Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension.

Primary pulmonary hypertension is a progressive and often fatal disorder in humans that results from an increase in pulmonary blood pressure associated with abnormal vascular proliferation. Dexfenfluramine increases the risk of pulmonary hypertension in humans, and its active metabolite is a selective serotonin 5-hydroxytryptamine 2B (5-HT(2B)) receptor agonist. Thus, we investigated the contribution of the 5-HT(2B)receptor to the pathogenesis of pulmonary hypertension. Using the chronic-hypoxic-mouse model of pulmonary hypertension, we found that the hypoxia-dependent increase in pulmonary blood pressure and lung remodeling are associated with an increase in vascular proliferation, elastase activity and transforming growth factor-beta levels, and that these parameters are potentiated by dexfenfluramine treatment. In contrast, hypoxic mice with genetically or pharmacologically inactive 5-HT(2B)receptors manifested no change in any of these parameters. In both humans and mice, pulmonary hypertension is associated with a substantial increase in 5-HT(2B) receptor expression in pulmonary arteries. These data show that activation of 5-HT(2B) receptors is a limiting step in the development of pulmonary hypertension.

Modulation of bleomycin-induced lung fibrosis by serotonin receptor antagonists in mice.

Serotonin (5-hydroxytryptamine; 5-HT) is known to increase proliferation and collagen synthesis by fibroblasts. Two receptor subtypes, 5-HT2A and 5-HT2B, have been shown to play the most important roles in the lung. In the present study, the role of serotonin in lung fibrosis was investigated using the bleomycin mouse model. Serotonin concentrations in lung homogenates increased significantly over the time course of bleomycin-induced fibrosis, with a maximum at day seven. The expression of serotonin receptors 5-HT2A and 5-HT2B increased in the lung after bleomycin treatment, as assessed by PCR, specific binding and immunohistochemistry. Blockage of 5-HT2A receptors by ketanserin and 5-HT2B receptors by SB215505 reduced bleomycin-induced lung fibrosis, as demonstrated by reduced lung collagen content and reduced procollagen 1 and procollagen 3 mRNA expression. Serotonin antagonists promoted an antifibrotic environment by decreasing the lung mRNA levels of transforming growth factor-beta1, connective growth factor and plasminogen activator inhibitor-1 mRNA, but had minimal effects on lung inflammation as assessed by bronchoalveolar lavage cytology analysis. Interestingly, the 5-HT2B receptor was strongly expressed by fibroblasts in the fibroblastic foci in human idiopathic pulmonary fibrosis samples. In conclusion, the present study showed involvement of serotonin in the pathophysiology of bleomycin-induced lung fibrosis in mice and identified it as a potential therapeutic target in lung fibrotic disorders.

Increased delta aminolevulinic acid and decreased pineal melatonin production. A common event in acute porphyria studies in the rat.

Tryptophan (TRP) is the precursor of melatonin, the primary secretory product of the pineal gland. Hepatic heme deficiency decreases the activity of liver tryptophan pyrrolase, leading to increased plasma TRP and serotonin. As a paradox, patients with attacks of acute intermittent porphyria (AIP), exhibit low nocturnal plasma melatonin levels. This study using a rat experimental model was designed to produce a pattern of TRP and melatonin production similar to that in AIP patients. Pineal melatonin production was measured in response to: (a) a heme synthesis inhibitor, succinylacetone, (b) a heme precursor, delta-aminolevulinic acid (Ala), (c) a structural analogue of Ala, gamma-aminobutyric acid. Studies were performed in intact rats, perifused pineal glands, and pinealocyte cultures. Ala, succinylacetone, and gamma-aminobutyric acid significantly decreased plasma melatonin levels independently of blood TRP concentration. In the pineal gland, the key enzyme activities of melatonin synthesis were unchanged for hydroxyindole-O-methyltransferase and decreased for N-acetyltransferase. Our results strongly suggest that Ala overproduced by the liver acts by mimicking the effect of gamma-aminobutyric acid on pineal melatonin in AIP. They also support the view that Ala acts as a toxic element in the pathophysiology of AIP.

High plasma serotonin levels in primary pulmonary hypertension. Effect of long-term epoprostenol (prostacyclin) therapy.

Elevated plasma serotonin is associated with primary pulmonary hypertension (PPH). To test whether this elevation could be related to platelet activation, the 2 pools of blood serotonin (platelets and plasma) and plasma 5-hydroxyindoleacetic acid (5-HIAA) as well as markers of platelet activation (alpha(IIb)beta(3), CD36, P-selectin, and CD63 membrane epitopes) were measured in 16 patients with severe PPH (group 1) before and at days 10 and 40 of treatment with a continuous infusion of epoprostenol (prostacyclin). The same biological parameters were also measured in 19 healthy subjects (group 2) and in 10 patients after cardiovascular surgery with extracorporeal circulation (group 3), a condition known to profoundly activate the platelets. Twelve PPH patients showed hemodynamic and clinical improvement, 3 remained stable, and 1 had the treatment stopped because of clinical aggravation. At day 0, mean plasma serotonin (5-hydroxytryptamine [5-HT]) concentration was much higher in PPH patients than in normal subjects (34.4+/-21.2 versus 9.1+/-6.0 nmol/L, respectively; P:<0.001) and positively correlated with total pulmonary resistance. The mean platelet 5-HT content was not significantly different in PPH compared with normal individuals. Mean plasma 5-HIAA concentrations were much higher in PPH than in normal patients (162+/-57 versus 61+/-7 nmol/L, respectively; P<0.001). These parameters did not significantly change during epoprostenol treatment. There was no correlation between the changes in plasma 5-HT during treatment and clinical or hemodynamic improvement. In PPH patients, the mean platelet volume significantly decreased (ANOVA, P<0.01) during treatment. Positive correlations were evidenced between the size of platelets and the number of alpha(IIb)beta(3) and CD36 epitopes. When compared with control platelets, the number of alpha(IIb)beta(3) epitopes detected on PPH platelets at day 0 tended to be higher, but this difference did not reach a statistical significance (41 300+/-7140 for PPH patients versus 36 010+/-3930 for control subjects, P=0.069). The number of CD36 epitopes, in the range of controls at day 0 (11 590+/-5080 for PPH patients versus 11 900+/-1790 for control subjects), decreased during treatment (ANOVA, P=0.038) and became significantly low at day 40 (8660+/-3520, P<0.001). The number of CD63 epitopes was not elevated, and P-selectin was never detected at any time point on PPH platelets. This glycoprotein profile indicates that the platelets of PPH patients were not highly activated but had an accelerated turnover and returned to normal under epoprostenol treatment without change of the elevated plasma serotonin, characteristic of PPH. In conclusion, neither platelet activation nor a significant alteration of the 5-HT endothelial metabolism explains the high level of plasma 5-HT in PPH patients. The 5-HT plasma concentration is not a predictive marker of the severity of PPH, and its evolution is independent of the clinical and hemodynamic status. Treatment by a potent antiaggregating agent, epoprostenol, does not affect the increase of plasma 5-HT, despite a therapeutic benefit.

L-arginine and L-NAME have no effects on the reendothelialization process after arterial balloon injury.

OBJECTIVE: Growth regulatory properties of nitric oxide (NO) in cultured endothelial cells is controversial. The aim of our study was to investigate the effect of L-arginine, the endogenous NO precursor, and L-NAME, an inhibitor of NO synthase on the reendothelialization process after angioplasty. METHODS: Fifty-five New Zealand White rabbits underwent denudation of the left iliac artery. After injury the rabbits were randomized in three groups: L-arginine 2.25% (L-arginine, n = 19); NG-nitro-L-arginine methyl ester 15 mg/kg/day (L-NAME, n = 19); and placebo (controls, n = 17). Treatment was solubilized in drinking water. Reendothelialization was evaluated at 4 weeks by macroscopic evaluation of Evans blue staining and endothelial-specific immunostaining (CD-31) on cross sections. Intimal hyperplasia was evaluated by morphometric analysis. RESULTS: Despite a significant increase in plasma arginine (P = 0.001) and a reduction in intimal hyperplasia (P = 0.003) with L-arginine, neither agent had a significant effect on reendothelialization at 4 weeks (controls = 36 +/- 4%, L-arginine = 43 +/- 3%, L-NAME = 33 +/- 4%; NS). CONCLUSION: These results suggest that, in spite of previously demonstrated effects on neointimal hyperplasia, the NO pathway does not influence the regrowth of macrovascular endothelial cells in vivo.

Short therapeutic window for MK-801 in transient focal cerebral ischemia in normotensive rats.

The present study investigates the role of N-methyl-D-aspartate (NMDA) receptors in a model of transient focal cerebral ischemia in normotensive rats. The left middle cerebral artery and both common carotid arteries were occluded for 60 min. Preliminary studies indicated that this gave reproducible infarctions of the cortex and striatum. These infarctions were the result of severe ischemia followed by complete reperfusion after clamp removal, as showed by striatal tissue Po2 monitoring. Microdialysis indicated that glutamate concentration increased immediately after occlusion and returned to the baseline value 40 min after clamp removal. MK-801 (1 mg kg-1 i.v.), an antagonist of the NMDA glutamatergic receptor, reduced the cortical infarct volume by 29% (p < 0.001) and the striatal infarct volume by 14% (p < 0.05) when given just prior to ischemia, but had no neuroprotective activity when given 30 min after the onset of ischemia. This short therapeutic window for MK-801 suggests that NMDA receptors play only a transient role in reversible focal ischemia in rats.

alpha-Phenyl-N-tert-butylnitrone attenuates excitotoxicity in rat striatum by preventing hydroxyl radical accumulation.

Various in vitro experiments have indicated that oxygen-derived free radicals may contribute to excitotoxic neuronal death. In the present study we induced excitotoxicity in rat striatum by perfusing glutamate at a high concentration through a microdialysis probe. We observed an increased formation of hydroxyl radicals (.OH) during the perfusion of the excitotoxin and an extensive striatal lesion 24 h after the insult. The spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), attenuated both hydroxyl radical levels and the volume of the lesion. This result suggests that the neuroprotection may be due to a free radical scavenging mechanism. It also implies that PBN may be used in pathological situations involving excitotoxicity such as stroke, brain trauma, and chronic neurologic diseases.

Effects of monoamine oxidase A inhibition on barrel formation in the mouse somatosensory cortex: determination of a sensitive developmental period.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

Decreased whole blood 5-hydroxytryptamine (serotonin) in AIDS patients.

Significantly (p less than 0.001) decreased whole blood unconjugated serotonin levels were detected in AIDS patients as compared to patients with advanced cancers and to healthy individuals.

Long-term superior cervical sympathectomy induces mast cell hyperplasia and increases histamine and serotonin content in the rat dura mater.

Nerve fibres and mast cells are often described in close morphological and functional interactions in various organs such as the dura mater. The respective roles of mast cell activation and sympathetic impairment in cluster headache and migraine attacks have been repeatedly suggested. We have thus investigated the long-term effects of sympathectomy on mast cell morphology and content in the rat dura mater. Fifteen to 60 days after either sham, unilateral or bilateral superior cervical ganglionectomy, dura were removed for either histochemical or biochemical analysis. In the first case, they were fixed and mast cell heparin was stained by fluorescein isothiocyanate-conjugated avidin. Microscopic examination was followed by digital acquisitions using a tomographic process to assess mast cell density in the whole depth of the dura mater. Unilateral ganglionectomy induced a progressive and significant increase in mast cell density 15-60 days post-surgery in contralateral hemi-dura and 30 days post-surgery in ipsilateral hemi-dura. This increase was significant in both dura 60 days after bilateral ganglionectomy. Following perfusion with saline, we also examined the content of histamine and serotonin, pre-formed amines stored in mast cell granules. Biochemical analysis of dura serotonin and histamine content using high-pressure liquid chromatography and radioenzymatic assays, respectively, revealed under all conditions a serotonin tissue concentration lower than that of histamine. After sham ganglionectomy, the dura serotonin content increased from 15 to 60 days post-surgery, whereas the histamine content remained stable over the same period. After unilateral ganglionectomy, the histamine content increased progressively and significantly 30-60 days post-surgery in both hemi-dura, whereas the serotonin content became significantly different from that of sham only 60 days post-surgery in the ipsilateral dura. After bilateral ganglionectomy, the histamine level significantly increased in both hemi-dura 15-60 days post-surgery, whereas the serotonin level had significantly increased at 60 days post-surgery. These results clearly demonstrate, for the first time, a long-term trophic effect of sympathetic nerve degeneration on mast cells in the dura mater. Since mast cell activation has been described previously on the painful side of cluster headache patients during attack periods, we propose that the sympathetic impairment reported in these patients could be prominent, directly or indirectly inducing mast cell hyperplasia and changes in amine contents in the tissue concerned.

Absence of serotonergic innervation from raphe nuclei in rat cerebral blood vessels--II. Lack of tryptophan hydroxylase activity in vitro.

Neurochemical studies performed in vivo have suggested that serotonin-containing and -synthesizing nerves, originating in the raphe nuclei, directly innervate pial blood vessels. Nerve fibres of these vessels have been shown by immunocytochemistry to contain tryptophan hydroxylase (the rate-limiting enzyme of serotonin synthesis) but no serotonin. The present study examines this contradiction by measuring in vitro the tryptophan hydroxylase activity of rat cerebral vessels and femoral arteries (which also contain tryptophan hydroxylase-immunopositive nerves), and comparing them to the tryptophan hydroxylase activity of the rat pineal body, raphe nuclei and brain cortex under identical conditions. Oxygenated incubation solutions contained either [14C]- or "cold" L-tryptophan (2 x 10(-5) to 5 x 10(-4) M) and NSD-1015 (3-hydroxybenzylhydrazine) which inhibits the decarboxylation of 5-hydroxytryptophan, the second step of serotonin synthesis. Tissue fragments were incubated for 35-60 min. High-performance liquid chromatography (on tissue extracts and incubation solutions) as well as determination of 14C activity in the 5-hydroxytryptophan fraction of elution from tissue extracts showed that the pineal body, the raphe nuclei and cortical slices synthesize various amounts of 5-hydroxytryptophan under our experimental conditions. All these tissues contained serotonin. Femoral arteries, but not cerebral vessels, also contained small amounts of serotonin stored before incubation, probably in mast cells. In contrast to brain tissues, no measurable amounts of "cold" or [14C]5-hydroxytryptophan were found in cerebral blood vessel and femoral artery extracts or incubation solutions. Under identical experimental conditions, sympathetic nerves of both types of vessels were able to synthesize large amounts of L-DOPA when incubation solutions contained L-tyrosine instead of L-tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of sepsis on mast cells in rat dura mater: influence of L-NAME and VIP.

1. The influence of lipopolysaccharide (LPS)-induced sepsis on the various mast cell phenotypes of rat dura mater were examined both by immunohistochemical and biochemical methods. 2. Three different populations of mast cells were identified in control rats: connective tissue type mast cells (CTMC) which contain rat mast cell protease1 (RMCP1), histamine, serotonin and heparin, mucosal type mast cells (MMC) which contain RMCP2, histamine and serotonin, and intermediate type which contains both RMCP1 and RMCP2 and probably various proportions of amines and heparin. 3. LPS (25 mg kg(-1) i.p.) caused changes in the proportions of the various types of mast cells. The number of MMC and intermediate type mast cells significantly increased and the number of mast cells immunopositive for both heparin and serotonin significantly decreased. Biochemical analysis showed that the histamine concentration of dura increased while its serotonin concentration decreased. 4. While vasoactive intestinal peptide (VIP) (25 ng kg(-1) i.p.) appears to potentiate LPS effects on dura mater mast cells, non-selective inhibition of nitric oxide (NO) synthase by N(g)-nitro-L-arginine methyl ester (L-NAME) (30 mg kg(-1) i.p.) did not influence sepsis-induced mast cell changes. 5. These findings suggest that mast cells of dura mater may play a role in brain protection during sepsis.

Competitive NMDA receptor blockers reduce striatal glutamate accumulation in ischaemia.

Our previous studies have shown that kynurenic acid, a broad-spectrum antagonist of excitatory amino acid receptors, depressed the ischaemia-induced accumulation of glutamate and aspartate in rat striatum. In the present experiments we examined the effect of two competitive N-methyl-D-aspartate (NMDA) receptor antagonists on striatal extracellular glutamate concentrations induced by a 30 min '4-vessel occlusion' ischaemia in rats. Local perfusion with 2-amino-5-phosphonovalerate (AP5; 300 microM) and with 2-amino-7-phosphonoheptanoate (AP7; 300 microM), using a microdialysis fibre markedly reduced the ischaemia-induced increase in glutamate concentrations. These results indicate that, during forebrain ischaemia the NMDA receptor type mediates glutamate and aspartate accumulation in rat striatum.

Effect of kynurenic acid on the ischaemia-induced accumulation of glutamate in rat striatum.

We examined the effect of kynurenic acid, a broad spectrum antagonist of excitatory amino acid receptors, on striatal extracellular glutamate and aspartate accumulation induced by a 30 min forebrain ischaemia in rats. Kynurenic acid, given systemically (500 mg kg-1, i.p.) or administered in situ through the dialysis probe (10 mM), markedly depressed the ischaemia-induced increase in glutamate and aspartate concentrations. These results indicate that, during forebrain ischaemia, local glutamate receptors play a major role in glutamate and aspartate accumulation in the striatum. Ischaemia-induced increase in extracellular concentrations of these excitatory amino acids may be due in part to a positive glutamatergic feedback loop via activation of NMDA and/or non-NMDA receptors.

Striatal dopamine participates in glutamate-induced hydroxyl radical generation.

Using a microdialysis technique we showed that the exposure of the rat striatum to glutamate yields hydroxyl radicals and results in striatal damage. We postulated that dopamine release is enhanced by glutamate perfusion and that the enzymatic metabolism of dopamine may account for this hydroxyl radical formation. The inhibition of monoamine oxidases by i.p. co-administration of clorgy-line and deprenyl reduced hydroxyl radical production induced by glutamate perfusion, but significantly increased the striatal damage. Our results suggest that the enzymatic metabolism of dopamine participates in glutamate-induced hydroxyl radical generation but that other by-products of dopamine may be responsible for the aggravation of the striatal injury.

The mouse 5-HT2B receptor: homologous subtype or species variant?

The recently characterized 5-HT2B subfamily of serotonin receptors has now been reported from three different species: human, rat and mouse. Their genomic structures include 2 introns present at identical positions. Despite this similarity, their respective protein sequences show some diversities. In addition, the pharmacology of these receptors is distantly related, and their sites of expression vary amongst species. Thus, it appears difficult at present to unambiguously classify these receptors into the same subfamily, raising the possibility of the existence of other 5-HT2B-like receptors, yet to be discovered.

Enhancement of endogenous excitatory amino acids by theophylline does not modify the behavioral and histological consequences of forebrain ischemia.

The neuroprotective role of endogenous adenosine during forebrain ischemia elicited by 4-vessel occlusion in rats was assessed using the adenosine antagonist, theophylline (32 mg/kg). Despite an increase in the release of glutamate in the hippocampus during ischemia, theophylline did not alter the neurological and histological outcomes. These results indicate that endogenous adenosine does not act as an endogenous neuroprotector by modulating glutamate release in this model.

Inhibition of glutamate release in rat hippocampus by kynurenic acid does not protect CA1 cells from forebrain ischemia.

We assessed the effect of a broad spectrum glutamatergic receptor antagonist, kynurenic acid (500 mg/kg) on ischemia-induced hippocampal glutamate release and neuronal damage. Kynurenic acid significantly decreased glutamate release during ischemia but had no effect on the hippocampal lesion. Some protection was observed in the cortex and in the striatum. These data suggested that the extracellular accumulation of glutamate during forebrain ischemia does not play a major role in the hippocampus.

A microdialysis study investigating the mechanisms of hydroxyl radical formation in rat striatum exposed to glutamate.

Considerable evidence has linked hydroxyl radicals (.OH) to excitotoxicity. Glutamate infused through a microdialysis probe into rat striatum induced a massive .OH production, which was completely blocked by PBN and attenuated by dizocilpine, 2-amino-5-phosphonopentanoic acid (AP-5), NG-nitro-L-arginine methyl ester (L-NAME) and mepacrine. Thus, we suggest that the neurotoxic effects of glutamate in vivo may derive from an increased formation of .OH resulting from excessive activation of NMDA receptors and downstream enzymes such as NOS and PLA2.

Comparison of selective and complete inhibitors of enkephalin-degrading enzymes on morphine withdrawal syndrome.

We investigated the effects of thiorphan, a selective inhibitor of endopeptidase 24.11 'enkephalinase', kelatorphan ((R)-3-(N-hydroxy-carboxamido-2-benzylpropanoyl)-L-alanine), and RB 38 A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) two almost complete inhibitors of enkephalin metabolism, on the naloxone-precipitated morphine withdrawal syndrome in rats. Inhibitors administered intracerebroventricularly reduced several symptoms of the withdrawal syndrome. Jumping, chewing and tooth chattering were decreased by all drugs. The rise in plasma corticosterone and the hypothermia were reduced by kelatorphan and RB 38 A whereas rhinorrhea was blocked by thiorphan, tremor by kelatorphan and diarrhoea by RB 38 A. Other signs remained unchanged. These data suggest that an increase in opioid receptor occupancy by endogenous opioid peptides, protected from biotransformation specially by mixed inhibitors reduced the severity of the morphine abstinence symptoms in rats.