The role of P2Y(14) 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 P2Y(14) receptor (P2Y(14)R) 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, P2Y(1)R, P2Y(12)R, and P2Y(13)R, were similar, and P2Y(11)R and P2Y(4)R were highly expressed at 5.8- and 3.8-fold of P2Y(1)R, respectively. Least expressed P2Y(2)R was 40-fold lower than P2Y(1)R, and P2Y(6)R and P2Y(14)R were ≤50 % of P2Y(1)R. None of the native P2YR agonists alone induced ß-hexosaminidase (ß-Hex) release, but some nucleotides significantly enhanced ß-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y(11)R and P2Y(4)R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y(4)R agonist UTP nor P2Y(11)R agonists ATPγS and NF546 had a substantial effect. P2Y(1)R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y(1)R potency, and the effect of P2Y(6)R 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 P2Y(14)R, MRS2690, enhanced C3a-induced ß-Hex release, which was inhibited by a P2Y(14)R antagonist, specific P2Y(14)R siRNA and pertussis toxin, suggesting a role of P2Y(14)R activation in promoting human mast cell degranulation.

Synthesis, characterization, and direct intracellular imaging of ultrasmall and uniform glutathione-coated gold nanoparticles.

Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and how they behave in the body. Here we report the synthesis of ultrasmall, uniform 144-atom AuNPs protected by p-mercaptobenzoic acid followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH-coated nanoparticles (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles' intracellular fate, the cell-penetrating peptide TAT is attached noncovalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)-TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells.

Molecular Structure of P2Y Receptors: Mutagenesis, Modeling, and Chemical Probes.

There are eight subtypes of P2Y receptors (P2YRs) that are activated, and in some cases inhibited, by a range of extracellular nucleotides. These nucleotides are ubiquitous, but their extracellular concentration can rise dramatically in response to hypoxia, ischemia, or mechanical stress, injury, and release through channels and from vesicles. Two subclasses of P2YRs were defined based on clustering of sequences, second messengers, and receptor sequence analysis. The numbering system for P2YR subtypes is discontinuous; i.e., P2Y(1-14)Rs have been defined, but six of the intermediate-numbered cloned receptor sequences (e.g., P2y(3), P2y(5), P2y(7-10)) are not functional mammalian nucleotide receptors. Of these two clusters, the P2Y(12-14) subtypes couple via Gα(i) to inhibit adenylate cyclase, while the remaining subtypes couple through Gα(q) to activate phospholipase C. Collectively, the P2YRs respond to both purine and pyrimidine nucleotides, in the form of 5'-mono- and dinucleotides and nucleoside-5'-diphosphosugars. In recent years, the medicinal chemistry of P2Y receptors has advanced significantly, to provide selective agonists and antagonists for many but not all of the subtypes. Ligand design has been aided by insights from structural probing using molecular modelling and mutagenesis. Currently, the molecular modelling of the receptors is effectively based on the X-ray structure of the CXCR4 receptor, which is the closest to the P2Y receptors among all the currently crystallized receptors in terms of sequence similarity. It is now a challenge to develop novel and selective P2YR ligands for disease treatment (although antagonists of the P2Y(12)R are already widely used as antithrombotics).

Pyrimidine nucleotides with 4-alkyloxyimino and terminal tetraphosphate δ-ester modifications as selective agonists of the P2Y(4) receptor.

P2Y(2) and P2Y(4) receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We structurally varied phosphate and uracil moieties in analogues of pyrimidine nucleoside 5'-triphosphates and 5'-tetraphosphate esters. P2Y(4) receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N(4)-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5'-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N(4)-(Phenylpropoxy)-CTP 16 (MRS4062), Up(4)-[1]3'-deoxy-3'-fluoroglucose 34 (MRS2927), and N(4)-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y(4) over P2Y(2) and P2Y(6) receptors (EC(50) values 23, 62, and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up(4) activated P2Y(2) but not P2Y(4) receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y(2) and P2Y(4) receptor models indicated greater steric tolerance of N(4)-phenylpropoxy group at P2Y(4). Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y(4) receptor-selective agonists.

Adenine nucleotide control of coronary blood flow during exercise.

The adenine nucleotide hypothesis postulates that the ATP released from red blood cells is broken down to ADP and AMP in coronary capillaries and that ATP, ADP, and AMP act on purinergic receptors on the surface of capillary endothelial cells. Purinergic receptor activation initiates a retrograde conducted vasodilator signal to the upstream arteriole that controls coronary blood flow in a negative feedback manner. A previous study (M. Farias 3rd, M. W. Gorman, M. V. Savage, and E. O. Feigl, Am J Physiol Heart Circ Physiol 288: H1586-H1590, 2005) demonstrated that coronary venous plasma ATP concentration increased during exercise and correlated with coronary blood flow. The present experiments test the adenine nucleotide hypothesis by examining the balance between oxygen delivery (via coronary blood flow) and myocardial oxygen consumption during exercise before and after purinergic receptor blockade. Dogs (n = 7) were chronically instrumented with catheters in the aorta and coronary sinus and a flow transducer around the circumflex coronary artery. During control treadmill exercise, myocardial oxygen consumption increased and the balance between oxygen delivery and myocardial oxygen consumption fell as indicated by a declining coronary venous oxygen tension. Blockade of P1 and P2Y(1) purinergic receptors combined with inhibition of nitric oxide synthesis significantly decreased the balance between oxygen delivery and myocardial oxygen consumption compared with control. The results support the hypothesis that ATP and its breakdown products ADP and AMP are part of a negative feedback control mechanism that matches coronary blood flow to myocardial oxygen consumption at rest and during exercise.