Intronic polymorphism of tryptophan hydroxylase and serotonin transporter: indication for combined effect in predisposition to suicide.

Indices of disturbed serotonergic neurotransmission are the most robust biological findings in suicide. Tryptophan hydroxylase (TPH) and 5-hydroxytryptamine transporter (5HTt) are the main regulators of 5HT signaling. Owing to the assumed functionality of intronic polymorphisms of TPH (218AC) and 5HTt (VNTR-2) genes, we investigated frequencies of concurrence of the TPH and 5HTt genotypes containing "lower activity" alleles (CC and 1010, respectively), in 192 suicide victims and 377 controls. Significant differences in frequencies of 5HTt and TPH genotype combinations were found between suicide victims and control subjects (p = 0.0156), with a clear dose-effect of the suspected ("lower activity") genotypes (p = 0.0046). Concurrent presence of the two, allegedly transcriptionally less active, variants of these genes seems to be in some kind of relation to the increased susceptibility to suicide.

Serotonin and immune response: effect of the amine on in vitro proliferation of rat lymphocytes.

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.

Serotonin transporter in rat platelets. Level of protein expression underlies inherited differences in uptake kinetics.

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.

Support for allelic association of a polymorphic site in the promoter region of the serotonin transporter gene with risk for alcohol dependence.

OBJECTIVE: An association between the 5-HTTLPR short variant polymorphism in the promoter region of the serotonin transporter gene and risk for alcohol dependence has been reported from case-control studies that are, however, prone to chance findings related to artifacts of population structure. The authors sought additional evidence for this association from a family-based study. METHOD: Ninety-two alcohol-dependent probands and their parents were tested for nonrandom transmission of alleles from heterozygous parents to affected probands. RESULTS: Preferential transmission of the short allele was found (65 of 102 transmissions from heterozygous parents). CONCLUSIONS: The results suggest allelic association between a variant in the promoter region of the serotonin transporter gene and the risk for alcohol dependence. However, it remains to be seen whether the functional properties of this variant are directly responsible for the increased risk to alcohol dependence.

mRNA expression of serotonin receptors in cells of the immune tissues of the rat.

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.

Physiological characteristics of platelet/circulatory serotonin: study on a large human population.

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.

Platelet monoamine oxidase activity, ego strength, and neuroticism in soldiers with combat-related current posttraumatic stress disorder.

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.

Posttraumatic stress disorder and platelet serotonin measures.

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.

Serotonin transporter gene and schizophrenia: evidence for association/linkage disequilibrium in families with affected siblings.

The serotonergic (5-HT) system has been implicated in the etiopathogenesis of psychoses. Since the 5-HT transporter plays an important role in regulation of 5-HT transmission, its gene can be considered as a candidate for vulnerability to psychiatric disorders. Two polymorphic sites of the 5-HT transporter gene-5-HTTLPR, a VNTR in the 5' regulatory region, and a VNTR in the second intron-were studied in a sample of 61 families with schizophrenia for transmission disequilibrium. Each family contained at least two siblings affected with schizophrenia or schizoaffective disorder (mainly schizophrenic). One hundred and thirty-nine affected offspring with parental information for genotyping, were available for analysis. No preferential transmission of either short or long alleles of the promoter polymorphism was observed. However, a transmission distortion was detected for alleles of the intronic VNTR polymorphism (chi2TDT max =14.33; P = 0.0002; corrected P value = 0.0003) resulting in more frequent than expected transmission of the 12 repeat allele. This finding adds additional evidence to the idea that the serotonergic system may be involved in development of psychoses. Molecular Psychiatry (2000) 5, 91-95.

Diazenes as modificators of drug-resistance in tumor cells.

To overcome the drug resistance, which is the major obstacle in the successful treatment of cancer patients, various compounds have been tested. Glutathione is one of the most promising targets for modulation. In the present study, we examined the influence of five new synthesized compounds--diazenes on the reduction of the intracellular level of GSH. Further, we investigated their ability to increase the cytotoxicity of cisplatin, vincristine and doxorubicin. In experiments human parental cervical (HeLa) and laryngeal (HEp2) carcinoma cells and their drug-resistant cell sublines (HeLaCA and CK2, respectively) were used. Intracellular GSH content was examined spectrophotometrically by the procedure developed by Tietze. The cell sensitivity to drugs was determined using a modified colorimetric MTT assay. Results showed that the rate of reduction of GSH concentration was dependent on the cell type and the type of diazenes. We did not find a correlation between the reduction in GSH level and increased cytotoxicity to selected anticancer drugs. Nevertheless, we found that: a) diazenes LV-35 and VZ-19 increased the cytotoxicity of cisplatin in HEp2 cells, b) diazene MG-19 potentiated the cytotoxicity of vincristine in HEp2 cells, and c) diazene VZ-19 in HeLaCA cells. These data suggest that specific combination of diazene and anticancer drug may be useful in the treatment of certain tumor types.

Diazene JK-279: potential anticancer drug.

The aim of this study was to examine the cytotoxic effect of 10 newly synthesized diazenecarboxamides (diazenes). Using a modified colorimetric MTT assay, their cytotoxicity was determined on 10 human cell lines: cervical carcinoma parental and cisplatin-resistant cells, laryngeal carcinoma parental and cisplatin- and vincristine-resistant cells, glioblastoma parental and cisplatin-resistant cells, breast adenocarcinoma parental and doxorubicin-resistant cells, and mammary carcinoma cells. Results show that diazene JK-279 was most effective, reducing significantly the cell survival of all 10 cell lines examined, including five drug-resistant cell lines. A cytotoxic effect was observed also on nine from 10 cell lines for diazene JK-835. A small reduction in cell survival was obtained (mainly for highest drug concentrations) for diazenes LV-57 and MG-19 on two cell lines, and JK-429 and JK-913 on one cell line. Other diazenes did not demonstrate any cytotoxic activity. The results encourage further research on diazene JK-279 as a potential anticancer drug.

Physiological characteristics of serotonin transporters on rat platelets.

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.

Basal and stimulated extracellular serotonin concentration in the brain of rats with altered serotonin uptake.

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.

Serotonin transporter on rat platelets: levels of mRNA underlie inherited differences in uptake kinetics.

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.

Characterization of human breast adenocarcinoma SK-BR-3 cells resistant to doxorubicin.

Doxorubicin shows a wide spectrum of activities in solid tumors, especially against breast carcinoma. The aim of this study was to examine if doxorubicin, when given at lower concentrations than applied in clinic, may induce changes in treated cells. With this purpose we developed human breast adenocarcinoma SK-BR-3 cell line resistant to doxorubicin. The sensitivity of these cells to doxorubicin and to some other cytostatics used in cancer treatment was determined by colorimetric MTT assay. Some parameters which may be of importance as prognostic factors in treatment of breast cancer were analyzed as well. The expression of genes involved in mitotic signal pathway (EGF, TGF alpha, EGF-R, erbB-2, erbB-3, c-myc and c-H-ras) was determined immunocytochemically. The concentrations of cathepsins were determined using quantitative immunoreactive assays (cathepsins B and L) or immunoradiometric assay (cathepsin D). The results revealed that even low doses of doxorubicin can induce numerous changes in treated cells: they become resistant to doxorubicin, and cross-resistant to several other cytostatics. The expression of the above mentioned genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells.

Arteriovenous fistula of the internal mammary artery (combat injury): transcatheter intravascular coil occlusion.

A case of arteriovenous fistula of the left internal mammary artery occluded by Gianturco coil is reported. A 26-year-old male suffered multiple explosive injuries. Right heart failure developed postoperatively. Digital subtraction angiography demonstrated arteriovenous fistulas between the left internal mammary artery and left subclavian and innominate veins. Considering previous multiple surgical interventions and severe general condition of the patient, both arteriovenous fistulas were successfully occluded by transcatheter placement of a Gianturco coil.

Reduction of gastrointestinal serotonin in alloxan-diabetic rats: reversal by 5-hydroxytryptophan treatment.

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.

Identification of serotonin transporter mRNA in rat platelets.

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.

Characterization of carboplatin-resistant sublines derived from human larynx carcinoma cells.

In human larynx carcinoma cells, resistance to carboplatin (CBDCA) was induced by continuous five-day exposure of parental lines to the increasing CBDCA concentrations in culture medium, reaching the clinical level of 9.23 micrograms/ml. Three clones were selected and characterized: CBP-3, CBP-6 and CBP-7, CBP-3 clone was 2.0-fold, CBP-6 2.1-fold, and CBP-7 2.9-fold more resistant to carboplatin. The response of these sublines to different cytostatics was compared to the response of the parental cell lines to the same drug. CBP-7 and CBP-6 clones exhibited cross-resistance to cisplatin (cis-DDP), CBP-7 clone became markedly more sensitive and CBP-3 slightly more sensitive to 5-fluorouracil (5-FU), CBP-6 became sensitive to etoposide (Et), CBP-6 became sensitive and CBP-7 resistant to vinblastine (VBL). Other clones did not change change their sensitivity to cis-DDP, 5-FU, Et or VBL. None of the three clones did alter the sensitivity to mitomycin C, doxorubicin (Dox) or vincristine (VCR). There was no change in the growth rate. Glutathione (GHS) levels were elevated in all three clones, but the increase was significant only for CBP-7 clone. Similarly, the activity of glutathione transferase (GST) was elevated in all clones, but this increase was not significant for CBP-7 clone. The analysis of the of c-myc, c-Ha-ras and c-fos genes reveal no change in the c-myc expression, induction of the c-Ha-ras oncogene in CBP-6 and CBP-7 cells, and biochemistry and oncogene expression indicate that the acquired resistance to carboplatin is a complex, multifactorial process in these cells.

Alteration of NK- and ADCC-activities in rats genetically selected for low or high platelet serotonin level.

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.