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OBJECTIVE--The aim of this analysis was to assess the prospective association of serum testosterone and dehydroepiandrosterone sulfate (DHEAS) levels with incident metabolic syndrome (MetS) in men. RESEARCH DESIGN AND METHODS--Data were obtained from the Study of Health in Pomerania (SHIP), a population-based prospective cohort of adults aged 20-79 years. Analyses were conducted in 1,004 men without baseline MetS defined by National Cholesterol Education Program Adult Treatment Panel III guidelines. Testosterone and DHEAS were categorized by age-specific quartiles and Poisson regression models with relative risks (RRs) and 95% CIs were estimated. RESULTS--After a median follow-up time of 5.0 years, 480 men (47.8%) developed MetS. Testosterone levels decreased with increasing number of MetS components. Testosterone in the lowest quartile predicted MetS (RR 1.38 [95% CI 1.13-1.69]), particularly among men aged 20-39 years (2.06 [1.29-3.29]), even after adjustment for age, smoking, alcohol consumption, physical activity, waist circumference, self-related health, and time of blood sampling. DHEAS levels were not related to incident MetS (0.99 [0.83-1.19]). CONCLUSIONS--Low testosterone but not DHEAS predicts development of MetS in a population-based cohort of 1,004 men aged 20-79 years. Especially in young men aged 20-39 years, results suggest low testosterone as a strong predictor for incident MetS. Assessment of testosterone in young and middle-age men may allow early interventions in the general population., In men, a decline in serum total testosterone and adrenal androgens like dehydroepiandrosterone sulfate (DHEAS) with increasing age is well documented (1) and has been linked to a variety of [...]

Objective: There has been debate whether polymorphisms within the serotonin transporter-linked polymorphic region (5-HTTLPR) moderate susceptibility to posttraumatic stress disorder (PTSD). The authors investigated 5-HTTLPR genotypes and their interaction with the number of traumatic events in the prediction of PTSD in a general population sample. Method: Analyses were based on data from 3,045 subjects who participated in the Study of Health in Pomerania. All participants were assessed with the PTSD module of the Structured Clinical Interview for DSM-IV. The short (S)/long (L) polymorphism of 5-HTTLPR (rs4795541) and the A-G polymorphism (rs25531) were genotyped. Results: Among the participants, 1,663 had been exposed to at least one traumatic event, and 67 (4.0%) developed PTSD. Among those who had experienced less than three traumatic events, the life-time prevalence of PTSD was 2.6%, 3.5%, and 4.3% for those with zero, one, and two [L.sub.A] alleles, respectively, but the life-time prevalence was 0%, 7.3%, and 19.6%, respectively, among those with three or more traumatic experiences. This finding suggests that there is an additive excess risk for frequent trauma in the LA/LA genotype, which was confirmed by the relative excess risk due to interaction (RERI). In allelic analysis, RERI was 3.3. Thus, the odds ratio for PTSD in [L.sub.A] allele carriers exposed to three or more traumas was 3.3 times higher as a result of the interaction between PTSD and the [L.sub.A] allele. Conclusions: An additive gene-environment interaction with the high expression [L.sub.A] allele of 5-HTTLPR and frequent trauma in PTSD was found. The attributable proportion indicated that more than 60% of all [L.sub.A] allele carriers who were exposed to three or more traumas developed PTSD as a result of an interaction between genotype and exposure.

The impact of the promoter polymorphisms of the serotonin transporter (5‐HTTLPR) on mood has been studied by two‐way interaction models comprising one environmental factor and genotype variants. However, childhood abuse is assumed to be associated with different psychobiological long‐term effects than adult traumatic events. Both types of trauma may interact on an individual basis throughout the lifespan moderating the impact of the 5‐HTTLPR sallele on depressive disorders. Therefore, the hypothesis of a three‐way interaction among the 5‐HTTLPR, childhood abuse and adult traumatic experience was tested. Caucasian subjects (1,974) from the general population in Germany (Study of Health in Pomerania (SHIP)) were analyzed. Depressive symptoms were measured with the Beck Depression Inventory (BDI‐II). Childhood abuse was assessed with the Childhood Trauma Questionnaire. Adult traumatic events were derived from the SCID interview (DSM‐IV) on posttraumatic stress disorder (PTSD). Global three‐way interactions among the 5‐HTTLPR, adult traumatic experiences and childhood abuse (P= 0.0007) were found. Carriers of the ssor slgenotypes who had been exposed to childhood abuse and to more than two adult traumatic events had higher mean BDI‐II scores (16.0 [95% CI 8.4–23.6]) compared to those carrying the llgenotype (7.6 [4.5–10.7]). These results were supported using a second, more severe definition of childhood abuse (P= 0.02). No two‐way interactions were observed (P> 0.05). Childhood abuse and adult traumatic events may act synergistically in interaction with the sallele of the 5‐HTTLPR to increase the risk for depressive symptoms independently from the lifetime diagnosis of PTSD. © 2012 Wiley Periodicals, Inc.

CYP2C19 is a polymorphic enzyme that plays a pivotal role in the metabolism of 10 of clinically used drugs worldwide. The CYP2C193allele is characterized by a premature stop codon that leads to a truncated nonfunctional protein and consequently a poor metabolizer phenotype. Aminoglycoside antibiotics have been shown to induce readthrough of premature stop codons and partial restoration of protein function. We investigated the ability of the aminoglycosides gentamicin and G418 to induce readthrough of CYP2C193premature stop codon in human cells.

Pancreatic adenocarcinoma is one of the malignancies that is highly resistant to therapy and among the leading causes of cancer-related death. Several factors may influence pancreatic cancer resistance, and expression of ATP-binding cassette transport proteins is one of the major mechanisms of drug resistance. Members of this family's C-branch, also referred to as multidrug resistance-associated proteins (MRPs), might be of particular interest because they are able to efflux nucleoside analogs used in the treatment of pancreatic cancer. Expression of MRP1, MRP3, MRP4, and MRP5 in human pancreas and pancreatic carcinoma has been reported. However, contributions of MRPs to chemoresistance of pancreatic cancer are not fully understood. MRP5 mRNA expression in pancreatic adenocarcinoma cell lines correlated significantly with cellular sensitivity to 5-fluorouracil (5-FU) (r = 0.738, p < 0.05). Long-term treatment with 5-FU increased expression of MRP5 by 2.4-fold and was associated with significant drug resistance [IC50values for control and 5-fluorouracil (5-FU)-resistant Patu-T cell lines were 11.3 ± 5.3 and 33.2 ± 6.9 µM, respectively (p < 0.05)]. Consequently, overexpression of MRP5 in Colo-357 cells resulted in significantly reduced accumulation of 5-FU related radioactivity and 5-FU cytotoxicity. Knockdown of MRP5 significantly increased cellular cytotoxicity of 5-FU to Patu-02 cells and enhanced accumulation of radioactivity related to 5-FU and its metabolites. Our results suggest that MRP5 is expressed and functionally active and contributes to variable sensitivities of pancreatic adenocarcinoma cell lines to 5-FU. Further investigations using models that resemble human pancreas tumors are necessary to prove a causative relation between expression and activity of MRP5 and tumor resistance to 5-FU.

We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet δ-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet δ-storage pool deficiency (δ-SPD)–like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4gene did not show any mutation that explained the lack of expression. In platelets with “classic” δ-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other δ-granules membrane marker proteins (eg, LAMP2), was mostly displaced from δ-granules to patches at the plasma membrane, suggesting that platelets with classic δ-SPD have an abnormality that impairs the assembly of normal δ-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new δ-SPD phenotype remains to be elucidated.

Statins are widely used to treat dyslipidemia. Effects of statins in addition to low-density lipoprotein lowering include altered platelet aggregation, requiring drug uptake into platelets. Possible candidates for mediating intraplatelet accumulation of statins include members of the organic anion-transporting polypeptide family such as OATP2B1 (SLCO2B1), a high-affinity uptake transporter for atorvastatin. Therefore, we analyzed OATP expression, localization, and function in human platelets. OATP2B1, but not OATP1B1, was detected in platelets and megakaryocytes on transcript and protein levels. Protein localization was almost exclusively confined to the plasma membrane. Moreover, we could demonstrate significant inhibition of estrone sulfate uptake into platelets by atorvastatin as well as direct transport of atorvastatin into platelets using a liquid chromatography-tandem mass spectrometry method. As a consequence of OATP2B1-mediated uptake of atorvastatin, we observed significant atorvastatin-mediated reduction of thrombin-induced Ca2+mobilization in platelets (37.3 ± 6.7% of control at 15 µM atorvastatin), mechanistically explainable by reduced lipid modification of signal proteins. This effect was reversed by addition of mevalonate. Finally, we demonstrated expression of HMG-CoA reductase, the primary target of atorvastatin, in platelet cytosol. In conclusion, OATP2B1 is an uptake transporter expressed in platelets and is involved in statin-mediated alteration of platelet aggregation.

Agonists and antagonists of G protein-coupled receptors are important drugs for the treatment of cardiovascular disease, but the therapeutic response of any given patient remains difficult to predict because of large interindividual variability. Among the factors potentially contributing to such variability, we have reviewed the evidence for a role of pharmacodynamic pharmacogenetics (i.e., polymorphisms in the cognate receptors for such drugs as well as other proteins potentially modifying their action). Based upon the availability of data and the prevalence of use, we have focused on ligands at adrenergic and angiotensin II receptors (including the indirectly acting angiotensin-converting enzyme inhibitors). The vast majority of gene polymorphisms reviewed here have shown only inconsistent effects on drug action, which does not make these polymorphisms useful genetic markers to predict treatment responses. We conclude that considerable additional research, partly involving other types of study than those available now, will be necessary to allow a definitive judgment whether pharmacodynamic pharmacogenetic markers are useful in an individualized approach to cardiovascular therapy. Moreover, we predict that even such additional research will result in only few cases where the promise of tailored treatment can be fulfilled; however, some of these few cases may be of major clinical relevance.

Genetic variability in hepatic uptake was recently shown to influence the disposition and cholesterol-lowering effects of statins. Ezetimibe, an inhibitor of the intestinal cholesterol uptake protein Niemann-Pick C 1 like 1, is another drug for which genetic polymorphisms of hepatic organic anion transporting polypeptides (OATPs) are expected to be of clinical relevance because ezetimibe undergoes intensive enterohepatic circulation for which hepatic uptake transporters may be rate-limiting determinants.

The T-allele of a polymorphism (C825T) in the gene for the G-protein β3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of Gβ3s, a splice variant of GNB3. Gβ3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional Gβ3 splice variant referred to as Gβ3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. Gβ3s2 is generated by alternative splicing using non-canonical splice sites. Gβ subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for Gβ3s2 are lacking 129bp of the coding sequence of the wild-type Gβ3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of Gβ3s. Co-immunoprecipitation analyses indicated that Gβ3s2 dimerizes with different Gγ subunits, e.g. Gγ5, Gγ8C and Gγ12. In Sf9 insect cells, expression of Gβ3s2 together with Gγ12 enhances receptor-stimulated activation of Gαi2. Expression of Gβ3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that Gβ3s2 is a biologically active Gβ variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.

The 825T-allele of the gene GNB3encoding the G protein β3 subunit is associated with hypertension and obesity, and identifies individuals highly responsive to diuretic therapy. Gβ3s, a Gβ3 protein variant generated by alternative splicing in carriers of the 825T-allele, is linked to increased signal transduction and is a potential cause for the observed pathophysiology. Here, we searched the entire GNB3gene for additional polymorphisms and analysed their prevalence in Caucasian, black African and Asian populations. We detected six novel single nucleotide polymorphisms which were termed according to their location as G76A, G1906T, G2906A, A3882C, G5177A, and G5249A. Furthermore, we found a CACA-insertion–deletion polymorphism at position 6496. Genotyping and association studies resulted in the definition of two major GNB3haplotypes, termed ‘C-haplotype’ (alleles 825C, 3882A, 5249G, 6496CACA −) and ‘T-haplotype’ (alleles 825T, 3882C, 5249A, 6496CACA). Molecular modelling studies revealed that the pre-mRNA structures of both haplotypes exhibit marked differences which may account for the alternative splicing predominantly observed with the T-haplotype. The prevalence of these haplotypes in major ethnic populations differs considerably. Furthermore, we detected additional frequent GNB3polymorphisms. These variants were restricted to one or two major ethnic populations. Our results will aid future studies on population-specific effects of the GNB3variants on risk and course of frequent diseases, including hypertension, obesity, stroke and myocardial infarction. Furthermore, they will contribute to the understanding of GNB3-related population-specific pharmacogenetic differences in the response to major drugs, as already shown for diuretics and antidepressants.

Wir konnten kürzlich einen C825T-Polymorphismus im Gen GNB3 nachweisen, das für die Gβ3-Untereinheit heterotrimerer G-Proteine kodiert. Das 825T-Allel verusacht ein alternatives Spleißen des Gens und die Expression einer funktionell aktiven Deletionsvariante von Gβ3, welche als Gβ3s bezeichnet wird. Eine Genotypisierung bezüglich des C825T-Polymorphismus ermöglicht eine Vorhersage über die Stärke der Aktivierbarkeit von G-Proteinen und nachgeschalteter Zellfunktionen.

Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the p3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3–S (encoding Gβ3–s), in which the nucleotides 498–620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the Gβ subunit. By western-blot analysis, Gβ3–s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPγS binding to Sf9 insect cells expressing Gβ3–s together with Ga 2 and Gy5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.

Abstract:  We examined the functional properties of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE) upon stable transfection into PS120 fibroblasts which lack endogenous Na+/H+ exchange. In contrast to untransfected cells, XL-NHE-transfected cells displayed Na+-dependent alkalinization upon acidification with nigericin. XL-NHE activity was inhibited by amiloride, ethylisopropylamiloride, HOE694 [(3-methylsulphonyl-4-piperidinobenzoyl)-guanidine methanesulphonate] and HOE642 [4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulphonate], K i values being calculated at 5 µmol/l, 25 nmol/l, 300 nmol/l and 180 nmol/l, respectively. The Na+ dependence of pHi recovery was compatible with simple Michaelis–Menten kinetics, the K m for Na+ being 22.0±3.2 mmol/l and the Hill coefficient for Na+ being approximately 1. XL-NHE was activated by phorbol ester, whereas forskolin exerted no effect, suggesting the involvement of phospholipase C/protein kinase C signalling pathways rather than protein kinase A signalling pathways in XL-NHE stimulation. Using reverse transcription polymerase chain reaction, XL-NHE message could be detected in various Xenopus tissues including heart, brain, skeletal muscle, reticulocytes, A6-kidney cells and oocytes.

The 825T allele of a common C825T polymorphism in the gene encoding the β3 subunit of heterotrimeric G proteins is associated with enhanced activation of pertussis toxin (PTX)‐sensitive G proteins. We investigated responses of human platelets upon stimulation with epinephrine, which activates PTX‐sensitive G proteins, and with agonists which activate additionally, or exclusively PTX‐insensitive pathways. Slopes and maximum of the secondary aggregation were significantly enhanced in platelets from 825T allele carriers after epinephrine, and after combined epinephrine/ADP. This effect was more pronounced after inhibition of the cyclooxygenase‐2 pathway by acetylsalicylic acid. This phenomenon appeared independent of platelet secretion, or inhibition of the adenylyl cyclase.

Using the reverse polymerase chain reaction (RT-PCR), we have examined the expression of Na +/H +exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection or low abundance Na +/H +antiporter specific transcripts by RT-PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na +/H +exchanger mRNA expression in human cells and tissue samples.

Using the reverse polymerase chain reaction (RT‐PCR), we have examined the expression of Na+/H+exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection or low abundance Na+/H+antiporter specific transcripts by RT‐PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na+/H+exchanger mRNA expression in human cells and tissue samples.

Accumulating evidence suggests an increased activity of the Na—Hexchanger in essential hypertension. The present investigation aimed at developing a test for routine measurements. Platelet-rich plasma was added directly to a cuvette placed into an aggregometer containing 140mmol/l sodium propionate medium (pH 6.7, 37°C). The accumulation of intracellular sodium due to activation of Na- H exchange results in an osmotic cell swelling, which is detectable as a decrease in optical density (OD). This reaction reflects activation of the Na—Hexchanger since we observed (1) a dose-dependent inhibition by amiloride (inhibition constant, Ki= 10µ,mol/l) and ethylisopropylamiloride (Ki= 0.07 µ,mol/l) and (2) a dependence on extracellular sodium of the OD changes. Electron microscopy of sodium propionate-treated platelets revealed a general swelling and a distinct decrease in electron density of the cytosol without other significant alterations. Quantification of Na—Hexchange activities was accomplished by calculating rate constants of the recorded changes in OD. Application of this assay to 20 essential hypertensives and 32 normotensives demonstrated an increased activity of the Na—Hexchanger in essential hypertensives (rate constants 29.8 x 1O-3pers versus 21.7 x 1O-3per s)

Objective: The aim was to investigate the effect of ethylisopropylamiloride (EIPA), an inhibitor of Na+/H+ exchange, and of extracellular pH on the proliferation of immortalised lymphoblasts derived from patients with essential hypertension and normotensive controls. Methods: Na+/H+ exchange activity was determined in cells loaded with the fluorescent pH indicator BCECF. Cell proliferation was determined by FACS analysis, by cell counting, and from the incorporation of [₃H]-thymidine. Results: EIPA inhibited the Na+/H+ exchanger with an average K₁ value of 14 nM in all cell lines. Cell growth and DNA synthesis were only inhibited at EIPA concentrations >10 µM suggesting a non-specific effect independent of Na+/H+ exchanger blockade. When extracellular pH was varied from 7.1 to 7.7 by changing the HCO₃– concentration at constant Pco₂, cell proliferation was optimal at pH 7.4, but reduced at acidic and alkaline pH in cells from normotensive and hypertensive subjects. The increased proliferation of lymphoblasts from hypertensive subjects persisted over the whole pH range. Comparable results were obtained when pH was altered by varying the Pco₂ at constant HCO₃–. Preincubation of cells with pertussis toxin inhibited serum stimulated DNA synthesis by 14.5% and 23.5% (P = 0.02) in cell lines from normotensive and hypertensive subjects. Conclusions: The enhanced Na+/H+ exchanger activity in lymphoblasts from patients with essential hypertension is obviously not the major determinant of the enhanced proliferation of these cells. The increased sensitivity of the growth of "hypersensitive" cell lines to pertussis toxin suggests a cellular alteration which resides upstream of Na+/H+ exchange activity and proliferation control.

Primary hypertension has been reported to be associated with an enhancement of Na−-Hexchange. However, details of the kinetic properties of the Na-Hexchanger in hypertensives and its dependence upon age and gender in normotensives are unknown.