The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Inhibition of pig kidney dopa decarboxylase by coenzyme-5-hydroxytryptophan adducts

The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridoxyl)-D-5-hydroxytryptophan and N-(5'-phosphopyridoxyl)-5-hydroxytryptamine on the reactivation of apoDopa decarboxylase to holoenzyme has been investigated. The different degree of inhibition exerted by these adducts has been interpreted on the basis of a different orientation of the 2 isomers of 5-HTP at the active of Dopa decarboxylase.     

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.     

Serotonin levels in fish brain: Effects of hydrostatic pressure and water temperature

Summary The contents of serotonin (5 HT) and its metabolite 5 hydroxy indoleacetic acid (5 HIAA) have been measured (HPLC technique) in the brains of eels exposed to different conditions of hydrostatic pressure and temperature (HP=1 or 101 ATA in winter, Tw=14°C, and in summer, Tw=19°C). It appears that an increase of Tw induces a significant increase of the 5 HT/5 HIAA ratio. In contrast, eels exposed at 101 ATA of HP for 1 h do not exhibit any modification in the 5 HT/5 HIAA brain ratio at a given temperature. The involvement of 5 HT under the conditions studied is discussed.     

The molecular mechanisms of congenital hypofibrinogenaemia

Congenital hypofibrinogenaemia is characterized by abnormally low levels of fibrinogen and is usually caused by heterozygous mutations in the fibrinogen chain genes (, and ). However, it does not usually result in a clinically significant condition unless inherited in a homozygous or compound heterozygous state, where it results in a severe bleeding disorder, afibrinogenaemia. Various protein and expression studies have improved our understanding of how mutations causing hypo- and afibrinogenaemia affect secretion of the mature fibrinogen molecule from the hepatocyte. Some mutations can perturb chain assembly as in the 153 Cys Arg case, while others such as the B Leu Arg and the B414 Gly Ser mutations allow intracellular hexamer assembly but inhibit protein secretion. An interesting group of mutations, such as 284 Gly Arg and 375 Arg Trp, not only cause hypofibrinogenaemia but are also associated with liver disease. The nonexpression of these variant chains in plasma fibrinogen is due to retention in the endoplasmic reticulum, which in turn leads to hypofibrinogenaemia.Received 17 December 2003; received after revision 19 January 2004; accepted 21 January 2004     

Comparison of diurnal and nocturnal rates of 5-hydroxytryptamine turnover in the rat mediobasal hypothalamus

Rates of 5-hydroxytryptamine (5-HT) turnover in the mediobasal hypothalamus of male rats were estimated using pharmacological methods during the daytime and at night. Concentrations of 5-HT in this hypothalamic area were higher nocturnally than diurnally; this was apparently due to increased 5-HT synthesis and decreased 5-HT catabolism at night.     

Radioprotective effectiveness of some serotonin-like compounds

Zusammenfassung Bei Mäusen wurde die Strahlenschutzwirksamkeit des 5-Ethoxytryptamins und 5-Methoxytryptamins mit derjenigen von 5-Hydroxytriptamin, SAS (Benzothiophenverbindung) und 5-Methoxy-SAS verglichen. Die Ergebnisse zeigen, dass die Schutzwirksamkeit des 5-Ethoxytryptamins der des SAS sehr ähnlich ist.     

Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents

Somatostatin and cortistatin exert multiple biological actions through five receptors (sst1-5); however, not all their effects can be explained by activation of sst1-5. Indeed, we recently identified novel truncated but functional human sst5-variants, present in normal and tumoral tissues. In this study, we identified and characterized three novel truncated sst5 variants in mice and one in rats displaying different numbers of transmembrane-domains [TMD; sst5TMD4, sst5TMD2, sst5TMD1 (mouse-variants) and sst5TMD1 (rat-variant)]. These sst5 variants: (1) are functional to mediate ligand-selective-induced variations in [Ca²⁺]_i and cAMP despite being truncated; (2) display preferential intracellular distribution; (3) mostly share full-length sst5 tissue distribution, but exhibit unique differences; (4) are differentially regulated by changes in hormonal/metabolic environment in a tissue- (e.g., central vs. systemic) and ligand-dependent manner. Altogether, our results demonstrate the existence of new truncated sst5-variants with unique ligand-selective signaling properties, which could contribute to further understanding the complex, distinct pathophysiological roles of somatostatin and cortistatin.     

Serotonin and 5-hydroxyindoleacetic acid concentrations in individual hypothalamic nuclei and other brain areas of rat

Summary Serotonin and 5-hydroxyindoleacetic acid (5-HIAA) were measured in individual nuclei of rat hypothalamus and other brain areas using HPLC with electrochemical detection. 5-HIAA levels were first demonstrated in hypothalamic and some discrete brain areas. The 5-HIAA/5-HT ratio was highest in the n. caudatus putamen, high in the n. ventromedialis and lowest in the n. suprachiasmaticus.     

Action of reserpine and imipramine on intracellular storage of 5-hydroxytryptamine in blood platelets

Zusammenfassung In isolierten Blutplättchen von Katzen vermindert Imipramin im Gegensatz zu Reserpin das endogene 5-Hydroxytryptamin (5HT) und die 5HT-Speicherorganellen nicht wesentlich, obwohl es wie Reserpin die Aufnahme von exogenem 5HT herabsetzt. Hemmung des Membrantransportes von 5HT durch Imipramin scheint also das intrazellulär gespeicherte 5HT nur relativ wenig zu beeinflussen.     

Human papillomavirus 16 E5 up-regulates the expression of vascular endothelial growth factor through the activation of epidermal growth factor receptor, MEK/ ERK1,2 and PI3K/Akt

The E5 oncoprotein of human papillomavirus (HPV) 16 plays an important role in early cervical carcinogenesis. Vascular endothelial growth factor (VEGF) plays a central role in switching on the angiogenic phenotype during early cervical carcinogenesis. However, the relationship between E5 and VEGF has not previously been examined. To clarify the regulatory role of E5 in VEGF expression, we transferred the E5 gene into various cell types. E5 increased VEGF expression. The addition of epidermal growth factor receptor (EGFR) inhibitor significantly suppressed VEGF expression, demonstrating that E5 stimulates VEGF expression through the activation of EGFR. E5-mediated EGFR activation was accompanied by phosphorylation of Akt and ERK1/2, which are also involved in VEGF expression. Furthermore, the mRNA stability of VEGF was not affected by E5, but VEGF promoter activity could be modulated by inhibitors of the EGFR, MEK-ERK1/2 and PI3K/Akt pathways in E5-expressing cells. Collectively, these novel results suggest that HPV 16 E5 increases VEGF expression by activating EGFR, MEK/ERK1/2 and PI3K/Akt. Received 23 November 2005; received after revision 10 January 2006; accepted 9 February 2006     

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC₅₀ = 37.5 μM; K _i = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.     

Comparison of diurnal and nocturnal rates of 5-hydroxytryptamine turnover in the rat mediobasal hypothalamus

Summary Rates of 5-hydroxytryptamine (5-HT) turnover in the mediobasal hypothalamus of male rats were estimated using pharmacological methods during the daytime and at night. Concentrations of 5-HT in this hypothalamic area were higher nocturnally than diurnally; this was apparently due to increased 5-HT synthesis and decreased 5-HT catabolism at night.Supported by NIH grant RR07187 (TSK) and NIH grant HD10202 (Neuroendocrine Core).     

Tryptophan (Trp), serotonin (5-HT), monoamino oxidase (MAO) and 5-hydroxyindole acetic acid (5-HIAA) in brain and subesophagic ganglions of earthworms. Effects of Parathion

Summary Tryptophan, 5-HT, MAO and 5-HIAA were determined in the first 5 segments of earthworms (Oligochaetae) where the brain and subesophagic ganglions are located. Tranylcypromine (IMAO) decreased MAO activity increasing 5-HT and decreasing 5-HIAA. Motility and survival of worms were disturbed. InAllolobophora species (young worms), parathion fumigation decreased acetylcholinesterase (AChE) activity and increased Trp, 5-HT and 5-HIAA. Motility was diminished after 24 h: it worsened after 72, but returned to normal levels 40/50 days later.Publication of the National Institute of Agrarian Technology (INTA), Argentina.Acknowledgments. We are most grateful to Prof. Zlatko Tomsic (from the Lillo Institute, Tucumán) and to Leticia Alvarado (from INTA) and to Prof. Mirta P. de Matosian for earthworm classification.     

Effect of des-Tyr1-gamma-endorphin on serotonin metabolism in rat brain regions

Intracerebroventricular (i.c.v.) administration of des-Tyr1-gamma-endorphin (0.1 and 1.0 microgram) caused a decrease of the serotonin (5-HT) concentration of the hippocampus. The concentration of 5-HIAA and the pargyline-induced alterations in 5-HT and 5-HIAA were not affected. No effects were noticed in other brain regions.     

Defensive steroids from a carrion beetle (Silpha americana)

The defensive anal effluent discharged by Silpha americana in response to disturbance contains a mixture of steroids stemming from a glandular annex of the rectum. The compounds have been characterized as 15 beta-hydroxyprogesterone (1, principal component), 5 beta-pregnan-15 beta-ol-3,20-dione (2), 5 beta-pregnan-3 alpha, 15 beta-diol-20-one (3), 5 beta-pregnan-7 beta, 15 beta-diol-3,20-dione (4), 5 beta-pregnan-3 alpha, 7 beta, 15 beta-triol-20-one (5), 5 beta-pregnan-16 alpha-ol-3,20-dione (6), and 5 beta-pregnan-3 alpha, 16 alpha-diol-20-one (7), none previously found in insects. Bioassays with jumping spiders showed compounds 1 and 6 to be feeding deterrents at the 1 microgram level.     

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo

We previously showed that, in vitro, hyperforin from St. John’s wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B₄ formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.     

Elucidating the principles of the molecular organization of heteropolymeric tight junction strands

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5?>?Cld5/Cld1?>?Cld3/Cld1?>?Cld3/Cld3?>?Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.