Non-synonymous polymorphisms in the P2RX 4 are related to bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

In the present study we investigated whether single nucleotide polymorphisms (SNPs) in the P2RX₄, which alter the P2X₄R function, are associated with the development of osteoporosis and whether an interaction between the P2X₄R and P2X₇R confer a synergistic effect of these two receptors on osteoporosis risk. Patients with fracture (690 females and 231 males, aged ≥50 years) were genotyped for three non-synonymous P2X₄R SNPs. Bone mineral density (BMD) was measured at the total hip, lumbar spine, and femoral neck. Subject carrying the variant allele of the Tyr315Cys polymorphism showed a 2.68-fold (95 % CI, 1.20–6.02) higher risk of osteoporosis compared with wild-type subject. Furthermore, significant lower lumbar spine BMD values were observed in subjects carrying the Cys315 allele as compared with wild-type (0.85 ± 0.17 and 0.93 ± 0.17 g/cm², respectively; p < 0.001). Assuming a recessive model, carriers of the variant allele of the Ser242Gly polymorphism showed increased BMD values at the lumbar spine compare to wild-type subject (1.11 ± 0.35 and 0.92 ± 0.17 g/cm², respectively; p = 0.0045). This is the first study demonstrating an association of non-synonymous polymorphisms in the P2RX₄ and the risk of osteoporosis, suggesting a role of the P2X₄R in the regulation of bone mass.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9337-0) contains supplementary material, which is available to authorized users.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

CYP2C9 genotype and association with bone mineral density: A pilot study

Background

In recent years reduced bone mineral density (BMD) and osteoporosis have become major public health problems. Single nucleotide polymorphisms (SNPs) in the cytochrome P450 2C9 (CYP2C9) gene influence the response to oral anticoagulant drugs, which are positively associated with the risk to develop osteoporosis. The aim of the present investigation was to clarify a potential role of CYP2C9 sequence variations and susceptibility to develop osteoporosis.

Subjects and methods

Ninety two consecutive angiologic outpatients, mean age: 60.3 ± 14.4, without secondary causes of bone loss were genotyped and classified as patients with normal BMD, osteopenia and osteoporosis according to WHO criteria by dual-energy X-ray absorptiometry at the lumbar spine and/or the femoral neck. Potential association between the CYP2C9 genotype and BMD was tested.

Results

59% of the patients (n = 54) presented with reduced BMD and were compared to 38 age-matched persons with normal BMD. The genotype distribution showed 15% heterozygous for CYP2C9*2 p.Arg144Cys, 14% for CYP2C9*3 p.IIe359Leu, 2% for both polymorphisms, and 69% had wildtype genotypes. Patients with CYP2C9 mutations had significantly lower BMD values at the femoral neck and displayed a four-fold higher adjusted risk to suffer from reduced BMD than individuals with wildtype genotypes (p = 0.02).

Discussion

Oral anticoagulant treatment is common in angiologic outpatients. The gene variants CYP2C9*2 and CYP2C9*3 have been shown to require lower maintenance doses of oral anticoagulant drugs. An association between oral anticoagulant drugs and the susceptibility to develop osteoporosis in relation to sequence variations in the CYP2C9 gene is suggested to be mediated via the glucocorticoid synthesis pathway.

Conclusion

The CYP2C9*2/CYP2C9*3 variants were significantly associated with femoral BMD in a selected elderly Austrian population. These variants could contribute to the complex risk to develop osteoporosis.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y₁₄ receptor (P2Y₁₄R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y₁R, P2Y₁₂R, and P2Y₁₃R, were similar, and P2Y₁₁R and P2Y₄R were highly expressed at 5.8- and 3.8-fold of P2Y₁R, respectively. Least expressed P2Y₂R was 40-fold lower than P2Y₁R, and P2Y₆R and P2Y₁₄R were ≤50 % of P2Y₁R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y₁₁R and P2Y₄R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y₄R agonist UTP nor P2Y₁₁R agonists ATPγS and NF546 had a substantial effect. P2Y₁R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y₁R potency, and the effect of P2Y₆R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y₁₄R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y₁₄R antagonist, specific P2Y₁₄R siRNA and pertussis toxin, suggesting a role of P2Y₁₄R activation in promoting human mast cell degranulation.     

The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss

Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y₁₃ receptor in lipid-induced enteric neuropathy. Littermate P2Y₁₃^+/+ and P2Y₁₃^−/− mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y₁₃^+/+ or P2Y₁₃^−/− mice were exposed to palmitic acid (PA), the P2Y₁₃ receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y₁₃^+/+, but not in P2Y₁₃^−/− mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y₁₃^−/− mice compared with P2Y₁₃^+/+ mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y₁₃^+/+ HFD mice. HFD also caused ileal mucosal thinning in P2Y₁₃^+/+ and P2Y₁₃^−/− mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y₁₃^+/+ mice while neurons from P2Y₁₃^−/− mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y₁₃^+/+ mice. 2meSADP caused no change in survival of cultured neurons. P2Y₁₃ receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y₁₃ receptor stimulation.     

Genetics of the P2X7 receptor and human disease

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.     

SNP combinations in chromosome-wide genes are associated with bone mineral density in Taiwanese women

Osteoporosis is a major public health problem, mainly quantified by low BMD. Eleven polymorphisms were investigated in this study; TNFalpha-857 (rs1799724), TGFbeta1-509 (rs1800469), osteocalcin (rs1800247), TNFalpha-308 (rs1800629), PTH BstB I (rs6254), PTH Dra II (rs6256), IL-1ra (VNTR), HSP70 hom (rs2227956), HSP 70-2 (rs1061581), CTR (rs1801197), and BMP-4 (rs17563). The relationship between the combined polymorphisms in different genomic regions and BMD variation was investigated. Among the female subjects, the proportion of subjects with low BMD in low BMI group (< or = 18.50) was significantly higher than that of the middle (18.51-22.99) and high (> or = 23.00) BMI groups (P < 0.05). In post-menopausal women, there was a significant association between low BMD and genotypes ranging from 2 to approximately 7 SNPs. For two combined SNPs, the portion of subjects with low BMD was significantly higher in those with CC-AA genotypes in rs1799724-rs1800629, compared to those with non-CC-AA genotypes in post-menopausal women and the combination of all women. Similarly, part of the combined SNPs with rs1799724-rs1800629-rs6254-rs6256-IL-1ra-rs2227956-rs1801197 was significantly associated with reduced BMD. After controlling for age and BMI, post-menopausal women with certain specific SNP combination had a 3.54- to 4.68-fold increased risk for low BMD, comparing to other SNP combinations. In conclusion, our data suggest that several gene polymorphisms may be cooperatively involved in the development of osteoporosis.     

The cannabinoid receptor type 2 (<Emphasis Type="Italic">CNR2</Emphasis>) gene is associated with hand bone strength phenotypes in an ethnically homogeneous family sample

Genetic variants within the CNR2 gene encoding the cannabinoid receptor CB2 have been shown to be associated with osteoporosis and low bone mineral density (BMD) in case-control studies. We now examined the association of polymorphisms in CNR2 with hand bone strength in an ethnically homogeneous healthy family sample of European origin (Chuvashians) living in Russia. We show that non-synonymous CNR2 SNPs are significantly associated with radiographic hand BMD and breaking bending resistance index (BBRI) by two different transmission disequilibrium tests. For both tests highly significant p values (ranging from 0.007 to 0.008 for hand BMD, and from 0.001 to 0.003 for BBRI) were also obtained with additional SNPs at the CNR2 locus. The associations remained significant after correction for multiple testing. In conclusion, in addition to the association of CNR2 polymorphisms with low BMD at selected clinically relevant skeletal sites, we now report their significant association with hand bone strength phenotypes using a family-based study design implying an even broader impact of genetic variation at the CNR2 locus on bone structure and function.     

Functional role of the conserved proline in helix 6 of the human bradykinin B2 receptor

Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B₂ receptor (B₂R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B₂R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B₂R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism.     

Combined effects of p53 and MDM2 polymorphisms on susceptibility and surgical prognosis in hepatitis B virus-related hepatocellular carcinoma

The p53 signaling pathway works as a potent barrier to tumor progression. Two single nucleotide polymorphisms (SNPs) in the gene loci of p53 pathway, p53 codon 72 Arg72Pro and MDM2 SNP309 (T>G), have been shown to cause perturbation of p53 function, but the effect of the two SNPs on the risk of hepatocellular carcinoma (HCC) remains inconsistent. This study investigated the influence of combined p53 Arg72Pro and MDM2 SNP309 on the risk of developing HCC in patients with chronic hepatitis B virus infection, and evaluated the significance of the two combined SNPs on patient prognosis. In total, 350 HCC patients, 230 non-HCC patients, and 96 healthy controls were genotyped for the p53 Arg72Pro and MDM2 SNP309. The combined p53 Pro/Pro and MDM2 G/G genotype was significantly associated with HCC risk (P= 0.047). Multivariate analysis indicated that combined p53 Pro/Pro and MDM2 G/G genotype was an independent factor affecting recurrence and survival (P<0.05). Patients with combined p53 Pro/Pro and MDM2 G/G genotypes had a poorer prognosis than other genotypes, P<0.01 for both disease-free survival (DFS) and overall survival (OS). DFS and OS rates also differed significantly between Barcelona Clinic Liver Cancer (BCLC) stage A patients with combined p53 Pro/Pro and MDM2 G/G and other genotypes (P<0.05). Thus, the combined p53 Pro/Pro and MDM2 G/G genotype is associated with increased risk of developing HCC and is an independent adverse prognostic indicator in early stage HCC.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Association analysis of IL-17A and IL-17F polymorphisms in Chinese Han women with breast cancer

Background

Research into the etiology of breast cancer has recently focused on the role of the immunity and inflammation. The proinflammatory cytokines IL-17A and IL-17F can mediate inflammation and cancer. To evaluate the influences of IL-17A and IL-17F gene polymorphisms on the risk of sporadic breast cancer, a case-control study was conducted in Chinese Han women.

Methodology and Principal Findings

We genotyped three single-nucleotide polymorphisms (SNPs) in IL-17A (rs2275913, rs3819025 and rs3748067) and five SNPs in IL-17F (rs7771511, rs9382084, rs12203582, rs1266828 and rs763780) to determine the haplotypes in 491 women with breast cancer and 502 healthy individuals. The genotypes were determined using the SNaPshot technique. The differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed with the Chi-square test for trends. For rs2275913 in IL-17A, the frequency of the AA genotype was higher in patients than controls (P = 0.0016). The clinical features analysis demonstrated significant associations between IL-17 SNPs and tumor protein 53 (P53), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2) and triple-negative (ER-/PR-/Her-2-) status. In addition, the haplotype analysis indicated that the frequency of the haplotype A_rs2275913G_rs3819025G_rs3748067, located in the IL-17A linkage disequilibrium (LD) block, was higher in patients than in controls (P = 0.0471 after correction for multiple testing).

Conclusions and Significance

Our results suggested that SNPs in IL-17A but not IL-17F were associated with the risk of breast cancer. Both IL-17A and IL-17F gene polymorphisms may provide valuable information for predicting the prognosis of breast cancer in Chinese women.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A_2A and A₃ receptor expression was observed in the arterial wall and A_2A-immunoreactivity was identified in the adventitia–media junction and endothelium. A₁ and A_2B receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA) = {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A_2A antagonist, reduced NECA relaxations that were not modified by A₁, A_2B, and A₃ receptor antagonists. Neuronal voltage-gated Ca²⁺ channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK_Ca)- and small (SK_Ca)-conductance Ca²⁺-activated K⁺ channels. Inhibition of cyclooxygenase (COX), large-conductance Ca²⁺-activated-, ATP-dependent-, and voltage-gated-K⁺ channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A_2A purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK_Ca and SK_Ca channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.     

CFH, VEGF, and PEDF genotypes and the response to intravitreous injection of bevacizumab for the treatment of age-related macular degeneration

We determined whether there is an association between complement factor H (CFH), high-temperature requirement A-1 (HTRA1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF) genotypes and the response to treatment with a single intravitreous injection of bevacizumab for age-related macular degeneration (AMD). Eighty-three patients with exudative AMD treated by bevacizumab injection were genotyped for three single nucleotide polymorphisms (SNPs; rs800292, rs1061170, rs1410996) in the CFH gene, a rs11200638-SNP in the HTRA1 gene, three SNPs (rs699947, rs1570360, rs2010963) in the VEGF gene, and four SNPs (rs12150053, rs12948385, rs9913583, rs1136287) in the PEDF gene using a TaqMan assay. The CT genotype (heterozygous) of CFH-rs1061170 was more frequently represented in nonresponders in vision than TT genotypes (nonrisk allele homozygous) at the time points of 1 and 3 months, while there was no CC genotype (risk allele homozygous) in our study cohort (p = 7.66 × 10⁻³, 7.83 × 10⁻³, respectively). VEGF-rs699947 was also associated with vision changes at 1 month and PEDF-rs1136287 at 3 months (p = 5.11 × 10⁻³, 2.05 × 10⁻², respectively). These variants may be utilized for genetic biomarkers to estimate visual outcomes in the response to intravitreal bevacizumab treatment for AMD.     

Relationship between leptin receptor and polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders, which is involved in the multi-system disease, and its etiology is still not clearly understood. It is currently considered that not only the genetic factors but also the environment factors play a crucial role in the pathogenesis of PCOS. Obesity plays an important role through the insulin, leptin and endocannabinoid system in the pathological process of PCOS, leading to more severe clinical manifestations. The aim of our present study is to investigate whether there is association between single nucleotide polymorphisms (SNPs) of Gln223Arg and Pro1019Pro in the leptin receptor gene (LEPR) and PCOS in a Korean population. Interestingly, a significant association was found between the Pro1019Pro in LEPR gene and PCOS, and a highly significant association was found between the Gln223Arg in LEPR gene and PCOS (P = 0.033, OR = 1.523, 95% confidence interval and P < 0.0001, OR = 0.446, 95% confidence interval). Moreover, genotype combination and haplotype analyses indicate that Gln223Arg and Pro1019Pro polymorphisms of LEPR are significantly associated with the risk of PCOS.     

The role of vitamin D receptor gene polymorphisms in the bone mineral density of Greek postmenopausal women with low calcium intake

The aim of this study was to investigate the effect of common vitamin D receptor (VDR) gene polymorphisms on the bone mineral density (BMD) of Greek postmenopausal women. Healthy postmenopausal women (n=578) were recruited for the study. The BMD of the lumbar spine and hip was measured using dual-energy X-ray absorptiometry with the Lunar DPX-MD device. Assessment of dietary calcium intake was performed with multiple 24-h recalls. Genotyping was performed for the BsmI, TaqI and Cdx-2 polymorphisms of the VDR gene. The selected polymorphisms were not associated with BMD, osteoporosis or osteoporotic fractures. Stratification by calcium intake revealed that in the low calcium intake group (<680 mg/day), all polymorphisms were associated with the BMD of the lumbar spine (P<.05). After adjustment for potential covariates, BsmI and TaqI polymorphisms were associated with the presence of osteoporosis (P<.05), while the presence of the minor A allele of Cdx-2 polymorphism was associated with a lower spine BMD (P=.025). In the higher calcium intake group (>680 mg/day), no significant differences were observed within the genotypes for all polymorphisms. The VDR gene is shown to affect BMD in women with low calcium intake, while its effect is masked in women with higher calcium intake. This result underlines the significance of adequate calcium intake in postmenopausal women, given that it exerts a positive effect on BMD even in the presence of negative genetic predisposition.     

Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries

Receptors for purines and pyrimidines are expressed throughout the cardiovascular system. This study investigated their functional expression in porcine isolated pancreatic arteries. Pancreatic arteries (endothelium intact or denuded) were prepared for isometric tension recording and preconstricted with U46619, a thromboxane A₂ mimetic; adenosine-5′-diphosphate (ADP), uridine-5′-triphosphate (UTP) and MRS2768, a selective P2Y₂ agonist, were applied cumulatively, while adenosine-5′-triphosphate (ATP) and αβ-methylene-ATP (αβ-meATP) response curves were generated from single concentrations per tissue segment. Antagonists/enzyme inhibitors were applied prior to U46619 addition. ATP, αβ-meATP, UTP and MRS2768 induced vasoconstriction, with a potency order of αβ-meATP > MRS2768 > ATP ≥ UTP. Contractions to ATP and αβ-meATP were blocked by NF449, a selective P2X1 receptor antagonist. The contraction induced by ATP, but not UTP, was followed by vasorelaxation. Endothelium removal and DUP 697, a cyclooxygenase-2 inhibitor, had no significant effect on contraction to ATP but attenuated that to UTP, indicating actions at distinct receptors. MRS2578, a selective P2Y₆ receptor antagonist, had no effect on contractions to UTP. ADP induced endothelium-dependent vasorelaxation which was inhibited by MRS2179, a selective P2Y₁ receptor antagonist, or SCH58261, a selective adenosine A_2A receptor antagonist. The contractions to ATP and αβ-meATP were attributed to actions at P2X1 receptors on the vascular smooth muscle, whereas it was shown for the first time that UTP induced an endothelium-dependent vasoconstriction which may involve P2Y₂ and/or P2Y₄ receptors. The relaxation induced by ADP is mediated by P2Y₁ and A_2A adenosine receptors. Porcine pancreatic arteries appear to lack vasorelaxant P2Y₂ and P2Y₄ receptors.