The nucleotide-sugar-activated P2Y 14 receptor (P2Y 14 -R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by Gauthier et al. ) that exhibited antagonist activity at the P2Y 14 -R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y 14 -R was shifted to the right in a concentration-dependent manner by PPTN.Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a K B of 434 pM observed. In contrast, 1 mM PPTN exhibited no agonist or antagonist effect at the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , or P2Y 13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the K B determined with recombinant P2Y 14 -R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y 14 -R that is useful for interrogating the action of this receptor in physiologic systems.

The P2Y 14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y 14 receptor, allowing facile examination of its coupling to native G i family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330: [162][163][164][165][166][167][168] 2009). In the current study, we examined P2Y 14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y 14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC 50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y 14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC 50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y 14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y 14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y 14 receptor over the UDP-activated P2Y 6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y 14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y 14 receptor.The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, that primarily signal through G q -activated signaling pathways and a subgroup of three receptors, the P2Y 12 , P2Y 13 , and P2Y 14 receptors, that primarily signal by activating heterotrimeric G proteins of the G i family (Abbracchio et al., 2006;Burnstock, 2007). The human P2Y 1 , P2Y 11 , P2Y 12 , and P2Y 13 receptors are activated by adenine nucleotides. The human P2Y 4 and P2Y 6 receptors are activated by uridine nucleotides, and the P2Y 2 receptor is activated by both ATP and UTP.

The P2Y14 receptor (P2Y14R), one of eight P2Y G protein-coupled receptors (GPCR), is involved in inflammatory, endocrine, and hypoxic processes and is an attractive pharmaceutical target. The goal of this research is to develop high-affinity P2Y14R fluorescent probes based on the potent and highly selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (6, PPTN). A model of hP2Y14R based on recent hP2Y12R X-ray structures together with simulated antagonist docking suggested that the piperidine ring is suitable for fluorophore conjugation while preserving affinity. Chain-elongated alkynyl or amino derivatives of 6 for click or amide coupling were synthesized, and their antagonist activities were measured in hP2Y14R-expressing CHO cells. Moreover, a new Alexa Fluor 488 (AF488) containing derivative 30 (MRS4174, Ki = 80 pM) exhibited exceptionally high affinity, as compared to 13 nM for the alkyne precursor 22. A flow cytometry assay employing 30 as a fluorescent probe was used to quantify specific binding to P2Y14R. Known P2Y receptor ligands inhibited binding of 30 with properties consistent with their previously established receptor selectivities and affinities. These results illustrate that potency in this series of 2-naphthoic acid derivatives can be preserved by chain functionalization, leading to highly potent fluorescent molecular probes for P2Y14R. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. This approach demonstrates the predictive power of GPCR homology modeling and the relevance of newly determined X-ray structures to GPCR medicinal chemistry.

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.

We previously synthesized a series of potent and selective A 3 adenosine receptor (AR) agonists (North-methanocarba nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A 3 AR activation was preserved in these multivalent conjugates, which bound with apparent K i 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A 3 AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A 3 and P2Y 14 receptors (via amide-linked uridine-5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

The P2Y 6 receptor is a cytoprotective G protein-coupled receptor (GPCR) activated by UDP (EC 50 , 0.30 μM). We compared and combined modifications to enhance P2Y 6 receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as α,β-methylene and extension of the terminal phosphate group into γ-esters of UTP analogues. The conformationally constrained (S)-methanocarba UDP is a full agonist (EC 50 0.042 μM). 4-Methoxyimino modification of pyrimidine enhanced P2Y 6 , preserved P2Y 2 and P2Y 4 , and abolished P2Y 14 receptor potency, in the appropriate nucleotide. N 4 -Benzyloxy-CDP (15, MRS2964) and N 4 -methoxy-Cp 3 U (23, MRS2957) were potent, selective P2Y 6 receptor agonists (EC 50 0.026 μM and 0.012 μM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-γ-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y 6 receptor potency, but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N 4 -methoxy-cytidine 5′-triphospho-γ-[1]glucose were active (EC 50 of 2.47 μM and 0.18 μM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y 6 receptor agonists may be enhanced by modest structural changes.

Background and Purpose Previous research suggests that patients receiving inpatient stroke rehabilitation are sedentary although there is little data to confirm this supposition within the Canadian healthcare system. The purpose of this cross-sectional study was to observe two weeks of inpatient rehabilitation in a tertiary stroke center to determine patients' activity levels and sedentary time. Methods Heart rate (HR) and accelerometer data were measured using an Actiheart monitor for seven consecutive days, 24 h/day, on the second week and the last week of admission. Participants or their proxies completed a daily logbook. Metabolic equivalent (MET) values were calculated and time with MET < 1.5 was considered sedentary. The relationship between patient factors (disability, mood, and social support) and activity levels and sedentary time were analyzed. Results Participants (n = 19; 12 males) spent 10 h sleeping and 4 h resting each day, with 86.9% of their waking hours sedentary. They received on average 8.5 task-specific therapy sessions; substantially lower than the 15 h/week recommended in best practice guidelines. During therapy, 61.6% of physical therapy and 76.8% of occupational therapy was spent sedentary. Participants increased their HR about 15 beats from baseline during physical therapy and 8 beats during occupational therapy. There was no relationship between sedentary time or activity levels and patient factors. Discussion Despite calls for highly intensive stroke rehabilitation, there was excessive sedentary time and therapy sessions were less frequent and of lower intensity than recommended levels. Conclusions In this sample of people attending inpatient stroke rehabilitation, institutional structure of rehabilitation rather than patient-related factors contributed to sedentary time.

Background: Phospholipase C-␤ (PLC-␤) isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate to propagate signals for several physiological responses. Results: Membranes are essential for the allosteric release of autoinhibition of PLC-␤ isozymes. Conclusion: Activators of PLC-␤ release autoinhibition by orientating the isozymes at the membrane. Significance: The model described provides a better understanding of PLC-␤ regulation and potential mechanisms to inhibit their activation.