial reduction in different brain areas in mood disorders. This study was aimed to test whether glial cell line-derived neurotrophic factor (GDNF), a member of transforming growth factor (TGF)-beta superfamily, in blood levels was associated with mood disorders. We measured GDNF and TGF-beta levels in whole blood in remitted patients with mood disorders [n=56; major depressive disorders (MDD) 39, bipolar disorders (BD) 17] and control subjects (n=56). GDNF and TGF-beta were assayed with the sandwich ELISA method. Total GDNF levels were significantly lower in MDD and in BD than in control subjects (MDD, p=0.0003; BD, p=0.018), while no significant difference in total TGF-beta1 or total TGF-beta2 levels was found in these groups. Our study suggests that lower GDNF levels might be involved in the pathophysiology of mood disorders, although this preliminary study has several limitations.

[Mechanisms of antidepressants and serotonin (5-HT)-induced glial cell line-derived neurotrophic factor (GDNF) releases in rat C6 gliobrastoma cells].

Recent studies show that neuronal and glial plasticity are important for the therapeutic action of antidepressants. Here, we demonstrated that amitriptyline, a tricyclic antidepressant, significantly increased GDNF mRNA and GDNF release in C6 cells. Furthermore, different classes of antidepressants increased GDNF release, but non-antidepressant psychotropic drugs did not. The amitriptyline-induced GDNF release was completely inhibited by U0126, a mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89, a protein kinase A inhibitor or calphostin C, a protein kinase C inhibitor. These results suggest that the amitriptyline-induced GDNF release may be regulated through a MEK/MAPK pathway. Next, we examined the effects of monoamines on GDNF release, because antidepressants are known to increase monoamines. 5-HT increased GDNF mRNA and GDNF release, but noradrenaline and dopamine did not. The 5-HT-induced GDNF release was partially, but significantly, blocked by ketanserin, a 5-HT2A receptor antagonist. The 5-HT-induced GDNF release was completely inhibited by U0126, but was not inhibited by H-89 or calphostin C. These results suggest that the 5-HT-induced GDNF release was mediated through a MEK/MAPK pathway and, at least, 5-HT2A receptors. GDNF, as well as other neurotrophic factors, may contribute to explain the therapeutic action of antidepressants and suggest a novel strategy of pharmacological intervention.

Serotonin increases glial cell line-derived neurotrophic factor release in rat C6 glioblastoma cells.

Antidepressants, which increase monoamine levels, induce glial cell line-derived neurotrophic factor (GDNF) release in C6 cells. Thus, we examined whether monoamines affect on GDNF release in C6 cells. We found that serotonin (5-HT) specifically increased GDNF mRNA expression and GDNF release in a dose- and time-dependent manner. The 5-HT-induced GDNF release was mediated through the MEK/mitogen-activated protein kinase (MAPK) pathway and, at least, 5-HT(2A) receptors. The action of 5-HT on GDNF release may provide important insights into the mechanism of antidepressants.

Antidepressant drugs and cytokines in mood disorders.

This article reviews recent developments in cytokine research that pertain to pharmacological treatment of mood disorders such as antidepressants and lithium. We review the possible involvement of cytokines in mood disorders and their role in the therapeutic effects of antidepressant drugs. Growing evidence suggests that specific cytokines signal the brain to generate neurochemical, neuroimmune, neuroendocrine and behavior changes. An imbalance of cytokines within the central nervous system (CNS), or even systemically, may play a role in the pathophysiology of mood disorders. Modulation of these cytokines by chronic antidepressant treatment may result in restored balance. However, the effect of antidepressants on cytokines is still unclear both in clinical and preclinical research due to limited data. Further research is needed to clarify the involvement of cytokines in mood disorders. Understanding this relationship may lead to rational, therapeutic improvements in antidepressant and mood stabilizing drugs.

5-HT-induced, 5-HT3 receptor-mediated, and ruthenium red- and capsaicin-sensitive positive chronotropic effects in the isolated guinea pig atrium.

We investigated the mechanisms of 5-HT-induced tachycardia, which we reported previously to be triggered by 5-HT3 receptor stimulation, in the isolated guinea pig atrium in comparison with that induced by isoproterenol and histamine. We found that 5-HT-induced tachycardia was completely inhibited by ruthenium red. 5-HT-induced tachycardia was reduced in the capsaicin pre-treated atrium as well as in the presence of capsaicin. The effects of isoproterenol and histamine were not affected by ruthenium red or capsaicin treatment. Furthermore, 5-HT-induced tachycardia was found to be potentiated by thiorphan, an inhibitor of peptide degeneration. Calcitonin gene-related peptide (CGRP) (1-37), a full agonist of CGRP1-like receptors, was found to act selectively as a potent stimulator of chronotropic action. CGRP (8-37), an antagonist of CGRP1-type receptors, inhibited 5-HT-induced tachycardia as well as effects induced by CGRP (1-37). The observation that tetrodotoxin failed to affect 5-HT-induced tachycardia excluded the involvement of 5-hydroxytryptaminergic interneurons. Thus, we confirmed that the mechanism of 5-HT-induced tachycardia is distinct from that induced by isoproterenol and histamine. In conclusion, the activation of 5-HT3 receptors on the sensory nerve terminals brought about ruthenium red-sensitive Ca2+ influx and resulted in the release of CGRP from capsaicin-sensitive stores, and then CGRP stimulated CGRP1-like receptors to produce 5-HT-induced tachycardia.