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Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ (1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We tested the hypothesis that gestational diabetes mellitus (GDM) alters the DNA methylation pattern of the fetal serotonin transporter gene (SLC6A4), and examined the functional relevance of DNA methylation for regulation of the SLC6A4 expression in the human placenta. The study included 50 mother-infant pairs. Eighteen mothers were diagnosed with GDM and 32 had normal glucose tolerance (NGT). All neonates were of normal birth weight and born at term by planned Cesarean section. DNA and RNA were isolated from samples of tissue collected from the fetal side of the placenta immediately after delivery. DNA methylation was quantified at 7 CpG sites within the SLC6A4 distal promoter region using PCR amplification of bisulfite treated DNA and subsequent DNA sequencing. SLC6A4 mRNA levels were measured by reverse transcription-quantitative PCR (RT-qPCR). Functional SLC6A4 polymorphisms (5HTTLPR, STin2, rs25531) were genotyped using standard PCR-based procedures. Average DNA methylation across the 7 analyzed loci was decreased in the GDM as compared to the NGT group (by 27.1%, p = 0.037) and negatively correlated, before and after adjustment for potential confounder/s, with maternal plasma glucose levels at the 24th to 28th week of gestation (p<0.05). Placental SLC6A4 mRNA levels were inversely correlated with average DNA methylation (p = 0.010) while no statistically significant association was found with the SLC6A4 genotypes (p>0.05). The results suggest that DNA methylation of the fetal SLC6A4 gene is sensitive to the maternal metabolic state in pregnancy. They also indicate a predominant role of epigenetic over genetic mechanisms in the regulation of SLC6A4 expression in the human placenta. Longitudinal studies in larger cohorts are needed to verify these results and determine to which degree placental SLC6A4 changes may contribute to long-term outcomes of infants exposed to GDM.

Disturbed brain and peripheral serotonin homeostasis is often found in subjects with autism spectrum disorder (ASD). The role of the serotonin receptor 2A (HTR2A) in the regulation of central and peripheral serotonin homeostasis, as well as its altered expression in autistic subjects, have implicated the HTR2A gene as a major candidate for the serotonin disturbance seen in autism. Several studies, yielding so far inconclusive results, have attempted to associate autism with a functional SNP -1438 G/A (rs6311) in the HTR2A promoter region, while possible contribution of epigenetic mechanisms, such as DNA methylation, to HTR2A dysregulation in autism has not yet been investigated. In this study, we compared the mean DNA methylation within the regulatory region of the HTR2A gene between autistic and control subjects. DNA methylation was analysed in peripheral blood leukocytes using bisulfite conversion and sequencing of the HTR2A region containing rs6311 polymorphism. Autistic subjects of rs6311 AG genotype displayed higher mean methylation levels within the analysed region than the corresponding controls (P < 0.05), while there was no statistically significant difference for AA and GG carriers. Our study provides preliminary evidence for increased HTR2A promoter methylation in leukocytes of a portion of adult autistic subjects, indicating that epigenetic mechanisms might contribute to HTR2A dysregulation observed in individuals with ASD., (© 2015 International Society for Autism Research, Wiley Periodicals, Inc.)

The major obstacle to successful chemotherapy of cancer patients is drug resistance. Previously we explored the molecular mechanisms of curcumin cross-resistance in carboplatin resistant human laryngeal carcinoma 7T cells. Following curcumin treatment we found a reduction in curcumin accumulation, and reduced induction of reactive oxygen species (ROS) and their downstream effects, compared to parental HEp-2 cells. In order to shed more light on mechanisms involved in drug resistance of 7T cells, in the present study we applied Fourier transform infrared (FTIR) spectroscopy, a technique that provides information about the nature and quantities of all molecules present in the cell. By comparing the spectra from parental HEp-2 cells and their 7T subline, we found an increase in the intensity of ester vibrational bands in 7T cells. This implied an increase in the amount of cholesteryl esters in resistant cells, which we confirmed by an enzymatic assay. Since cholesteryl esters are localized in lipid droplets, we confirmed their higher quantity and serum dependency in 7T cells compared to HEp-2 cells. Moreover, treatment with oleic acid induced more lipid droplets in 7T when compared to HEp-2 cells, as shown by flow cytometry. We can conclude that along with previously determined molecular mechanisms of curcumin resistance in 7T cells, these cells exhibit an increased content of cholesteryl esters and lipid droplets, suggesting an alteration in cellular lipid metabolism as a possible additional mechanism of drug resistance. Furthermore, our results suggest the use of FTIR spectroscopy as a promising technique in drug resistance research.

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral ("brain parenchymal") compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-β-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPβ levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid β (Aβ) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aβ levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aβ levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aβ levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

The combinatory effect of polymorphisms in serotonin transporter and monoamine oxidase-A genes on the aetiopathogenesis of alcoholism was investigated in a sample of 714 individuals. Increased frequency of subjects having three 'suspected' genotypes (5-HTTLPR-LL, STin2-1010 and MAO-A 3-repeat allele) was found among type-2 alcoholic patients (P=0.0189). Results highlight serotonergic/genetic contribution to early-onset alcoholism., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Altered folate levels may play an important role in colon carcinogenesis. The aim of this study was to investigate the association of polymorphisms in key folate-metabolizing genes with susceptibility to sporadic colon cancer. Six common polymorphisms (two in MTHFR and one each in MTR, MTRR, RFC1, and DHFR genes) were genotyped in 300 healthy subjects and 300 colon cancer patients from Croatia. Obtained results indicate possible protective role of MTRR 66 AA in sporadic colon cancer (OR=0.655; 95% CI=0.441-0.973; p=0.04). Maximum-likelihood analysis of haplotypes revealed a linkage disequilibrium (LD) between the two investigated polymorphisms of the MTHFR gene (C677T and A1298C), both in the control and patient groups (p<0.01 for both). LD was also detected between MTRR A66G and MTHFR A1298C polymorphisms but only in a group of patients (p<0.01). A haplotype of A66G and A1298C polymorphisms, A/A, proved to be protective (OR=0.775; 95% CI=0.603-0.996; p=0.04), whereas haplotype A/G was a risk factor for colon cancer (OR=1.270; 95% CI=1.007-1.602; p=0.04). Contrary to some previous studies, single-locus analyses identified no polymorphisms associated with risk for colon cancer, but demonstrated a possible protective effect of MTRR 66 AA genotype. The detected significant LD between two loci (MTHFR A1298C and MTRR A66G) located on different chromosomes indicates a strong selective force as a mechanism for the maintenance of their linkage. Specific combinations of alleles of these two polymorphisms showed a protective but also a risk effect on colon cancer susceptibility.

The association between suicide and G-703T polymorphism of the tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, was studied in a sample of 291 suicide victims and 280 healthy subjects of Croatian origin. No significant differences were found between the groups. Obtained results do not support involvement of the investigated polymorphism in the susceptibility to suicide completion., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Altered activity of brain serotonergic (5HT) system has been implicated in a wide range of behaviours and behavioural disorders, including anxiety. Functioning of 5HT-1A receptor has been suggested as a modulator of emotional balance in both, normal and pathological forms of anxiety. Here, we studied serotonergic modulation of anxiety-like behaviour using a genetic rat model with constitutional differences in 5HT homeostasis, named Wistar-Zagreb 5HT (WZ-5HT) rats. The model, consisting of high-5HT and low-5HT sublines, was developed by selective breeding of animals for extreme activities of peripheral (platelet) 5HT transporter, but selection process had affected also central 5HT homeostasis, as evidenced from neurochemical and behavioural studies. Anxiety-like behaviour in WZ-5HT rats was evaluated by two commonly used paradigms: open field and elevated-plus maze. The involvement of 5HT-1A receptors in behavioural response was assessed by measuring mRNA expression in cell bodies (raphe nuclei) and projection regions (frontal cortex, hippocampus) by use of RT-PCR and in situ hybridization, and by measuring functionality of cortical 5HT-1A receptors by use of [(3)H]8-OH-DPAT radioligand binding. Animals from the high-5HT subline exhibit increased anxiety-like behaviour and decreased exploratory activity when exposed to novel environment. No measurable differences in constitutional (baseline) functionality or expression of 5HT-1A receptors between sublines were found. The results support contribution of increased serotonergic functioning to the anxiety-like behaviour. They also validate the high-5HT subline of WZ-5HT rats as a potential model to study mechanisms of anxiety, especially of its nonpathological form, while the low-5HT subline may be useful to model sensation seeking phenotype., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Purpose: Temporal lobe epilepsy (TLE) is the most common epilepsy and about 30% of patients have poorly controlled seizures. Neurobiology underlying responsiveness to medical treatment in TLE patients is unclear and there are currently no biological tests to predict course of the disease. Animal and human studies repeatedly suggested serotonergic dysfunction in subjects with TLE. We investigated association of serotonin transporter (5-HTT) gene polymorphisms with medical treatment response in patients with TLE., Methods: We analyzed 5-HTT gene linked polymorphic region (5-HTTLPR) in promoter and variable number of tandem repeats in the second intron of the 5-HTT gene (VNTR-2) in 101 consecutive subjects with TLE., Results: TLE patients with the combination of transcriptionally more efficient genotypes, i.e. 5-HTTLPR L/L and VNTR-2 12/12, had increased seizure refractoriness to antiepileptic medication therapy and shorter periods of seizure freedom, than subjects with other combinations of the 5-HTT genotypes. There were no other clinical or demographic differences among patient groups based on the 5-HTT genotypes., Conclusion: Combination of the 5-HTT genotypes linked with higher 5-HTT gene expression was found to be associated with worse response to optimal drug therapy. Further studies should determine potential role of this 5-HTT genotype polymorphism in epileptogenesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Several lines of evidence point to the role of serotonin (5HT) neurotransmission in the epileptogenesis. The present preliminary study investigated possible association of the temporal lobe epilepsy (TLE) with the polymorphisms in several 5HT-related genes, including serotonin transporter (5HTT), monoamine oxidase A (MAO-A) and serotonin receptors 5HT-1A, 5HT-1B and 5HT-2C. All participants (101 TLE patients and 170 healthy controls) were unrelated individuals of Croatian origin. 5HT-1B allele 861G was found to be slightly overrepresented in the patient group (p=0.0385). No significant differences between groups were observed for the other tested polymorphisms. Within the limitations imposed by the size of our sample, negative findings suggest that the respective loci do not make considerable contribution to the etiopathogenesis of TLE. Further examination of 5HT-1B gene, which yielded positive result at a trend level, is possibly warranted., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Although maternal-fetal cholesterol transfer may serve to compensate for insufficient fetal cholesterol biosynthesis under pathological conditions, it may have detrimental consequences under conditions of maternal hypercholesterolemia leading to preatherosclerotic lesion development in fetal aortas. Maternal cholesterol may enter fetal circulation by traversing syncytiotrophoblast and endothelial layers of the placenta. We hypothesized that endothelial cells (ECs) of the fetoplacental vasculature display a high and tightly regulated capacity for cholesterol release. Using ECs isolated from human term placenta (HPECs), we investigated cholesterol release capacity and examined transporters involved in cholesterol efflux pathways controlled by liver-X-receptors (LXRs). HPECs demonstrated 2.5-fold higher cholesterol release to lipid-free apolipoprotein (apo)A-I than human umbilical vein ECs (HUVECs), whereas both cell types showed similar cholesterol efflux to high-density lipoproteins (HDLs). Interestingly, treatment of HPECs with LXR activators increased cholesterol efflux to both types of acceptors, whereas no such response could be observed for HUVECs. In line with enhanced cholesterol efflux, LXR activation in HPECs increased expression of ATP-binding cassette transporters ABCA1 and ABCG1, while not altering expression of ABCG4 and scavenger receptor class B type I (SR-BI). Inhibition of ABCA1 or silencing of ABCG1 decreased cholesterol efflux to apoA-I (-70%) and HDL(3) (-57%), respectively. Immunohistochemistry localized both transporters predominantly to the apical membranes of placental ECs in situ. Thus, ECs of human term placenta exhibit unique, efficient and LXR-regulated cholesterol efflux mechanisms. We propose a sequential pathway mediated by ABCA1 and ABCG1, respectively, by which HPECs participate in forming mature HDL in the fetal blood.

Findings relating serotonin to suicidal behavior suggest the role of genes for tryptophan hydroxylase (TPH) in the genetic substrate of this disorder. Association of Tph1 gene and suicidality, despite considerable research efforts, remains controversial. Polymorphism A218C in intron 7 of Tph1 gene was studied in violent suicide victims (N = 247) and controls (N = 320) of Slavic (Croatian) origin, with specific consideration of the influence of subjects' age. The frequency of, allegedly less active, CC genotype was increased in older (above 65 years) victims as compared to controls (P = 0.0126 and 0.0008, for comparison with age-specific and integral control samples, respectively), while there were no differences between victims under 65 years and controls. Excess of the CC genotype in elderly victims of violent suicide points to the possible combined effect of the respective genetic factor and physiological changes during aging on the predisposition to this disorder., ((c) 2006 Wiley-Liss, Inc.)

Tryptophan hydroxylase (TPH), the enzyme controlling serotonin synthesis, is considered to be a potential contributor to the biological substrate of suicide. The association of the promoter (-7065CT) and intron 7 (218AC) polymorphisms, and the related haplotype, of the Tph1 gene with suicidal behavior was investigated in a sample of 160 victims of violent suicide and 284 healthy controls. All individuals were males of Croatian (Slavic) origin. Allele frequencies of both polymorphisms in Croatian controls were similar to control values reported for other European populations. Alleles at the two loci demonstrated highly significant linkage disequilibrium. No differences between controls and victims for the Tph1 genetic variation, either at single loci, or at a haplotypic level, were demonstrated, albeit there was a tendency, not reaching statistical significance, towards an increase of the intron 7CC genotype in the suicide group. Negative association results on the individual Tph1 loci, in accordance with the majority of previous reports, confirmed the lack of their major effect also in the Slavic ethnicity. Haplotypic results, on the other hand, opposing the previous positive finding, point to the possible influence of ethnicity (or gender) on the association between the Tph1 gene polymorphism and suicide.

Abnormal cortical activity and brainstem functioning are considered the possible etiopathogenetic factors of migraine. Monoamine oxidase A and B (MAO-A and -B) regulate the levels of monoamine neurotransmitters, so changes in their activity could participate in migraine pathogenesis. We have investigated the possible association of MAO-A and -B alleles and haplotypes with two common types of migraine, i.e. migraine without aura (MO) and migraine with aura (MA), on the sample of 110 migraineours (80 MO and 30 MA) and 150 controls. MAO-A promoter and MAO-B intron 13 polymorphisms were genotyped by the PCR-based methods. In addition, we have reevaluated the reported association between MAO-B intron 13 polymorphism and platelet MAO-B activity. The platelet MAO-B activity was determined fluorimetrically using kynuramine as a substrate. We have found a tendency toward association of the shorter variant of MAO-A gene promoter with migraine without aura in male subjects. Regarding investigated MAO-B polymorphism, no association with migraine or with platelet MAO-B activity was found. The suggestive association of the variant in MAO-A gene with migraine is considered worthy of independent replication. On the other hand, further studies on MAO-B polymorphism in migraine do not seem promising.

Background: Two polymorphic regions of serotonin transporter (5-HTT) gene: a 44 base pair (bp) insertion/deletion in the promoter region (5-HTTLPR) and a 17 bp variable number of tandem repeats in second intron (VNTR-2), seem to modulate the gene's transcription in allele-dependent manner., Methods: We have earlier demonstrated association with 5-HTT gene in families multiply affected by schizophrenia. Here, we investigated separate and combined effects of VNTR-2 and 5-HTTLPR on the rate of peripheral 5-HTT transcription in a sample of offspring from those families. Relative 5-HTT mRNA levels were determined in 53 permanent lymphoblast cell lines by semiquantitative real-time polymerase chain reaction using beta-actin as reference., Results: Since the low-expressing alleles (short [S], 10) appeared to act dominantly, genotypes were grouped as "high-expressing" (long [L]/L, 12/12) versus "low-expressing" (S, 10). At both loci, nonsignificant differences in 5-HTT mRNA levels ( approximately 30%) were observed between "high"- and "low-expressing" genotypes. In order to search for the potential combined effect of 5-HTTLPR and VNTR-2, levels of 5-HTT mRNA were compared among three groups of samples having "low-expressing" genotype at none, one, or both loci. Increase in number of "low-expressing" genotypes significantly reduced relative 5-HTT gene expression (p <.02)., Conclusions: Our results indicate weak individual influence, but possible combined effect, of 5-HTTLPR and VNTR-2 polymorphisms on 5-HTT gene expression.

Serotonergic dysfunction is believed to be involved in the susceptibility to suicide due to functional alternations in the serotonin-related genes. Serotonin 1B (5HT-1B) receptor mediates aggressive behavior in mice models and was proposed to be involved in the control of aggression and impulsivity in humans. In this study we have investigated the association of G861C polymorphism of the 5HT-1B receptor gene with suicide commitment. Study was based on two independent samples, one of German (245 suicide victims vs. 248 controls) and the other of Slavic/Croatian (118 suicide victims vs. 192 controls) ethnicity. No significant differences in allele or genotype frequencies between victims and controls were demonstrated either in German or Croatian sample. There were no differences in allele frequencies between German and Croatian population, and the combined sample, having high statistical power, also did not demonstrate significant differences between victims and controls. Results provide evidence that the investigated 5HT-1B receptor gene variants are not implicated in the susceptibility to suicide., (Copyright 2004 Wiley-Liss, Inc.)

Background: Disturbances in serotonin (5HT) transmission are the most frequently reported neurobiological substrates of suicidal behavior. Because 5HT transporter plays a central role in the regulation of 5HT synaptic function and its gene contains two functional polymorphisms (5-HTTLPR in the promoter region and VNTR in the second intron), it represents an interesting candidate for association studies in suicidal behavior., Methods: In this study, a possible association of 5-HTTLPR and intron 2-VNTR polymorphisms of the 5HT transporter gene with suicidal behavior was investigated in a sample of 135 suicide victims and 299 healthy control subjects of Croatian/southern Slavic origin., Results: There were no significant differences in 5-HTTLPR and intron 2-VNTR genotype- and allele- frequency distributions between suicide victims and healthy control subjects; however, a tendency toward an increase of 5-HTTLPR allele L and VNTR-allele 10 were observed in suicide group. Analysis of distribution of estimated haplotype frequencies revealed differences between suicide victims and control subjects, with an excess of haplotype L10 among suicide victims (p =.0112)., Conclusions: Our results provide modest evidence for a possible association of the 5HT transporter gene with a completed suicide. Further studies are needed to determine whether alterations in 5HTt gene expression are involved in suicidal behavior.

Objective: The apoptogenic potential of serotonin (5-hydroxytryptamine) on mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells, in two sets of experiments., Methods: Apoptosis was measured by morphologic criteria and flow cytometry., Results: In both assays, serotonin, at a concentration producing half-maximal inhibition of lymphocyte proliferation, significantly increased the basal levels of apoptosis., Conclusion: An apoptotic mechanism should be taken into account when considering immunoinhibition induced by serotonin., (Copyright 2002 S. Karger AG, Basel)

Besides the pineal gland, melatonin is reported to be produced in a number of extrapineal sites, where it could act as an intracellular mediator or paracrine signal in addition to its endocrine effects. In view of the suggested immunoregulatory role of melatonin, we compared lymphoid organs and several other tissues of the rat for their potential to synthesize melatonin. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we determined the tissue-specific expression of mRNAs encoding two key enzymes of the melatonin biosynthesis: serotonin-N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The minimal number of PCR cycles required to obtain a positive signal served as a measure for the abundance of a given mRNA. NAT and HIOMT mRNAs were detected in all tested tissues at high numbers of PCR cycles (40 and 45, respectively). At 35 cycles, only gut, testis, spinal cord, raphe nuclei, stomach fundus and striatum yielded positive signals for both enzymes. In conclusion, the presence of NAT and HIOMT mRNAs in a wide range of tissues corroborates and extends the notion of extrapineal melatonin synthesis. Comparatively low levels of the HIOMT messages in lymphoid organs, however, indicate a limited significance of melatonin synthesis within the immune system.

Objective: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells., Methods: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10(-13) up to 10(-2) M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24-72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6-12 animals and was expressed as a percentage of the control values obtained with mitogen alone., Results: No significant effect of 5HT at concentrations from 10(-13) to 10(-5) M was found. At concentrations of > or =10(-4) M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 x 10(-4) M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells., Conclusion: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations., (Copyright 2001 S. Karger AG, Basel)

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a role in immunoregulation; however, little is known about specific subtypes of 5-HT receptors involved in peripheral immunomodulation. In the present study we used RT-PCR methods to examine the mRNA expression of 5-HT receptors in the cells of lymphoid tissues of the rat. All 13 rat 5-HT receptor genes cloned so far were examined in ex vivo isolated spleen, thymus, and peripheral blood lymphocytes, as well as in mitogen-stimulated spleen cells. Positive signals were obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 receptor mRNAs in all three compartments. Mitogen (ConA and PWM) stimulated cells additionally expressed mRNA corresponding to the 5HT-3 receptor subtype. In contrast, 5-HT1A, 5-HT1D, 5-HT2C, 5-HT4, 5-HT5A, and 5-HT5B mRNAs were not detected in any of the examined cell populations. These results may be useful as a starting point for future functional studies on immunomodulatory effects of 5-HT and may help to understand conflicting serotonergic effects on immune functions as found in the literature., (Copyright 2000 Academic Press.)