RESUMEN

Significantly lower platelet serotonin level (PSL) in patients with primary Sjogren's syndrome (pSS) than in healthy controls has been reported in our prior studies. In the present report, we demonstrated effect of functional polymorphisms in the serotonin transporter gene (5-HTT) on PSL. We describe a group of 61 pSS patients and 100 healthy individuals subjects, who received PSL measurement in our prior study. All subjects were genotyped for the promoter 5-HTTLPR (L/S), rs25531 (A/G) and intronic 5-HTTVNTRin2 (l/s) polymorphisms. Overall, the presence of 5-HTTVNTRin2 ss genotype was associated with significantly lower PSL in pSS patients, not in healthy controls. Reduced PSL in pSS patients is in line with hypothesis of association between chronic immunoinflammation and 5-HT system dysregulation, identifying additional mechanisms such as altered 5-HT transport as potential genetic factor contributing to PSL depletion.

Asunto(s)

RESUMEN

BACKGROUND: A role of the serotonin (5HT) transporter, a key regulator of serotonergic transmission, in the physiology, pharmacology and genetics of pain responses has been proposed recently. The present study aimed to explore the impact of constitutive differences in the activity of the serotonin transporter, and 5HT homeostasis in general, on the modulation on pain sensitivity and analgesic responses to drugs that utilize 5HT mechanisms. METHODS: A novel genetic animal model, Wistar-Zagreb 5HT rats, obtained by selective breeding of animals for extreme activity of the platelet serotonin transporter was used. As a consequence of breeding, two sublines of this model, termed high-5HT and low-5HT, differ in both central and peripheral serotonin homeostasis. Thermal pain sensitivity of 5HT sublines was assessed at baseline and following administration of analgesic drugs, as determined by paw withdrawal latency to radiant heat stimulation. RESULTS: Animals from 5HT sublines show differences in both basal pain sensitivity and analgesic responses. Rats with the low-5HT phenotype displayed decreased baseline paw withdrawal latencies (hyperalgesia) in comparison to their high-5HT counterpart (25%; p < 0.001). They also showed better analgesic response to acute and prolonged treatment with tramadol (p = 0.027) and clomipramine (p = 0.019), respectively, whereas administration of fluvoxamine did not produce an analgesic effect in either 5HT subline. CONCLUSIONS: These findings support the idea that functionality of the serotonin transporter is one of the physiological/genetic determinants of individual differences in pain responses and modulation. They also validate Wistar-Zagreb 5HT rats, with constitutionally up-regulated/down-regulated serotonin transporter, as a potential new genetic model for studying serotonergic modulation of pain responses.

Asunto(s)

RESUMEN

Due to their involvement in dependence pathways, opioid system genes represent strong candidates for association studies investigating alcoholism. In this study, single nucleotide polymorphisms within the genes for mu (OPRM1) and kappa (OPRK1) opioid receptors and precursors of their ligands - proopiomelanocortin (POMC), coding for beta-endorphin and prodynorphin (PDYN) coding for dynorphins, were analyzed in a case-control study that included 354 male alcohol-dependent and 357 male control subjects from Croatian population. Analysis of allele and genotype frequencies of the selected polymorphisms of the genes OPRM1/POMC and OPRK1/PDYN revealed no differences between the tested groups. The same was true when alcohol-dependent persons were subdivided according to the Cloninger's criteria into type-1 and type-2 groups, known to differ in the extent of genetic control. Thus, the data obtained suggest no association of the selected polymorphisms of the genes OPRM1/POMC and OPRK1/PDYN with alcoholism in Croatian population.

Asunto(s)

RESUMEN

The expression of Ikaros family transcription factors and consequently their signalling pathway is limiting for hematopoietic and lymphocyte development in mice and human. Due to their importance, these transcription factors are highly homologous between species. As an initial approach to examining the possible involvement of Ikaros transcription factors in pathogenesis of rat lymphoid development, we analyzed the expression of all known Ikaros family members, Ikaros, Aiolos, Helios, Eos and Pegasus in the rat thymus. We established a semi-quantitative RT-PCR to detect mRNA of each transcription factor. For the first time we give evidence of the expression of Ikaros family transcription factors in the rat thymus. Further, we evaluated whether their mRNA expression was succumbed to changes when the rats were exposed to ethanol, as a known debilitating agent during development. Therefore we analyzed the thymus of adult rats whose mothers were forced to drink ethanol during gestation, to detect possible changes in thymus mRNA expression levels of Ikaros, Aiolos, Helios, Eos and Pegasus. We found that rats prenatally exposed to ethanol show a slightly higher expression of Ikaros family transcription factors in the adult thymus when compared to control rats, but these differences were not statistically significant. We further studied the distribution of the major lymphocyte subpopulations in the rat thymus according to CD3, CD4 and CD8 expression by four color flow cytometry. We found a higher incidence of CD3 positive cells in the double positive, CD4+CD8+ thymic subpopulation of rats prenatally exposed to ethanol when compared to non-exposed animals. Our findings indicate that ethanol exposure of pregnant rats might influence the development of CD3 positive cells in the thymus of the offspring but this result should be further tackled at the level of transcription factor expression.

Asunto(s)

RESUMEN

The co-localization of serotonin (5-hydroxytryptamine, 5HT) and neuroactive peptides in the same neuron points to the importance of interactions between serotonergic and peptidergic systems in maintaining body homeostasis. In this work, we used an original genetic rat model to search for possible interrelations between 5HT system functioning and the activities of aminopeptidases, i.e. enzymes which are the key regulators of (neuro)peptides level/function. The activities of three cytosolic exopeptidases: alanyl aminopeptidase (alanyl-AP), arginyl aminopeptidase (arginyl-AP) and dipeptidyl peptidase III (DPP III) were measured in brains and peripheral tissues of the sublines of rats with constitutionally upregulated/downregulated 5HT transporter activity. These rat sublines, termed as high-5HT and low-5HT subline, have been obtained previously by selective breeding for the extreme values of platelet 5HT level and velocity of 5HT uptake. Besides in the periphery they show marked alterations also in brain 5HT function, indicating the differences in central 5HT transmission/homeostasis. In this study, we have found that animals from the high-5HT subline have significantly lower activity of brain alanyl-AP (p<0.05) and arginyl-AP (p<0.01) as compared to control animals. No other differences were noticed regardless of the 5HT subline, investigated organ or analyzed aminopeptidase. Results suggest that the constitutional upregulation of serotonergic activity may be related to a lowered brain cytosolic aminopeptidase activity which may have an influence on the cleavage of their physiological substrates.

Asunto(s)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

By breeding selection for the extreme values of platelet serotonin level (PSL), two sublines of Wistar-derived rats, with constitutionally high or low PSL and platelet serotonin uptake (PSU), have been developed. Searching for the basis of these differences, we performed quantitative western blot analysis of serotonin transporter (5HTt) in platelet membranes isolated from both rat sublines. A polyclonal anti-5HTt antibody labeled a single, 5HTt-related 94 kDa protein band in platelet membranes, with significantly stronger intensity in membranes from rats that exhibited a high PSL. We conclude that the inherited differences in PSL and PSU in rats, following breeding selection, are determined by the level of 5HTt expression in platelet membranes.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

AIM: To assess possible differences in platelet monoamino oxidase-B (MAO-B) activity, ego strength, and neuroticism in combat-experienced soldiers with or without current posttraumatic stress disorder (PTSD). METHOD: The soldiers with current PTSD (N=36) and a control group of 34 healthy soldiers were matched in combat experience, time passed between combat experience and the study, demographic variables (age, marital status, education), and smoking status. Platelet MAO-B was assayed fluorometrically, ego strength was measured by the Croatian version of the Ego Identity Scale, and neuroticism by the N-scale from Eysenck's EPQ-R questionnaire. RESULTS: Soldiers with combat-related current PTSD had lower platelet MAO-B activity than the control group (9.1+/-3.9 vs. 11.9+/-4.0; p<0.05), as well as lower ego-strength (86.3+/-8.3 vs. 108.6+/-13.4; p<0.05) and higher neuroticism (23.5+/-13.2 vs. 5. 9+/-4.7; p<0.05). There was no association of ego strength or neuroticism with platelet MAO-B activity. CONCLUSION: Ego identity strength and emotional stability are associated with successful coping with combat trauma. The involvement of platelet MAO-B activity in biological basis of ego strength and neuroticism could not be demonstrated.

Asunto(s)

RESUMEN

The aim of this work was the study of platelet/circulatory serotonin (5-hydroxytryptamine, 5-HT), specifically alternative ways of its measurement and main physiological characteristics. The study was performed on a large human population (N=500) of blood donors of both sexes over the course of a longer time period (17 months). Owing to the heterogeneity in measurement of circulatory serotonin encountered in the literature, three ways of expression were comparatively studied: per unit number of platelets, per unit mass of platelet protein and per unit volume of whole blood. Results demonstrated unimodal distribution of individual frequencies of platelet/circulatory serotonin in the human population with the mean values of 579+/-169 ng 5-HT/10(9) platelets; 332+/-89.9 ng 5-HT/mg protein and 130+/-42.3 ng 5-HT/ml blood (mean+/-S.D.). A progressive decrease of serotonin level with age (18-65 years) was demonstrated, reaching statistical significance between the extreme age groups. No significant differences in the serotonin level between the sexes were observed. No seasonal oscillations in platelet/circulatory serotonin were found. Platelet serotonin demonstrated intra-individual stability over time. Finally, regarding the methodology of measurement, our results demonstrated a good correlation among the above-mentioned ways of expression of platelet/circulatory serotonin. This indicates the possibility of intercomparison of the literature reports expressing this physiological parameter either as 5-HT concentration in platelets or as 5-HT level in the circulation.

Asunto(s)

RESUMEN

The role of serotonin (5HT) in the pathophysiology of posttraumatic stress disorder (PTSD) has been suggested by the overlap in clinical symptoms between PTSD and psychiatric conditions in which a serotonin dysfunction is implicated, as well as by the therapeutic efficiency of 5HT-related drugs (antidepressants, selective serotonin reuptake inhibitors and monoamine oxidase inhibitors) in alleviating symptoms in PTSD. In the present study, the blood platelet, which has been proposed as a peripheral model for the central serotonergic neurons, has been used to search for alterations in 5HT mechanisms in PTSD. Platelet serotonin level and kinetics of serotonin transporter and monoamine oxidase (MAO-B) were assessed in 63 combat-related PTSD patients and 43 sex and age-matched control subjects. A significant reduction in maximal velocity of platelet MAO-B (approx. 30%), with no changes in the enzyme affinity was observed in our patient sample. Conversely, no alterations in kinetic parameters (V(max), K(m)) of platelet serotonin transporter, as well as in platelet 5HT level, were found in the PTSD group.

Asunto(s)

RESUMEN

Physiological characteristics of serotonin (5-hydroxytryptamine, 5HT) transport through the platelet membrane was investigated in Wistar rats with our recently developed method permitting repetitive measurements of transporter kinetics in individual animals. Full kinetic analysis in the population of 91 animals revealed Michaelis constant (K(m)) of 0.158 +/- 0.025 microM and maximal velocity (Vmax) of 5HT uptake of 225 +/- 32 pmol per 10(8) platelets min-1 (mean +/- S.D.). Both kinetic parameters demonstrated normal distribution curves, which for Vmax were slightly skewed toward higher than average values. No gender effect was shown in frequency distributions, mean values and variability of kinetic parameters. A significant intraindividual correlation between kinetic parameters was found suggesting compensation at the level of the plasma membrane. Kinetic parameters were not influenced by age (until the middle age) or annual cycle (under laboratory conditions) and were shown to be fairly stable in time, supporting the view that platelet 5HT transport kinetics could be a useful biological trait marker.

Asunto(s)

RESUMEN

We examined the relationship between the density of serotonergic (5-hydroxytryptamine [5-HT]) uptake sites and extracellular 5-HT concentration in the rat brain using microdialysis with two different models, lesions with 5,7-dihydroxytryptamine (50 microg in the dorsal raphe nucleus (DRN) 15 days before) and sublines of rats genetically selected displaying extreme values of platelet 5-HT uptake. Compared to controls, lesioned rats had a reduced cortical concentration of 5-hydroxyindoles (45%), unchanged basal extracellular 5-HT in the DRN and ventral hippocampus (VHPC), and reduced basal 5-hydroxyindoleacetic acid (5-HIAA) concentrations (46%, DRN; 22%, VHPC). Yet the perfusion of 100 mmol/L KCl or 1 micromol/L citalopram elevated dialysate 5-HT significantly more in the DRN and VHPC of controls. In genetically selected rats, platelet 5-HT content and uptake were highly correlated (r2 = 0.9145). Baseline dialysate 5-HT (VHPC) was not different between high and low 5-HT rats and from normal Wistar rats. However, KCl or citalopram perfusion increased dialysate 5-HT significantly more in high 5-HT than in low 5-HT rats, and the former displayed a greater in vivo tissue 5-HT recovery. Significant but small differences in the same direction were noted in [3H]citalopram binding in several brain areas, as measured autoradiographically. Thus, basal extracellular 5-HT (but not 5-HIAA) concentrations are largely independent on the density of serotonergic innervation and associated changes in uptake sites. However, marked differences emerge during axonal depolarization or reuptake blockade. The significance of these findings for the treatment of mood disorders in patients with neurological disorders is discussed.

Asunto(s)

RESUMEN

By the breeding selection for the extreme values of platelet serotonin transporter activity, two sublines of Wistar-derived rats, with constitutionally high or low platelet serotonin uptake (PSU), were previously developed. In order to study the genetic background of these inherited differences, comparative Northern blot analysis of the platelet serotonin transporter messenger RNA levels of the animals from the two sublines was performed. If the values of animals from the high-PSU subline are taken as 100%, animals from the low-PSU subline demonstrated lower values of both platelet serotonin uptake and transporter mRNA levels (amounting to 62 and 76% respectively). Correlation between platelet serotonin uptake and the respective levels of messenger RNA for the serotonin transporter (r = 0.829, P < 0.01, N = 8) points to the same direction, indicating that the process of breeding selection for the extreme values of transporter kinetics has influenced transcription mechanisms of the serotonin transporter gene.

Asunto(s)

RESUMEN

Total RNA isolated from rat platelets by guanidinium-acid-phenol extraction, and mRNA for the serotonin (5-hydroxytryptamine) transporter (5HTt) was identified. From a typical starting sample of 20 mL of rat blood (approximately 9 x 10(9) platelets), 14 to 17 micrograms of total platelet RNA was obtained. Northern blot analysis, using 32P-labeled 5HTt cDNA as a probe, identified approximately 3.3 kb long 5HTt mRNA. After rehybridization with cyclophilin cDNA, approximately 1 kb long mRNA for cyclophilin, which could serve as a reference for 5HTt mRNA quantification, was also identified. Densitometric analysis demonstrated clearly measurable signals for both mRNAs. The possibility of quantification of rat platelet 5HTt mRNA should enable parallel studies on 5HTt gene expression in platelets and brain of the same animal, and the evaluation of the platelet model at the molecular genetic level.

Asunto(s)

RESUMEN

A rat model of alloxan-induced diabetes was used to investigate the effect of diabetic state on serotonin (5-HT) levels in peripheral body compartments, gastrointestinal (GI) and platelet, and the metabolic response of these compartments to serotonin precursor (5-hydroxytryptophan, 5-HTP) loading in diabetes. In all segments of diabetic gut a massive reduction in 5-HT concentration (to 45-64% at 6th week after induction of diabetes, with further progression to 30-52% at 14th week) was shown. After parenteral loading with 5-HTP for 6 days (30 mg/kg per day) 5-HT concentration in all parts of the GI tract returned to the control values (82-108%), indicating reduced serotonin precursor availability in diabetes. Platelet serotonin levels (PSL) in diabetic rats demonstrated a slight gradual reduction that became significant at 14th week of diabetic state. On the mentioned 5-HTP challenge only blunted response of PSL in diabetics, as contrasted to control animals (54% vs. 113%) was shown, indicating possible suppression of the membrane 5-HT transporter. The observed alterations in peripheral 5-HT homeostasis in diabetic rats as well as the possibility of their reversal by 5-HTP treatment could be of clinical interest.

Asunto(s)

RESUMEN

Serotonergic mechanisms have been implicated in pathophysiology of Parkinson's disease, an illness where the dopamine deficiency represents the prime biochemical deficit. Present interest centres on the possible involvement of serotonergic receptors in modulating dopamine transmission. In this paper the binding of the selective 5-HT3 antagonist [3H]GR 65630 was studied in rats with a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. The maximal density of specific [3H]GR 65630 binding was reduced in homogenates of entorhinal (17.1%, P < 0.05) and prefrontal cortex (27.5%, P < 0.05) on the lesioned side of the rat brain compared to the control tissues. An increase in affinity for [3H]GR 65630 binding was also found in homogenates of prefrontal cortex (33.8%, P < 0.05). No changes in the characteristics of [3H]GR 65630 binding to homogenates from the amygdala and hippocampus were observed. These data suggest that altered dopamine function may affect serotonergic mechanisms in the cortex in Parkinson's disease.

Asunto(s)

RESUMEN

By selective breeding we have recently obtained two discrete sublines of rats that differ in serotonin content in their platelets. As both serotonin and platelets may influence, or even take part, in immune reactions, we tested in this work the natural cytotoxicity in rats with constitutionally different platelet serotonin levels (PSL). Rats with low platelet serotonin level (mean +/- SD, 1.26 +/- 0.14 micrograms 5HT/mg protein; 81% vs. controls) had significantly higher (P less than 0.001) natural killer (NK) activity (mean +/- SD, 9.1 +/- 3.9%) than control rats with average PSL (1.57 +/- 0.18 micrograms 5HT/mg protein). On the contrary, rats with constitutionally high PSL (2.42 +/- 0.21 micrograms 5HT/mg protein, 154% vs. controls) had somewhat lower (P less than 0.02) NK activity (4.1 +/- 1.7%) than control animals (5.7 +/- 1.9%). Antibody-dependent cellular cytotoxicity (ADCC) against nucleated targets of the RCH line, detecting lymphoid effectors, as well as ADCC against chicken red blood cells (CRBC), detecting predominantly non-lymphoid effectors, were also significantly higher (P less than 0.001) in rats with low PSL (19.6 +/- 6.8% vs. 6.6 +/- 3.1% in controls for lymphoid effectors, and 71.8 +/- 6.1% vs. 48.7 +/- 8.8% in control rats for non-lymphoid effectors). However, no significant alteration of either ADCC was determined in rats with high PSL. The results suggest in vivo regulation of natural cytotoxicity by serotonin.

Asunto(s)

RESUMEN

It has recently been shown that platelet serotonin (5-hydroxytryptamine, 5HT) levels (PSL) in Wistar rats represent an individually stable biological parameter (neither subject to periodic oscillations nor markedly influenced by sex and age) that shows a unimodal frequency distribution within the population (range: 1.2-2.2 micrograms 5HT/mg platelet protein). To investigate the genetic background of PSL, selective breeding for the extreme values of this trait was performed. In the fourth generation, two discrete sublines of animals (statistically different from the unselected population) with constitutionally high or low PSL could be discerned: one with congenitally low PSL (1.1-1.6 micrograms 5HT/mg platelet protein; approximately normal distribution) and the other with congenitally high PSL (1.6-2.9 micrograms 5HT/mg platelet protein; irregular distribution). No difference in the pattern of inheritance between sexes could be discerned. The results demonstrated a marked heritable component underlying the expression of individual values of PSL in rats, suggesting that this parameter is a trait characteristic.

Asunto(s)