Stimulation of serotonin transport by the cyclic GMP phosphodiesterase-5 inhibitor sildenafil

The serotonin (5-hydroxtryptamine, 5-HT) transporter (SERT) plays a critical role in the inactivation of synaptic 5-HT and has been implicated in multiple psychiatric and peripheral disorders. SERT regulation studies demonstrate that activation of cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-linked pathways can increase SERT activity. As cGMP actions are limited by cGMP-specific phosphodiesterase (PDEs), we investigated whether the cGMP-specific PDE5 inhibitor sildenafil (Viagra) can stimulate 5-HT uptake and potentiate cGMP-mediated regulation. In RBL-2H3 cells, SERT activity was stimulated by sildenafil in a concentration- and time-dependent manner. Sildenafil also enhanced the stimulation of SERT triggered by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), effects blocked by the PKG inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8). Sildenafil stimulation of 5-HT uptake arises from an increase in 5-HT transport Vmax and is paralleled by elevated SERT surface antagonist binding, also H8-sensitive. These findings implicate cGMP-targeted PDEs in limiting the regulation of antidepressant-sensitive 5-HT transport.     

Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors

Activation of A? adenosine receptors (A?ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A?AR agonist stimulation of SERT activity is lost in A?AR knockout mice. A?AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A?ARs target SERTs among other potential effectors is unknown. Here we present evidence that A?ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A?AR/hSERT cotransfected cells. Treatment of A?AR/SERT-cotransfected Chinese hamster ovary cells with the A?AR agonist N?-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A?AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A?AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A?AR activation. Because PKGI and nitric-oxide synthetase are required for A?AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A?AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.     

The proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha activate serotonin transporters.

Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT Km for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1beta stimulated 5-HT uptake in a dose- and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 microM), a p38 MAPK inhibitor. TNF-alpha also dose- and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1beta and TNF-alpha activated p38 MAPK, in an SB203580-sensitive manner. IL-1beta induced an SB203580-sensitive decrease in 5-HT Km with no significant change in Vmax. In contrast, TNF-alpha stimulation decreased 5-HT Km and increased SERT Vmax. SB203580 selectively blocked the TNF-alpha-induced change in SERT Km. In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1beta, 10 ng/ml; TNF-alpha, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1beta and TNF-alpha were completely (IL-1beta) or partially (TNF-alpha) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.     

Validation of [(123)I]beta-CIT SPECT to assess serotonin transporters in vivo in humans: a double-blind, placebo-controlled, crossover study with the selective serotonin reuptake inhibitor citalopram.

Disturbances in the serotonin (5-HT) system are associated with various neuropsychiatric disorders. The 5-HT system can be studied in vivo by measuring 5-HT transporter (SERT) densities using (123)iodine-labeled 2beta-carbomethoxy-3beta(4-iodophenyl)tropane ([(123)I]beta-CIT) and single photon emission computed tomography (SPECT). Validation of this technique is important because [(123)I]beta-CIT does not bind selectively to SERTs. Some studies have validated this technique in vivo in the human brain in SERT-rich areas, but the technique has not been validated yet in SERT-low cortical areas. The aim of this study was to further validate [(123)I]beta-CIT SPECT in assessing SERTs in vivo in humans in both SERT-rich and SERT-low areas. A double-blind, placebo-controlled, crossover design was used with the selective 5-HT reuptake inhibitor (SSRI) citalopram. Six male subjects underwent two [(123)I]beta-CIT SPECT sessions: one after pretreatment with citalopram and one after placebo. Scans were acquired 4 h and 22-27 h p.i., and both region-of-interest and voxel-by-voxel analyses were performed. Citalopram reduced [(123)I]beta-CIT binding ratios in SERT-rich midbrain and (hypo)thalamus. Binding ratios were also lower after citalopram in SERT-low cortical areas, but statistical significance was only reached in several cortical areas using voxel-by-voxel analysis. In addition, citalopram increased binding ratios in the DAT-rich striatum and increased absolute uptake in the cerebellum. The results show that [(123)I]beta-CIT SPECT is a valid technique to study SERT binding in vivo in human brain in SERT-rich areas. Although we provide some evidence that [(123)I]beta-CIT SPECT may be used to measure SERTs in SERT-low cortical areas, these measurements must be interpreted with caution.     

Activation of p38 mitogen-activated protein kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils

The signaling pathways leading to p38 mitogen-activated protein kinase (MAPK) activation in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils were examined. Immunoblot analysis with antibodies against a phosphorylated form of p38 MAPK showed that fMLP-stimulated p38 MAPK activation was dependent on a pertussis toxin-sensitive G protein. Two phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), did not affect the p38 MAPK activation. Phosphorylation of p38 MAPK was concentration dependently attenuated by a tyrosine kinase inhibitor, genistein, and by a Ca(2+)-dependent protein kinase C inhibitor, 13-cyanoethyl-12-methyl-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3 , 4-c]carbazole-7-one (G?6976). However, the protein kinase C inhibitors with a broader spectrum, 2-[1-(3-dimethylaminopropyl)-5-methoxy-1H-indol-3-yl]-3-(1H-indol-3-y l)-maleimide (G?6983) and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF109203X), had no inhibitory effect. fMLP-stimulated p38 MAPK phosphorylation was also reduced in cells pretreated with a phospholipase C inhibitor, 1-[6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), or preloaded with an intracellular Ca(2+) chelator, 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). We conclude that phosphorylation of p38 MAPK by fMLP stimulation in rat neutrophils is dependent on G(i/o) protein, nonreceptor tyrosine kinase, phospholipase C/Ca(2+), and probably Ca(2+)-dependent protein kinase C pathways.     

Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells

The endogenous nucleoside adenosine is thought to play a role in the pathophysiology of asthma by stimulating mast cells. We previously showed that the human mast cell line HMC-1 expresses A2A and A2B receptors, and that both receptors activate adenylate cyclase via Gs-protein but that only A2B receptors are also coupled to phospholipase C via Gq proteins. Stimulation of A2B but not A2A receptors induced production of interleukin-8 (IL-8) from HMC-1 cells. The mechanism by which adenosine promotes IL-8 synthesis has not been defined. In this study, we tested the hypothesis that mitogen-activated protein kinase (MAPK) signaling pathways are involved in this process. Stimulation of HMC-1 with the stable adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) activated p21(ras) and both p42 and p44 isoforms of extracellular signal-regulated kinase (ERK). NECA (10 microM) induced a 1.9 +/- 0. 06-fold increase in ERK activity, whereas 10 microM of the selective A2A agonist CGS 21680 (4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid) had no effect. NECA, in parallel with the activation of ERK, also stimulated the p46 isoform of c-Jun N-terminal kinase (MEK) and p38 MAPK. Furthermore, the selective MAPK/ERK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), and p38 MAPK inhibitors SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole) and SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) blocked A2B receptor-mediated production of IL-8. These results indicate that extracellular adenosine can regulate ERK, c-Jun N-terminal kinase, and p38 MAPK signaling cascades and that activation of ERK and p38 MAPK pathways are essential steps in adenosine A2B receptor-dependent stimulation of IL-8 production in HMC-1.     

Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A(1) receptor in DDT(1)MF-2 cells

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. We have previously reported that the human adenosine A(1) receptor stimulates p42/p44 MAPK in transfected Chinese hamster ovary cells. In this study, we have investigated whether the endogenous adenosine A(1) receptor in the smooth muscle cell line, DDT(1)MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A(1) receptor agonist N(6)-cyclopentyladenosine stimulated time and concentration-dependent increases in p42/p44 MAPK and p38 MAPK phosphorylation in DDT(1)MF-2 cells. No increases in JNK phosphorylation were observed following adenosine A(1) receptor activation. N(6)-cyclopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphorylation were blocked by the selective adenosine A(1) receptor antagonist 1,3-dipropylcyclopentylxanthine and following pretreatment of cells with pertussis toxin. Furthermore, adenosine A(1) receptor-mediated increases in p42/p44 MAPK were sensitive to the MAPK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas p38 MAPK responses were blocked by the p38 MAPK inhibitor SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole). The broad range protein tyrosine kinase inhibitors genistein and tyrphostin A47 (alpha-cyano-(3,4-dihydroxy)thiocinnamide) did not block adenosine A(1) receptor stimulation of p42/p44 MAPK. For comparison, insulin-mediated increases in p42/p44 MAPK were blocked by genistein and tyrphostin A47. The Src tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) also had no effect on adenosine A(1) receptor stimulation of p42/p44 MAPK. Furthermore, the protein kinase C inhibitors Ro 31-8220 (3-[1-[3-(2-isothioureido) propyl]indol-3-yl]-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), chelerythrine and GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) were without effect on adenosine A(1) receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A(1) receptor stimulation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A(1) receptor stimulates p42/p44 MAPK through a pathway which appears to be independent of tyrosine kinase activation but involves phosphatidylinositol 3-kinase. Finally, adenosine A(1) receptor stimulation in DDT(1)MF-2 cells also activated p38 MAPK but not JNK via a pertussis toxin-sensitive pathway.     

Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK

Activation of adenosine receptors in folliculostellate (FS) cells of the pituitary gland leads to the secretion of IL-6 and vascular endothelial growth factor (VEGF). We investigated the action of adenosine A2 receptor agonists on IL-6 and VEGF secretion in two murine FS cell lines (TtT/GF and Tpit/F1), and demonstrated a rank order of potency, 5'-N-ethylcarboxamidoadenosine (NECA)>2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine>adenosine, suggesting mediation via the A2b receptor. NECA-mediated IL-6 release was inhibited by the PLC inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-tiene-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione, the PI3 kinase inhibitor wortmannin and the PKC inhibitors bisindolylmaleimide 1 and bisindolymaleimide X1 HCl (Ro-32-0432). NECA-mediated IL-6 release was attenuated (<50%) by the extracellular signal-regulated kinase MAPK inhibitor 2'-amino-3'-methoxyflavone, and completely (>95%) inhibited by the p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole. NECA stimulates p38 MAPK phosphorylation that is inhibited by Ro-32-0432 but not by wortmannin. Dexamethasone inhibits NECA-stimulated IL-6 and VEGF secretion. These findings indicate that adenosine can stimulate IL-6 secretion in FS cells via the A2b receptor coupled principally to PLC/PKC and p38 MAPK; such an action may be important in the modulation of inflammatory response processes in the pituitary gland.     

Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes.

The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1-100 microM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 microM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the beta-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 microM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.     

Control of survival of proliferating L1210 cells by soluble guanylate cyclase and p44/42 mitogen-activated protein kinase modulators.

Intracellular signaling pathways involved in the survival of proliferating L1210 leukemia cells were investigated by using specific modulators. Among the various inhibitors tested, only 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor, was found to induce a marked increase in caspase activity, which was associated with a loss of cell viability and a reduction in cGMP content. ODQ also provoked the processing of caspases-3 and -9, release of cytochrome c and, as early events, reduction of Bcl-2 content and dephosphorylation of Bad at Ser 112. Furthermore, YC-1, an sGC activator, and 8-Br-cGMP, a cell-permeant analogue of cGMP, exerted some protection against various apoptotic stimuli, such as serum deprivation or spermine accumulation. Although PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, did not increase basal caspase activity, and ODQ did not affect p44/42 MAPK phosphorylation significantly, phorbol myristate acetate stimulated p44/42 MAPK and reduced caspase activation induced by ODQ, serum deprivation, and spermine in a p44/42-dependent manner. SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole), a p38 MAPK inhibitor, also partially protected against ODQ-induced apoptosis by increasing p44/42 MAPK phosphorylation. In conclusion, these results suggest that sGC may be relevant both for survival of L1210 cells under basal growing conditions and for protection against various apoptotic stimuli. p44/42 MAPK activation may also confer some protection from apoptosis, but apparently through a pathway largely independent of cGMP.     

Interleukin-1 Receptor Activation by Systemic Lipopolysaccharide Induces Behavioral Despair Linked to MAPK Regulation of CNS Serotonin Transporters

Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.     

5-Hydroxytryptamine induces cyclooxygenase-2 in rat vascular smooth muscle cells: Mechanisms involving Src, PKC and MAPK activation [corrected

Considering the importance of 5-hydroxytryptamine (5-HT) and cyclooxygenase (COX) products in vascular pathology, we investigated the effects of 5-HT on COX expression in rat vascular smooth muscle cells (VSMCs), and to provide mechanistic insights into these effects. VSMCs were enzymatically isolated from aortic media of Wistar rats. Incubation of VSMCs with 5-HT for 24h stimulated prostaglandin I(2) production, but this stimulation was completely suppressed by NS-398, a selective COX-2 inhibitor. 5-HT induced transient COX-2, but not COX-1, protein and mRNA expression in concentration- and time-dependent manners. This effect of 5-HT was completely inhibited by sarpogrelate, a 5-HT(2A) receptor antagonist. 5-HT-induced COX-2 expression was markedly blunted by Ca(2+) depletion; GF 109203X, a protein kinase C (PKC) inhibitor; PP2, an inhibitor of Src-family tyrosine kinase (Src); PD 98059, an inhibitor of extracellular signal-regulated kinase (ERK) activation; SB 203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK); and SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK). 5-HT activated ERK and p38 MAPK, followed by JNK activation. PP2 inhibited these activations, while GF 109203X inhibited only JNK activation. Furthermore, PD 98059 inhibited JNK activation. These results suggest that 5-HT induces COX-2 expression in rat VSMCs, and that PKC, Src, and MAPK activation are each essential for the full expression of COX-2 pathways.     

Tyrosine phosphorylation of the human serotonin transporter: a role in the transporter stability and function

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant (32)P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.     

Involvement of p38 mitogen-activated protein kinase in PLL-AGE-induced cyclooxgenase-2 expression

In the present study, murine RAW 264.7 macrophages were incubated with poly-L-lysine-derived advanced glycosylation end products (PLL-AGEs) to examine cyclooxygenase-2 protein expression. Treatment of RAW 264.7 cells with PLL-AGEs caused the dose-dependent expression of cylooxygenase-2 but not cylooxygenase-1 and an increase in cylooxygenase activity. Increased cylooxygenase-2 expression was seen at 6 h and reached a maximum at 24 h. The tyrosine kinase inhibitor, genistein, and the p38 mitogen-activated protein kinase (MAPK) inhibitor, [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (SB 203580), inhibited PLL-AGE-induced cylooxygenase-2 expression, while the Ras inhibitor, FPT inhibitor II, and the MAP kinase kinase inhibitor, (2'-amino-3'-methoxyflavone) (PD 98059), had no effect on PLL-AGE-induced cylooxygenase-2 expression. Incubation of RAW 264.7 cells with PLL-AGEs resulted in activation of p38 MAPK, and this activation was suppressed by genistein and SB 203580. Taken together, our results suggest that activation of protein tyrosine kinase and p38 MAPK is involved in AGE-induced cyclooxygenase-2 expression in RAW 264.7 macrophages.     

Inability of Serotonin to Activate the c-Jun N-terminal Kinase and p38 Kinase Pathways in Rat Aortic Vascular Smooth Muscle Cells

Background

Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells.

Results

Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10^-9 – 10^-5 M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10^-5 M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin.

Conclusion

Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.     

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.     

Agonist properties of pindolol at h5-HT1A receptors coupled to mitogen-activated protein kinase

At h5-HT1A receptors, stably transfected into Chinese Hamster Ovary Cells (CHO-h5-HT1A), the selective 5-HT1A receptor agonist, (+)8-hydroxy-dipropyl-amino-tetralin, ((+)8-OH-DPAT), transiently activated mitogen-activated protein kinase (MAPK) with a pEC50 of 8.5. The arylalkylamine, (-)-pindolol, also behaved as an agonist with a maximal effect of 57% relative to (+)8-OH-DPAT (100%), and with a pEC50 of 7.2. The selective 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-hexane carboxamide (WAY100,635), blocked (+)8-OH-DPAT- and (-)-pindolol-induced MAPK activation with pK(B)s of 9.7 and 9.9, respectively, whereas the selective 5-HT(1B) receptor antagonist, 1'-Methyl-5-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5H-spiro[furo[2,3-f]indole-3,4'-piperidine] (SB224,289) was inactive. Pertussis toxin blocked the actions of (+)8-OH-DPAT and (-)-pindolol demonstrating implication of G(i)/G(o) proteins. Thus, stimulation of MAPK provides an intracellular marker and signal for expression of the agonist actions of (-)-pindolol at h5-HT1A receptors.     

Methylene blue inhibits function of the 5-HT transporter

BACKGROUND AND PURPOSE

Methylene blue (MB) is commonly employed as a treatment for methaemoglobinaemia, malaria and vasoplegic shock. An increasing number of studies indicate that MB can cause 5-HT toxicity when administered with a 5-HT reuptake inhibitor. MB is a potent inhibitor of monoamine oxidases, but other targets that may contribute to MB toxicity have not been identified. Given the role of the 5-HT transporter (SERT) in the regulation of extracellular 5-HT concentrations, the present study aimed to characterize the effect of MB on SERT.

EXPERIMENTAL APPROACH

Live cell imaging, in conjunction with the fluorescent SERT substrate 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP⁺), [³H]5-HT uptake and whole-cell patch-clamp techniques were employed to examine the effects of MB on SERT function.

KEY RESULTS

In EM4 cells expressing GFP-tagged human SERT (hSERT), MB concentration-dependently inhibited ASP⁺ accumulation (IC₅₀: 1.4 ± 0.3 µM). A similar effect was observed in N2A cells. Uptake of [³H]5-HT was decreased by MB pretreatment. Furthermore, patch-clamp studies in hSERT expressing cells indicated that MB significantly inhibited 5-HT-evoked ion currents. Pretreatment with 8-Br-cGMP did not alter the inhibitory effect of MB on hSERT activity, and intracellular Ca²⁺ levels remained unchanged during MB application. Further experiments revealed that ASP⁺ binding to cell surface hSERT was reduced after MB treatment. In whole-cell radioligand experiments, exposure to MB (10 µM; 10 min) did not alter surface binding of the SERT ligand [¹²⁵I]RTI-55.

CONCLUSIONS AND IMPLICATIONS

MB modulated SERT function and suggested that SERT may be an additional target upon which MB acts to produce 5-HT toxicity.     

(3)H] citalopram binding to serotonin transporter sites in minnow brains

Mammalian serotonin (SERT) and norepinephrine transporters (NET) are target sites for antidepressants and are affected by pesticide exposures. Herein, we examined whether golden shiner (Notemigonus crysoleucas) or fathead minnow (Pimphales promelas) SERTs and catecholamine transporters respond comparably to mammalian SERTs and NETs. We compared the pharmacological profiles of central SERT and NET binding sites of the golden shiner minnow to those of rats. Homogenate binding with the radioligand [(3)H] citalopram indicated that golden shiner SERT has a K(D) of 7 +/- 3 nM and a B(max) of 226 +/- 46 fmol/mg protein. These values are similar to those of rat cortical SERT (K(D) 1.4 +/- 0.1 nM and B(max) 240 +/- 48 fmol/mg protein). We also examined SERT binding in fathead minnow brain, and found it similar to that of the golden shiner. A putative golden shiner NET, measured using [(3)H] nisoxetine, had K(D) = 12 +/- 5 nM and B(max) = 187 +/- 49 fmol/mg protein, whereas rat hippocampal NET had K(D) = 5 +/- 2 nM and B(max) = 93 +/- 8 fmol/mg protein. Minnow SERT and NET binding is displaceable by selective reuptake inhibitors. Finally, we exposed zebrafish (Danio rerio) to the serotonin reuptake inhibiting antidepressant sertraline or the organophosphate chlorpyrifos for 21 days. After either treatment, SERT binding was reduced by 50% (n = 3-6, P < 0.05). In summary, minnow central SERT and NET express slightly lower affinity for antidepressants than rats. However, magnitudes of affinity are similar, and minnow SERT binding is decreased by chronic sertraline or chlorpyrifos administration.     

Binding of tritiated sildenafil, tadalafil, or vardenafil to the phosphodiesterase-5 catalytic site displays potency, specificity, heterogeneity, and cGMP stimulation

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC(50), K(D) (isotherm), K(D) (dissociation rate), and K(D) ((1/2) EC(50)), respectively, were the following: sildenafil (3.7 +/- 1.4, 4.8 +/- 0.80, 3.7 +/- 0.29, and 11.7 +/- 0.70 nM), tadalafil (1.8 +/- 0.40, 2.4 +/- 0.60, 1.9 +/- 0.37, and 2.7 +/- 0.25 nM); and vardenafil (0.091 +/- 0.031, 0.38 +/- 0.07, 0.27 +/- 0.01, and 0.42 +/- 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil > tadalafil > sildenafil. Binding of each (3)H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds. (3)H Inhibitors did not bind to isolated PDE5 regulatory domain. Close correlation of EC(50) values using all three (3)H inhibitors competing against one another indicated that each occupies the same site on PDE5. Studies of sildenafil and vardenafil analogs demonstrated that higher potency of vardenafil is caused by differences in its double ring. Exchange-dissociation studies revealed two binding components for each inhibitor. Excess unlabeled inhibitor did not significantly affect (3)H inhibitor dissociation after infinite dilution, suggesting the absence of subunit-subunit cooperativity. cGMP addition increased binding affinity of [(3)H]tadalafil or [(3)H]vardenafil, an effect presumably mediated by cGMP binding to PDE5 allosteric sites, implying that either inhibitor potentiates its own binding to PDE5 in intact cells by elevating cGMP. Without inhibitor present, cGMP accumulation would stimulate cGMP degradation, but with inhibitor present, this negative feedback process would be blocked.