α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca2+ homeostasis in human erythroleukemia cells.

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca2+ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca2+ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca2+-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin. Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca2+.

α1-Adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism.

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Decline of bone marrow-derived hematopoietic progenitor cell quality during aging in the rat.

Several studies have shown that aging is associated with quantitative and qualitative alterations of the stem and progenitor cell compartment. The current results indicate that there is a significant age-associated decline in the proliferative capacity of rat myeloid progenitor cells. In contrast, no difference was found in the frequency of myeloid progenitor cells in the bone marrow of young versus old rats. Furthermore, a significant shift towards higher proliferative capacity of myeloid progenitors was observed after lifelong voluntary exercise. These data emphasize that aging is accompanied by a loss of proliferative capacity and that voluntary exercise could retard this process.

The alpha1-adrenergic receptor antagonists, benoxathian and prazosin, induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells.

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.

Acute adrenergic stress inhibits proliferation of murine hematopoietic progenitor cells via p38/MAPK signaling.

Acute adrenergic stress is a cause of hematopoietic failure that accompanies severe injury. Although the communication between neuronal and immune system is well documented and catecholamines are known as important regulators of homeostasis, the molecular mechanisms of hematopoietic failure are not well understood. To study the influence of adrenergic stress on hematopoietic progenitor cells (HPCs), which recently have been found to express adrenergic receptors, Lin(-),Sca(+), cells were isolated and treated with alpha- and beta-adrenergic agonists in vitro. Indeed, this stimulation resulted in significantly decreased colony formation capacity using granulocyte/macrophage colony-forming unit assays. This decline was dependent on the formation of reactive oxygen species (ROS) and activation of the p38/mitogen-activated protein kinase (MAPK) pathway, since the addition of antioxidants or a p38 inhibitor restored CFU formation. DNA damage by adrenergically induced ROS, however, does not seem to account for the reduction of colonies. Thus, catecholamine/p38/MAPK is identified as a key signal transduction pathway in HPCs besides those dependent on Wnt, Notch, and sonic hedgehog. Furthermore, a well-known target of p38 signaling, p16 is transcriptionally activated after adrenergic stimulation, suggesting that cell cycle arrest might importantly contribute to hematopoietic failure and immune dysfunctions after severe injury. Since increased levels of catecholamines are also observed in other conditions, such as during aging which is linked with decline of immune functions, adrenergic stress might as well contribute to the lowered immune defence in the elderly.

lin-Sca-1+ cells and age-dependent changes of their proliferation potential are reliant on mesenchymal stromal cells and are leukemia inhibitory factor dependent.

Aging as a process is paralleled by a variety of hematological alterations. Characteristic features are a diminished homeostatic control of blood cell production and a decline in immune functions. It is generally accepted that stromal cells play a basal role in hematopoiesis by providing survival and differentiation signals, by secreting cytokines, or through direct contact with hematopoietic stem cells, thereby supporting the generation and replenishment of hematopoi- etic progenitor cells (HPC). Here we demonstrated that HPC-related colony formation is positively influenced by mesenchymal stromal cells (MSCs) when grown in co-culture, in particular regarding the number of primary granulocyte/macrophage colony-forming units as well as with respect to the average size of the formed colonies. These effects were more pronounced when the MSCs originated from young donors than from old ones. Because leukemia inhibitory factor (LIF) plays an important role during hematopoiesis, properties of lin- Sca-1+ cells and MSCs derived from LIF-deficient mice (LIF-/-) were determined both ex vivo and in vitro. LIF-/- animals contain a significantly reduced number of lin- Sca-1+ cells, nevertheless the replating capacity of LIF-/- HPCs was found to be generally unchanged when compared to those from LIF+/+ animals. However, when cocultured with MSCs, LIF-/- lin- Sca-1+ cells exhibited comparable characteristics to HPCs derived from old wild-type animals.

ROC analysis of subcutaneous adipose tissue topography (SAT-Top) in female coronary heart disease patients and healthy controls.

The aim of this study was to investigate whether subcutaneous adipose tissue topography (SAT-Top) is different in female CHD patients (n=26) and healthy controls (n=36) matched to age, body size, weight, and BMI. The thicknesses of SAT layers were measured by LIPOMETER at 15 specified body sites. To calculate the power of the different body sites to discriminate between CHD women and healthy controls, receiver operating characteristic (ROC) curve analysis was performed. For each parameter, sensitivity and specificity were calculated at different cutoff points. CHD women showed a significant decrease to 78.36% (p=0.012) at body site 11-front thigh, 73.10% (p=0.012) at 12-lateral thigh, 72.20% (p=0.009) at 13-rear thigh, 66.43% (p<0.001) at 14-inner thigh, and 49.19% (p<0.001) at 15-calf. The best discriminators analysed by ROC curves between female CHD patients and healthy controls turned out to be calf and inner thigh (optimal cut off values: calf: 3.85 mm and inner thigh: 11.15 mm). Stepwise discriminant analysis identified the body sites calf, lateral chest, and inner thigh as significant. In conclusion, information was obtained on the extent to which SAT thickness at each measured body site is able to discriminate between the two subject groups. The good discrimination results obtained for the present dataset are encouraging enough to recommend applying LIPOMETER SAT-Top measurements in further studies to investigate individual risks for CHD.

Haploinsufficiency of senescence evasion factor causes defects of hematopoietic stem cells functions.

The quality of hematopoietic stem cells (HSCs) is essentially defined by two characteristics, i.e., multilineage differentiation and self-renewal capacity. Thus, it is of high priority to clarify mechanisms that regulate these functions and to understand them at the molecular level. In the present study, we investigated the role of senescence evasion factor (synonymously hPrp19,hPSO4,hNMP200: SNEV), a multifunctional protein involved in pre-mRNA splicing, regulation of replicative life span, and DNA repair. Here we report that murine SNEV mRNA expression is high in lineage-depleted (Lin(-)) precursor cells of the bone marrow immediately after isolation as compared to fully differentiated peripheral blood lymphocytes (PBLs). Furthermore, the progenitor cell subset with highest colony-forming ability and self-renewal capacity (Lin(-), Sca-1(+)) showed also the highest SNEV expression. To test if the observed differences in SNEV mRNA levels cause stem cell defects, Lin(-) cells derived from heterozygous SNEV knockout mice were tested for primary as well as secondary colony-forming potential as a measure of self-renewal capacity. Interestingly, both, primary and secondary colonies were significantly less formed from SNEV(+/-) cells, a defect that was rescued by ectopic SNEV expression. Similarly, bone marrow cells derived from the short-lived Senescence-Accelerated-Mouse-Prone (SAMP8) model showed similar differences in comparison to the aging-resistant (SAMR1) control strain. These data suggest that the expression of SNEV is closely associated with the growth of murine HSCs and determines the proliferative and repopulating capacity of phenotypically defined HSC subsets.

Norepinephrine treatment and aging lead to systemic and intracellular oxidative stress in rats.

Reactive oxygen species (ROS) play important roles in cellular senescence and organismic aging. Furthermore, they have been implicated in some of the adverse effects of chronic stress due to elevated peripheral levels of catecholamines. Here, we applied three different techniques to individually compare the systemic and intracellular oxidative stress in aged (23 months) and young (5 months) Sprague-Dawley rats, and in young rats treated for 12 or 24 h with norepinephrine (NE). Thiol groups of blood serum proteins (RSH) were determined by means of Ellman's reaction. Intracellular ROS were assessed in spleen cells and peripheral blood lymphocytes (PBL) by carbonylation of cellular (spleen) proteins as determined by immunoblotting (Oxyblot) and/or by means of 2',7'-dichlorofluorescein (DCF) fluorescence. As compared to the young, untreated controls, both old rats and NE treated young rats showed similarly lowered RSH values paralleled by elevated intracellular ROS levels or enhanced Oxyblot signals. Individual RSH values were highly significantly, negatively correlated with respective Oxyblot data as well as with DCF fluorescence. The results confirm the roles of ROS in aging and adrenergic stress in the rat model, and suggest that the decrease in RSH of blood serum may be taken as a valid indicator for the enhanced oxidative stress in lymphocytes.

The non-competitive metabotropic glutamate receptor-1 antagonist CPCCOEt inhibits the in vitro growth of human melanoma.

Five decades ago, the dicarboxylic amino acid glutamate became recognized as the major excitatory neurotransmitter in the central nervous system. In recent years, the expression of glutamate receptors was detected also in peripheral, non-neuronal tissues. Furthermore, it was found that glutamate stimulated the proliferation and migration of several peripheral tumor cells, and that glutamate receptor antagonists limited tumor growth. Most of these studies, however, used broad spectrum compounds and/or group-specific antagonists. Here we report that a selective, non-competitive metabotropic glutamate receptor-1 antagonist, CPCCOEt (7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester), significantly inhibited the proliferation and modified the morphology of two human melanoma cell lines. These effects were independent of the external glutamate level in the culture medium. In addition, CPCCOEt significantly enhanced the tumoricidal effects of cytostatic drugs. Thus, selective non-competitive metabotropic glutamate receptor antagonists may be used alone and/or with the synergistic effects of chemotherapy, thus enhancing existing therapies of melanoma and possibly other malignancies.

Biocompatibility of ultra-high molecular weight polyethylene (UHMW-PE) stabilized with alpha-tocopherol used for joint endoprostheses assessed in vitro.

Adding the natural antioxidant alpha-tocopherol to ultra-high molecular weight polyethylene (UHMW-PE) can remarkably delay the oxidation of hip cups made thereof. However, alpha-tocopherol is likely to undergo different chemical transformations during manufacturing and sterilization of hip cups than in human metabolism. Therefore, the biocompatibility of the putative transformation products has to be investigated. In-vitro tests with L929 mice fibroblast-cells gave no evidence for cytotoxicity. To further ensure the biocompatibility, in-vitro tests with human cells were carried out in this study. Two different human cell lines, one adherent cell line, HF-SAR, and one suspension culture, GSJO, were tested on UHMW-PE-tablets (diameter: 15 mm; thickness: 2 mm; processed according to standard procedures for artificial hip-cups) with and without alpha-tocopherol with respect to cell viability, proliferation and morphology by means of cell counting, WSt-1 proliferation assay and scanning electron microscopy. Similar proliferation rates were found with both polyethylene samples. Further, we found intact morphology in light and electron microscopy on each substrate. The morphologic characteristics of skin fibroblasts were not changed by any material. Normal adherence and spreading of the fibroblasts was found on controls of glass, as well as on polystyrene and on stabilized and unstabilized polyethylene. The characteristic behaviour as suspension of the GSJO cells remained unchanged. The mitochondrial activity, as studied by WST-1 cell proliferation reagent, was identical on each substrate during the whole observation period of 7 days.

Immune reconstitution after purified autologous and allogeneic blood stem cell transplantation compared with unmanipulated bone marrow transplantation in children.

Immune reconstitution is critical for the long-term success of haematopoietic stem cell transplantation (HSCT). We prospectively analysed immune reconstitution parameters after transplantation of autologous (group 1; n = 10) and allogeneic (group 2; n = 12) highly purified CD34+ peripheral blood stem cells (PBSC) and unmanipulated allogeneic bone marrow (BM) (group 3; n = 9) in children. Median follow-up after HSCT was 56 (group 1), 61 (group 2), and 40.5 months (group 3). Median CD34-cell dose transplanted in the three groups was 9.4 x 10(6)/kg, 20.3 x 10(6)/kg, and 4.25 x 10(6)/kg recipient's body weight (BW) respectively. Complete haematopoietic engraftment was seen in all patients without any significant differences between the three groups. T-cell reconstitution at 6 months was significantly delayed in autologous peripheral blood stem cell transplantation (PBSCT) compared with allogeneic BM transplantation (P < 0.028) and allogeneic PBSCT (P < 0.034). At 3 months after transplantation numbers of CD56+/3- natural killer cells were higher in the allogeneic PBSC group (P < 0.01) compared with the BM group. The numbers of proven bacterial and viral infections were equally distributed between the three groups. In conclusion, recipients of allogeneic highly purified CD34+ PBSC or unmanipulated BM have higher lymphocyte subset counts at 6 months after transplantation than recipients of autologous CD34-selected PBSC. Infection rates and outcome, however, were not significantly different.

Role of free radicals in aseptic loosening of hip arthroplasty.

Fibrous pseudocapsule around hip implants is an invariable finding at revision operations and is believed to release inflammatory mediators that stimulate bone resorption. Reactive oxygen species have been proposed to be causative factors in various disorders with tissue fibrosis. We were interested in investigating whether aseptic loosening is connected with high oxidative stress, and in showing the underlying mechanism of periprosthetic fibrosis and its role in loosening. Levels of oxidative stress markers reduced (GSH) and oxidized (GSSG) gluthatione and malondialdehyde (MDA) were assayed in 28 loose hips and in 12 stable hips revised for high rate of wear and osteolysis. Collagen in the periprosthetic tissues was measured as hydroxyproline content. Osteolysis and polyethylene wear were graded. Increased oxidative stress measured by low GSH/GSSG ratio as well as by increased MDA level was established in patients compared to controls. Oxidative stress markers intercorrelated significantly. MDA and both GSH and GSSG levels correlated significantly with hydroxyproline level. Levels of GSSG and MDA were higher in hips with greater polyethylene wear. The results suggest that high oxidative stress may play a role in formation of a fibrous membrane observed at revision of loose hips. The fibrous pseudocapsule is probably related to high intraarticular pressure and expansion of the effective joint space. This study may elicit some aspects of the pathogenesis of aseptic hip loosening and aid in future investigations aiming at prevention of this complication.

Glutamate receptor-mediated effects on growth and morphology of human histiocytic lymphoma cells.

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and binds to a variety of receptors, which recently have also been detected in peripheral, non-excitable cells. New research suggests that this abundant amino acid might also be involved in the growth of tumor cells acting via novel receptor-mediated autocrine/paracrine signal transduction pathways. We report here that glutamate, as well as glutamate receptor reactive drugs, differentially modulate growth and morphology of human histiocytic lymphoma-derived U937 cells. These effects were different depending on the culture milieu: in glutamine-free medium the glutamate receptor agonists, kainate (KA), and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), but also the antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly decreased the proliferation of U937 cells. In contrast, in cultures devoid of glutamate, glutamine and serum, the agonists significantly increased cell proliferation whereas the antagonist CNQX showed no effect. These data point to a significant role of peripheral glutamate receptors in tumor cell proliferation.

Early atherosclerosis in obese juveniles is associated with low serum levels of adiponectin.

CONTEXT: There is growing evidence that adiponectin, the most abundant adipocytokine of adipose tissue cells, plays a crucial role in advanced atherosclerosis. OBJECTIVE: The objective of the study was to evaluate the role of adiponectin in early atherosclerosis. DESIGN: One hundred forty obese juveniles (mean age, 13.5 +/- 4.4 yr) and 100 age-matched, healthy, normal-weight controls from the STYrian Juvenile Obesity Study were investigated. We measured adipocytokines, inflammatory biomarkers, parameters of insulin resistance, and lipid subfractions. Intima-media thickness (IMT) of common carotid arteries was determined by ultrasonography. Furthermore, lipometric measurements were performed in obese juveniles to determine the topographic distribution of sc adipose tissue (SAT). RESULTS: Compared with controls, the group of obese juveniles exhibited a significantly increased IMT (P < 0.001) and elevated high-sensitive C-reactive protein (P < 0.001), indicating early stages of atherosclerosis. Serum levels of adiponectin were highly significantly negatively correlated with carotid IMT, even after controlling for common cardiovascular risk factors (P < 0.001; r = -0.34). Furthermore, adiponectin was positively correlated with high-density lipoprotein-free cholesterol and serum apolipoprotein-A1 and negatively with triglycerides, insulin resistance, uric acid, and serum transaminases. By a multiple regression analysis, adiponectin was shown to be the strongest predictive variable for carotid IMT. Finally, adiponectin was found positively correlated with SAT thickness of the rear and inner thigh in boys and negatively with the SAT thickness of the neck in girls. CONCLUSION: In summary, our study describes an influence of SAT topography on adiponectin serum levels and provides first evidence that incipient atherosclerosis is associated with low serum levels of this adipocytokine.

Effects of social stress on blood leukocyte distribution: the role of alpha- and beta-adrenergic mechanisms.

Social stress in mammals has repeatedly been shown to cause substantial alterations in the distribution pattern of immune cells in the peripheral blood. The studies described here investigated the role of adrenergic mechanisms in mediating stressor-induced changes in blood leukocyte numbers using a social confrontation procedure in the rat. Experimental manipulations were carried out to eliminate the stress-associated release of adrenal hormones or to block the binding of endogenous catecholamines to alpha- and beta-adrenergic receptors. Adrenalectomy completely abolished the stressor-induced decreases in circulating numbers of T helper cells (CD4+), cytotoxic T cells (CD8+) and B cells but was ineffective in preventing neutrophil granulocytosis, monocytosis and an increase in natural killer (NK) cells. Treatment with the alpha-adrenergic antagonist phentolamine (PHE) was highly effective in preventing granulocytosis and partially blocked lymphopenia, but failed to abolish monocytosis and an increase in NK cells. Treatment with the beta-adrenergic antagonists propranolol (PROP) or nadolol (NAD) entirely blocked the increases in monocytes and NK cells. In addition, beta-adrenergic blockade also significantly reduced neutrophilia, with PROP being more effective than NAD. The results presented here provide evidence that catecholamines play an important role in the redistribution of blood leukocytes during social stress. In particular, the mobilization of cells of the innate immune response seems to be regulated by adrenergic mechanisms.

Beyond cholesterol--inflammatory cytokines, the key mediators in atherosclerosis.

The development of atherosclerotic lesions encompasses a cascade of cellular and molecular responses that can at best be characterized as an inflammatory process, and exhibits striking similarities to autoimmune diseases, such as rheumatoid arthritis. Chemokines, cytokines and their receptors are critically involved in initiation and perpetuation of atherosclerosis, and they play important roles at all levels in the pathogenesis of this disease. In the present article, the currently available information on cytokines and chemokines as key mediators in atherosclerosis is reviewed. Furthermore, based on recent experiences of our own with very early stages of atherosclerosis, possible new ways to make use of these parameters toward improved early detection, prevention and treatment of this disease are indicated.

Dopamine receptor D3 mRNA expression in human lymphocytes is negatively correlated with the personality trait of persistence.

It has been proposed that neurotransmitter receptor expression in peripheral immune cells reflects expression of these receptors in the brain. To test this "peripheral marker hypothesis", we compared mRNA expression of the dopamine receptors D3 (DRD3) and D4 (DRD4) in peripheral blood lymphocytes (PBL) to personality traits assessed with the Temperament and Character Inventory (TCI) in 50 healthy and unmedicated Caucasian individuals. A shared variance of at least 17% (p=0.016) between DRD3 mRNA expression in PBL and the personality trait of persistence was found. As personality traits have been generally assumed polygenic with a single gene accounting for rarely more than 1-2% of observed variance in a trait, this result lends further support to the peripheral marker hypothesis for DRD3 mRNA expression in PBL. It may also suggest a significant role for the DRD3 in the neurobiology of persistence and point to an interesting link between personality and functioning of the immune system.

Reduced dopamine D4 receptor mRNA expression in lymphocytes of long-term abstinent alcohol and heroin addicts.

AIM: It has been repeatedly suggested that dopamine receptor expression in peripheral blood lymphocytes reflects, to some extent, brain status. The aim of the present study was to investigate dopamine receptor expression in peripheral blood lymphocytes of long-term abstinent alcohol and heroin addicts against the background of the hypothesis, that a persisting dysfunction of the dopaminergic system contributes a biological cause to the chronic character of addiction. DESIGN: Dopamine D3 and D4 receptor mRNA expression in peripheral blood lymphocytes was measured by real-time polymerase chain reaction (PCR) in 19 alcohol addicts, abstinent for 6.2 +/- 4.7 months (mean +/- SD), and 20 heroin addicts, abstinent for 6.7 +/- 3.7 months (mean +/- SD), and compared to a control group of 29 age- and sex-matched individuals with no life-time history of substance abuse. FINDINGS: One-way anova showed significant differences in D4 mRNA expression between the groups (P = 0.005): both groups of addicts showed an approximately 50% reduction in D4 receptor mRNA expression in peripheral blood lymphocytes (PBL) compared to controls. No differences were found for D3 mRNA expression between the groups. CONCLUSION: The results of the present study indicate a withdrawal-persisting dopaminergic imbalance in abstinent addicts as measured by a suggested peripheral marker.

Reduced dopamine D3 receptor expression in blood lymphocytes of smokers is negatively correlated with daily number of smoked cigarettes: a peripheral correlate of dopaminergic alterations in smokers.

The mesolimbic dopaminergic system is known to mediate rewarding effects of nicotine administration, and dysfunctions of this system may underlie failure to stop cigarette smoking. Expression of dopamine receptors in peripheral blood lymphocytes (PBLs) has been indicated as a peripheral correlate of brain status. Dopamine receptor D(3) (DRD3) and D(4) (DRD4) mRNA expression in PBLs was measured by real-time polymerase chain reaction in smokers (n=26) and former smokers (n=14), compared with nonsmoking control subjects (n=35). A significant (p=.032, Bonferroni corrected) 30% reduction of DRD3 mRNA expression in PBLs was found in smokers but not former smokers in comparison with controls. DRD3 mRNA expression in PBLs in smokers but not former smokers was negatively correlated with daily number of cigarettes consumed (Pearson correlation coefficient r=-.54, p=.005). These data suggest a selective inhibiting effect of smoking on DRD3 mRNA expression and, with the known involvement of DRD3 in reward mediation, indicates a vicious-cycle explanation for the motivation for continued smoking.