Role of Nitric Oxide on Motor Behavior

The present review paper describes results indicating the influence of nitric oxide (NO) on motor control. Our last studies showed that systemic injections of low doses of inhibitors of NO synthase (NOS), the enzyme responsible for NO formation, induce anxiolytic effects in the elevated plus maze whereas higher doses decrease maze exploration. Also, NOS inhibitors decrease locomotion and rearing in an open field arena.These results may involve motor effects of this compounds, since inhibitors of NOS, NG-nitro-L-arginine (L-NOARG), N^G-nitro-L-arginine methylester (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), and 7-Nitroindazole (7-NIO), induced catalepsy in mice. This effect was also found in rats after systemic, intracebroventricular or intrastriatal administration.Acute administration of L-NOARG has an additive cataleptic effect with haloperidol, a dopamine D2 antagonist. The catalepsy is also potentiated by WAY 100135 (5-HT1a receptor antagonist), ketanserin (5HT2a and alfa1 adrenergic receptor antagonist), and ritanserin (5-HT2a and 5HT2c receptor antagonist). Atropine sulfate and biperiden, antimuscarinic drugs, block L-NOARG-induced catalepsy in mice.L-NOARG subchronic administration in mice induces rapid tolerance (3 days) to its cataleptic effects. It also produces cross-tolerance to haloperidol-induced catalepsy. After subchronic L-NOARG treatment there is an increase in the density NADPH-d positive neurons in the dorsal part of nucleus caudate-putamen, nucleus accumbens, and tegmental pedunculupontinus nucleus. In contrast, this treatment decreases NADPH-d neuronal number in the substantia nigra compacta.Considering these results we suggest that (i) NO may modulate motor behavior, probably by interfering with dopaminergic, serotonergic, and cholinergic neurotransmission in the striatum; (ii) Subchronic NO synthesis inhibition induces plastic changes in NO-producing neurons in brain areas related to motor control and causes cross-tolerance to the cataleptic effect of haloperidol, raising the possibility that such treatments could decrease motor side effects associated with antipsychotic medications.Finally, recent studies using experimental Parkinsons disease models suggest an interaction between NO system and neurodegenerative processes in the nigrostriatal pathway. It provides evidence of a protective role of NO. Together, our results indicate that NO may be a key participant on physiological and pathophysiological processes in the nigrostriatal system.     

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.     

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT(4) receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca(2+)-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (I(sc)), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT(4) receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and I(sc), whereas methiothepine (5-HT(1) receptor antagonist), ketanserin (5-HT(2) receptor antagonist), and a low concentration of ICS-205930 (5-HT(3) receptor antagonist) had no effect. RS-67506 (partial 5-HT(4) receptor agonist) concentration-dependently increased bicarbonate secretion and I(sc), whereas 5-carboxamidotryptamine (5-HT(1) receptor agonist), alpha-methyl-5-HT (5-HT(2) receptor agonist), and phenylbiguanide (5-HT(3) receptor agonist) did not significantly increase bicarbonate secretion or I(sc). RT-PCR analysis confirmed the expression of 5-HT(4) receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca(2+)-dependent signaling pathways and 5-HT(4) receptors located in the duodenal mucosa and/or epithelial cells.     

Extracellular superoxide enhances 5-HT-induced murine pulmonary artery vasoconstriction

Accumulating evidence suggests that changes in both 5-hydroxytryptamine (5-HT) receptor activity and in the levels of reactive oxygen species (ROS) play an important role in regulating pulmonary artery (PA) vascular responsiveness, particularly in the setting of pulmonary hypertension. Therefore, we hypothesized that increased levels of superoxide enhance 5-HT-induced PA constriction. With the use of a small-vessel bioassay, 5-HT (0.01-10 microM) induced a concentration-dependent vasoconstriction in isolated wild-type murine intrapulmonary arteries (100-150 microm diameter) that was enhanced by both removal of the endothelium and by treatment with either N(G)-nitro-L-arginine methyl ester (30 microM) or xanthine (10 microM) + xanthine oxidase (0.005 U/ml). PA isolated from extracellular superoxide dismutase (EC-SOD) knockout mice also showed enhanced constriction. On the other hand, PA constriction to 5-HT was attenuated by either the addition of GR-127935 (0.1 microM, a selective inhibitor of 5-HT(1B/1D) receptor) or copper/zinc-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) and in PA isolated from transgenic mice overexpressing human EC-SOD. With the use of both oxidative fluorescent confocal microscopy and lucigenin-enhanced chemiluminescence, superoxide levels were increased significantly after 5-HT-induced PA vasoconstriction. This increase in superoxide levels could be blocked by the exogenous addition of Cu/Zn SOD (150 U/ml) or by apocynin (30 microM, an inhibitor of NADPH oxidase) but was not affected by gp91(phox) knockout mice. Overall, our results are consistent with 5-HT increasing vascular smooth muscle superoxide production via an NADPH oxidase pathway that is independent of gp91(phox), which leads to increases in extracellular superoxide levels, which in turn enhances 5-HT-induced murine pulmonary vasoconstriction.     

Exercise preserves endothelium-dependent relaxation in coronary arteries of hypercholesterolemic male pigs.

We tested the hypothesis that exercise training (Ex) attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in male porcine coronary arteries [left anterior descending coronary arteries (LAD)] by increasing nitric oxide (NO) release [due to increased endothelial NO synthase (NOS) expression] and/or increased bioactivity of NO. Adult male pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20-24 wk. Pigs were Ex or remained sedentary (Sed) for 16-20 wk, beginning after 4 wk on diet. Four groups of pigs were used: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. HF enhanced LAD contractions induced by KCl, aggregating platelets (AP), and serotonin (5-HT). AP and 5-HT produced EDR after blockade of cyclooxygenase with indomethacin (Indo) and smooth-muscle 5-HT(2) receptors with ketanserin. HF impaired EDR induced by AP, 5-HT, and bradykinin. Results indicate a decreased contribution of NO to EDR in HF-Sed LADs, because the percentage of bradykinin-induced EDR inhibited by N(G)-nitro-L-arginine methyl ester was 27% in NF-Sed and 34% in NF-Ex but only 17% in HF-Sed. Also, N(G)-nitro-L-arginine methyl ester + Indo results indicate that release of an Indo-sensitive vasoconstrictor contributes to blunted EDR in HF-Sed LAD. Immunoblot and immunohistochemistry results indicate the following: 1) LAD endothelial NOS protein content was similar among groups; 2) HF decreased LAD superoxide dismutase (SOD) but increased caveolin-1 content; and 3) Ex increased SOD content of HF LADs. We conclude that HF impairs EDR by impairing the contribution of NO released from NOS (due to decreased SOD and increased caveolin-1 protein content) and by production of an Indo-sensitive vasoconstrictor. Ex preserves EDR in HF LADs by decreasing the production of the constrictor and increasing NO-release by NOS and/or NO bioactivity and bioavailability.     

5-Hydroxytryptamine-Facilitated Release of Substance P from Rat Spinal Cord Slices Is Mediated by Nitric Oxide and Cyclic GMP

Abstract: The role of nitric oxide (NO) in the control of 5-hydroxytryptamine (5-HT)-induced release of substance P was investigated in rat spinal cord in vitro. 5-HT facilitated the 60 m M K⁺-evoked release of substance P-like immunoreactive materials (SPLI) from the superfused rat dorsal spinal cord slices without affecting spontaneous SPLI release. The facilitatory effect of 5-HT was significantly inhibited by ICS 205-930 or granisetron (potent and specific 5-HT₃ receptor antagonists), by N ^G-monomethyl- l -arginine (NMMA, a NO synthase inhibitor), and by methylene blue or 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxaline-1-one (MB or ODQ, respectively; both are inhibitors of soluble guanylyl cyclase) and was mimicked by 2-methylserotonin (2-m-5-HT, a selective 5-HT₃ receptor agonist), l -arginine (a precursor of NO), or 8-bromo-cyclic GMP. NMMA, MB, or ODQ inhibited the 2-m-5-HT-induced increase of cyclic GMP levels in the rat dorsal spinal cord slices. These data suggest that the facilitatory effect of 5-HT on the release of SPLI is mediated by the 5-HT₃ receptor and that the intracellular signaling is mediated via NO by an increase in cyclic GMP production.     

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.     

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.     

Functional study of rat 5-HT2A receptors using antisense oligonucleotides

We studied the effects in rats of a 6-day intracerebroventricular (i.c.v) infusion of four different end-capped phosphorothioate-modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5-hydroxytryptamine2A (5-HT2A) receptor mRNA, on central 5-HT2A receptor expression and 5-HT2A receptor-mediated behaviours. Only one of the AOs (sequence 4), directed against the 5'-untranslated region (from + 557 to + 577), specifically affected central 5-HT2A receptor expression and receptor-mediated behaviour. This AO (sequence 4) reduced binding of the 5-HT2A agonist 1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5-day infusion of AO (sequence 4) resulted in profound inhibition of the head-twitch response (HTR) to 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM-induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5-HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood-brain barrier and cause a stress-induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5-HT2A receptor-mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5-HT2A receptor activation. Labelling of central 5-HT2C receptors by [3H]mesulergine, and 5-HT2C receptor-mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down-regulation of functional 5-HT2A receptors may shift the balance between various 5-HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor-mediated behaviour can be identified.     

Inhibition of Nitric Oxide Synthase Activity Attenuates Striatal Malonate Lesions in Rats

Abstract: Mitochondrial inhibitors such as malonate are potent neurotoxins in vivo. Intrastriatal injections of malonate result in neuronal damage reminiscent of "excitotoxic" lesions produced by compounds that activate NMDA receptors. Although the mechanism of cell death produced by malonate is uncertain, overactivation of NMDA receptors may be involved; pretreatment of animals with NMDA antagonists provides neuroprotection against malonate lesions. NMDA receptor activation stimulates the enzyme nitric oxide (NO) synthase (NOS). Elevated tissue levels of NO may generate highly reactive intermediates that impair mitochondrial function. We hypothesized that NO may be a mediator of malonate toxicity. We investigated whether in vivo inhibition of NO production by the NOS inhibitor N ^ω-nitro- l -arginine (NLA) would attenuate lesions produced by intrastriatal injections of malonate. We found that systemic injections of 3 mg/kg of NLA significantly reduced the extent of histologic damage elicited by intrastriatal injections of 1.5 µmol of malonate in adult rats.     

Microparticles from preeclamptic women induce vascular hyporeactivity in vessels from pregnant mice through an overproduction of NO

Preeclampsia is associated with an increase of circulating levels of microparticles (MPs), but their role in vascular dysfunction during the course of preeclampsia is not understood. Inasmuch as preeclampsia is a gestational disease, we tested the effect of MPs from preeclamptic women (PrMPs) and MPs from normal pregnant women (CMPs) on vessels from pregnant mice. We exposed aortic rings from pregnant mice to circulating levels of PrMPs or CMPs for 24 h and evaluated their response to serotonin (5-HT). PrMPs, but not CMPs, were able to induce hyporeactivity in response to 5-HT in aortas from pregnant mice. The nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine strongly enhanced the response to 5-HT in PrMP-treated vessels but had no significant effect on CMP-treated vessels. The 5-HT-induced contraction in PrMP-treated vessels was completely abolished by the selective cyclooxygenase-2 (COX-2) inhibitor NS-398 but was only reduced in CMP-treated vessels, suggesting an increased participation of COX-2 vasoconstrictor products in the effect of PrMPs. Consistent with this hypothesis, PrMPs enhanced levels of 8-isoprostane and PGE(2) in vessels, despite reduction of thromboxane B(2). These results strengthen the main concept that MPs in preeclampsia could act as vectors to stimulate intracellular cascades in vascular cells, leading to an enhanced NO production to counteract increased COX-2 vasoconstrictor metabolites by taking into account pregnancy.     

NOS inhibition synchronizes calcium oscillations in human adipose tissue-derived mesenchymal stem cells by increasing gap-junctional coupling

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.     

PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor

Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5-HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK(-) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.     

Serotonin agonist and antagonist actions in hippocampal CA1 neurons

The actions of serotonin (5-HT) and its putative agonists and antagonists were examined in vitro on hippocampal CA1 neurons using intracellular recordings, demonstrating that the cellular pharmacological effects can not necessarily be predicted from binding characteristics alone. The first response following 5-HT application was often a long-lasting (several minutes) hyperpolarization associated with decreased input resistance. Subsequent 5-HT applications caused only brief hyperpolarizations (30-120 s) and associated decreased input resistance, often followed by membrane depolarization. The post-spike train afterhyperpolarization (AHP) was prolonged for several minutes following the 5-HT induced hyperpolarization. 5-HT1 agonists (8-hydroxy-2-(di-n-propylamino)tetralin, 5-methoxytryptamine, MK-212) caused a prolonged hyperpolarization, decreased input resistance, and enhancement of the AHP. 5-HT applied following agonist application elicited only short-lasting hyperpolarizations. The 5-HT2 antagonists, cyproheptadine and mianserin, and a nonspecific 5-HT antagonist, methysergide, also caused a prolonged hyperpolarization with decreased input resistance. Spiperone, a nonspecific 5-HT antagonist, and ritanserin, a putative specific 5-HT2 receptor antagonist, depolarized CA1 neurons with little or no change in input resistance. The 5-HT-induced short-lasting hyperpolarization was not affected by drop application of 5-HT antagonists, except for methysergide, but perfusion of methysergide, ritanserin, and spiperone attenuated this response. The long-lasting 5-HT hyperpolarization might be mediated by 5-HT1A receptor activation, and the short-lasting hyperpolarization by another serotonergic receptor subtype.     

Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells.     

Characteristics of GABA release modified by glutamate receptors in mouse hippocampal slices

The major part of hippocampal innervation is glutamatergic, regulated by inhibitory GABA-releasing interneurons. The modulation of [(3)H]GABA release by ionotropic and metabotropic glutamate receptors and by nitric oxide was here characterized in superfused mouse hippocampal slices. The ionotropic glutamate receptor agonists kainate, N-methyl-D-aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate potentiated the basal GABA release. These effects were blocked by their respective antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX), dizocilpine and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX), indicating receptor-mediated mechanisms. The NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), sodiumnitroprusside and hydroxylamine enhanced the basal GABA release. Particularly the sodiumnitroprusside-evoked release was attenuated by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) and the inhibitor of soluble guanylyl cyclase 1H-(1,2,4)oxadiazolo(4,3a)quinoxalin-1-one (ODQ), indicating the involvement of the NO/cGMP pathway. This inference is corroborated by the enhancing effect of zaprinast, a phosphodiesterase inhibitor, which is known to increase cGMP levels. The K(+)-stimulated hippocampal GABA release was reduced by the groups I and III agonists of metabotropic glutamate receptors (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4), which effects were abolished by their respective antagonists (RS)-1-aminoindan-1,5-dicarboxylate (AIDA) and (RS)-2-cyclopropyl-4-phosphonophenylglycine (CPPG), again indicating modification by receptor-mediated mechanisms.     

Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+/calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons

We have studied the regulation of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT(1A) receptor agonists and blocked by 5-HT(1A) antagonists, indicating the mediation by 5-HT(1A) receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT(1A) agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT(1A) receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice.     

Lipopolysaccharide induces relaxation in lung pericytes by an iNOS-independent mechanism

Lipopolysaccharide (LPS)-regulated contractility in pericytes may play an important role in mediating pulmonary microvascular fluid hemodynamics during inflammation and sepsis. LPS has been shown to regulate inducible nitric oxide (NO) synthase (iNOS) in various cell types, leading to NO generation, which is associated with vasodilatation. The purpose of this study was to test the hypothesis that LPS can regulate relaxation in lung pericytes and to determine whether this relaxation is mediated through the iNOS pathway. As predicted, LPS stimulated NO synthesis and reduced basal tension by 49% (P < 0.001). However, the NO synthase inhibitors N (omega)-nitro-L-arginine methyl ester, aminoguanidine, and N (omega)-monomethyl-L-arginine did not block the relaxation produced by LPS. In fact, aminoguanidine and N (omega)-monomethyl-L-arginine potentiated the LPS response. The possibility that NO might mediate either contraction or relaxation of the pericyte was further investigated through the use of NO donor compounds; however, neither sodium nitroprusside nor S-nitroso-N-acetylpenicillamine had any significant effect on pericyte contraction. The inhibitory effect of aminoguanidine on LPS-stimulated NO production was confirmed. This ability of LPS to inhibit contractility independent of iNOS was also demonstrated in lung pericytes derived from iNOS-deficient mice. This suggests the presence of an iNOS-independent but as yet undetermined pathway by which lung pericyte contractility is regulated.     

Pulmonary vascular effects of serotonin and selective serotonin reuptake inhibitors in the late-gestation ovine fetus

Maternal use of selective serotonin (5-HT) reuptake inhibitors (SSRIs) is associated with an increased risk for persistent pulmonary hypertension of the newborn (PPHN), but little is known about 5-HT signaling in the developing lung. We hypothesize that 5-HT plays a key role in maintaining high pulmonary vascular resistance (PVR) in the fetus and that fetal exposure to SSRIs increases 5-HT activity and causes pulmonary hypertension. We studied the hemodynamic effects of 5-HT, 5-HT receptor antagonists, and SSRIs in chronically prepared fetal sheep. Brief infusions of 5-HT (3-20 μg) increased PVR in a dose-related fashion. Ketanserin, a 5-HT 2A receptor antagonist, caused pulmonary vasodilation and inhibited 5-HT-induced pulmonary vasoconstriction. In contrast, intrapulmonary infusions of GR127945 and SB206553, 5-HT 1B and 5-HT 2B receptor antagonists, respectively, had no effect on basal PVR or 5-HT-induced vasoconstriction. Pretreatment with fasudil, a Rho kinase inhibitor, blunted the effects of 5-HT infusion. Brief infusions of the SSRIs, sertraline and fluoxetine, caused potent and sustained elevations of PVR, which was sustained for over 60 min after the infusion. SSRI-induced pulmonary vasoconstriction was reversed by infusion of ketanserin and did not affect the acute vasodilator effects of acetylcholine. We conclude that 5-HT causes pulmonary vasoconstriction, contributes to maintenance of high PVR in the normal fetus through stimulation of 5-HT 2A receptors and Rho kinase activation, and mediates the hypertensive effects of SSRIs. We speculate that prolonged exposure to SSRIs can induce PPHN through direct effects on the fetal pulmonary circulation.