Taller height as a risk factor for venous thromboembolism: a Mendelian randomization meta-analysis.

Essentials Observational data suggest taller people have a higher risk of venous thromboembolism (VTE). We used Mendelian randomization techniques to further explore this association in three studies. Risk of VTE increased by 30-40% for each 10 cm increment in height. Height was more strongly associated with deep vein thrombosis than with pulmonary embolism. SUMMARY: Background Taller height is associated with a greater risk of venous thromboembolism (VTE). Objectives To use instrumental variable (IV) techniques (Mendelian randomization) to further explore this relationship. Methods Participants of European ancestry were included from two cohort studies (Atherosclerosis Risk in Communities [ARIC] study and Cardiovascular Health Study [CHS]) and one case-control study (Mayo Clinic VTE Study [Mayo]). We created two weighted genetic risk scores (GRSs) for height; the full GRS included 668 single-nucleotide polymorphisms (SNPs) from a previously published meta-analysis, and the restricted GRS included a subset of 362 SNPs not associated with weight independently of height. Standard logistic regression and IV models were used to estimate odds ratios (ORs) for VTE per 10-cm increment in height. ORs were pooled across the three studies by the use of inverse variance-weighted random effects meta-analysis. Results Among 9143 ARIC and 3180 CHS participants free of VTE at baseline, there were 367 and 109 incident VTE events. There were 1143 VTE cases and 1292 controls included from Mayo. The pooled ORs from non-IV models and models using the full and restricted GRSs as IVs were 1.27 (95% confidence interval [CI] 1.11-1.46), 1.34 (95% CI 1.04-1.73) and 1.45 (95% CI 1.04-2.01) per 10-cm greater height, respectively. Conclusions Taller height is associated with an increased risk of VTE in adults of European ancestry. Possible explanations for this association, including taller people having a greater venous surface area, a higher number of venous valves, or greater hydrostatic pressure, need to be explored further.

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1.

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER(+) breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

BACKGROUND AND PURPOSE: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. EXPERIMENTAL APPROACH: The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. KEY RESULTS: The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. CONCLUSIONS AND IMPLICATIONS: Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

Novel interactions between the 5-HT transporter, 5-HT1B receptors and Rho kinase in vivo and in pulmonary fibroblasts.

BACKGROUND AND PURPOSE: While the 5-HT and Rho-kinase (ROCK) pathways have been implicated in the development of pulmonary arterial hypertension (PAH), the nature of any interactions between them remain unclear. This study investigated a role for ROCK in 5-HT-regulated proliferative responses in lung fibroblasts in vivo and in vitro. EXPERIMENTAL APPROACH: PAH was examined in mice over-expressing human 5-HT transporters (SERT+), from which pulmonary artery fibroblasts (PFs) were isolated to assess ROCK expression. In vitro analysis of 5-HT signalling employed CCL39 hamster lung fibroblasts. KEY RESULTS: ROCK inhibition ablated increased pulmonary remodelling and hypertension observed in SERT+ mice, and ROCK1/2 protein levels were elevated in SERT+ PFs. ROCK inhibition also reduced 5-HT-stimulated proliferation by suppressing MEK-stimulated ERK phosphorylation. While optimal 5-HT-stimulated proliferation required 5-HT(1B) and 5-HT(2A) receptors and SERT, receptor sensitivity to Y27632 was restricted to the 5-HT(1B) receptor. Also, while hypoxia-induced pulmonary vascular remodelling and hypertension were sensitive to Y27632 in WT and SERT+ animals, the proportions sensitive to ROCK inhibition were increased by SERT over-expression. CONCLUSIONS AND IMPLICATIONS: SERT over-expression increased ROCK-dependent pulmonary remodelling in normoxia and hypoxia and SERT over-expression was associated with elevated ROCK1/2 levels. ROCK also potentiated 5-HT(1B) receptor-stimulated ERK activation and proliferation in vitro by facilitating MEK-ERK interaction.

Suppression of inflammatory and immune responses by the A(2A) adenosine receptor: an introduction.

The purine nucleoside adenosine has been described as a 'retaliatory metabolite' by virtue of its ability to function in an autocrine manner to modify the activity of a range of cell types following its extracellular accumulation during cell stress or injury. These effects are largely protective and are triggered by the binding of adenosine to any of four G-protein-coupled adenosine receptors. Most of the anti-inflammatory effects of adenosine have been assigned to the adenosine A(2A) receptor subtype, which is expressed in many immune and inflammatory cells. In this brief article, we will outline the growing evidence to support the hypothesis that the development of agonists selective for the A(2A) receptor is an effective strategy for suppressing the exaggerated inflammatory responses associated with many diseases by virtue of the receptor's ability to inhibit multiple pro-inflammatory signalling cascades.

Inhibition of pro-inflammatory cytokine receptor signalling by cAMP in vascular endothelial cells.

The anti-inflammatory effects of the prototypical second messenger cAMP have been extensively documented in multiple cell types. However, in many instances, the molecular mechanisms by which cAMP elevation disrupts specific pro-inflammatory signalling cascades are unknown. In this review, we will describe the importance of the JAK-STAT (where JAK stands for Janus kinase and STAT for signal transducer and activator of transcription) signalling pathway in vascular endothelial cell function, outline key inhibitory processes that serve to reduce cytokine-stimulated tyrosine phosphorylation and activation of STAT proteins, and discuss possible mechanisms by which intracellular cAMP sensors could interact with these inhibitory processes to diminish cytokine receptor-mediated pro-inflammatory signalling.

The role of pit-and-fissure discoloration in caries assessment.

Occlusal dental caries often is not apparent using traditional diagnostic techniques. The clinical significance of pit-and-fissure discoloration in the absence of dietary and habit substances was examined. The study included 462 extracted teeth (216 with obvious pit-and-fissure discoloration, 216 with subtle discoloration, and 30 with no discoloration); 130 clinical teeth with varying degrees of pit-and-fissure discoloration; and 159 teeth in young adults, which had sealants placed > or = 10 years. All teeth studied were excavated very conservatively using air abrasion and/or the uniquely small H1 004 carbide bur. Presence of caries and its depth and extent were recorded, photographed, and measured with a custom made calibrated probe. Of the 721 teeth with discolored pits and fissures studied (432 extracted teeth and 289 clinical teeth), 660 (92%) had two or more of the four clinical criteria used to define dental caries in this study. Sixty-four percent of the lesions were > 2 mm in depth and 27% were > 3 mm in depth. Of the 159 teeth sealed for > or = 10 years, 47 (92%) were carious, and 26% had large, deep carious lesions penetrating > 3 mm. These data indicate: more effective methods are needed to diagnose pit-and-fissure caries and presence of pit-and-fissure discoloration in the absence of substances causing extrinsic staining should be a strong warning for clinicians to examine carefully for dental caries.

Identification of threonine residues controlling the agonist-dependent phosphorylation and desensitization of the rat A(3) adenosine receptor.

Activation of the A(3) adenosine receptor (A(3)AR) contributes to the cardioprotective, bronchoconstrictive, and hypotensive effects of adenosine. Agonist occupation of the A(3)AR results in a rapid desensitization of receptor function, which is associated with the phosphorylation of the receptor protein by one or more members of the G protein-coupled receptor kinase family of protein kinases. Although we demonstrated previously that phosphorylation of the C-terminal 14 amino acids of the rat A(3)AR is crucial for rapid desensitization to occur, the identity of the critical phosphorylation sites has remained unknown. Here, we demonstrate that the simultaneous mutation of Thr(307), Thr(318), and Thr(319) to Ala residues dramatically reduces agonist-stimulated phosphorylation and rapid desensitization of the rat A(3)AR. Individual mutation of each residue demonstrated that Thr(318) and Thr(319) are the major sites of phosphorylation. Phosphorylation at Thr(318) appeared to be necessary to observe phosphorylation at Thr(319), but not vice versa. However, the replacement of Thr(318) with a glutamate residue demonstrated that the simple addition of negative charge at position 318 was not sufficient to rescue phosphorylation at position 319. In addition, the mutation of two predicted palmitoylation-site cysteine residues proximal to the regulatory domain resulted in the appearance of an agonist-independent basal phosphorylation. Therefore, G protein-coupled receptor kinase-mediated phosphorylation of the C-terminal tail of the A(3)AR in situ appears to follow a sequential mechanism, perhaps involving receptor depalmitoylation, with phosphorylation at Thr(318) being particularly important.

Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases.

Despite coupling to the same class of inhibitory G proteins and binding the same physiological ligand, the human A(1) and rat A(3) adenosine receptors (ARs) desensitize at different rates in response to sustained agonist exposure. This is due to the ability of the A(3)AR, but not the A(1)AR, to serve as a substrate for rapid phosphorylation and desensitization by members of the G protein-coupled receptor kinase (GRK) family. The aim of this study was to investigate whether these differences were also manifested in their abilities to undergo agonist-dependent receptor internalization. For the first time, we report that A(3)ARs internalize profoundly in response to short-term exposure to agonist but not activators of second messenger-regulated kinases. The A(3)AR-selective antagonist MRS1523 blocked both A(3)AR phosphorylation and internalization. Moreover, in contrast to the A(1)AR, which internalized quite slowly (t(1/2) = 90 min), A(3)ARs internalized rapidly (t(1/2) = 10 min) over a time frame that followed the onset of receptor phosphorylation. A nonphosphorylated A(3)AR mutant failed to internalize over a 60-min time course, suggesting that receptor phosphorylation was essential for rapid A(3)AR internalization to occur. In addition, fusion onto the A(1)AR of the A(3)AR C-terminal domain containing the sites for phosphorylation by GRKs conferred rapid agonist-induced internalization kinetics (t(1/2) = 10 min) on the resulting chimeric AR. In conclusion, these data suggest that GRK-stimulated phosphorylation of threonine residues within the C-terminal domain of the A(3)AR is obligatory to observe rapid agonist-mediated internalization of the receptor.

Short-term dynamics of an acacia ant community in Laikipia, Kenya.

In monospecific stands of Acacia drepanolobium in Laikipia, Kenya, virtually all but the smallest trees are occupied by one of four species of ants. Although trees are a limiting resource, all four ant species are maintained in this system. Three separate lines of evidence confirm a linear dominance hierarchy among these four ants: (1) experimentally staged conflicts, (2) natural transitions among 1773 tagged trees over a 6-month period, and (3) the average sizes of trees occupied by ants of different species. Short-term dynamics during a drying period reveal that many smaller trees (<1 m) occupied by dominant ants were subsequently abandoned, and that abandoned trees had grown more slowly than those that were not abandoned. Height growth increments over 6 months were generally independent of ant occupant, but increased with tree height. Among taller trees (>1 m), changes in ant occupation congruent with the dominance hierarchy (i.e., transitions from more subordinate ant species to more dominant ant species) occurred on trees that grew faster than average. In contrast, the (less frequent) changes in ant occupation "against" the direction of the dominance hierarchy occurred on trees that grew more slowly than average. Observed correlations between tree vigor and takeover direction suggest that colony growth of dominant ant species is either favored in more productive microhabitats, or that such colonies differentially seek out healthier trees for conquest. Colonies of dominant species may differentially abandon more slowly growing trees during (dry) periods of retrenchment, or suffer higher mortality on these trees. Subordinate ant species appear to move onto these abandoned trees and, to a lesser extent, colonize new recruits in the sapling class. These data reveal that within a simple linear dominance hierarchy, short-term variations exist that may reveal underlying mechanisms associated with coexistence.

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.

Resin polymerization problems--are they caused by resin curing lights, resin formulations, or both?

Negative effects of rapid, high-intensity resin curing have been predicted for both argon lasers and plasma-arc curing lights. To address these questions, six different resin restorative materials were cured with 14 different resin curing lights representing differences in intensities ranging from 400 mW/cm2 to 1,900 mW/cm2; delivery modes using constant, ramped, and stepped methods; cure times ranging from 1 second to 40 seconds; and spot sizes of 6.7 mm to 10.9 mm. Two lasers, five plasma-arc lights, and seven halogen lights were used. Shrinkage, modulus, heat generation, strain, and physical changes on the teeth and resins during strain testing were documented. Results showed effects associated with lights were not statistically significant, but resin formulation was highly significant. Microfill resins had the least shrinkage and the lowest modulus. An autocure resin had shrinkage and modulus as high as or higher than the light-cured hybrid resins. Lasers and plasma-arc lights produced the highest heat increases on the surface (up to 21 degrees C) and within the resin restorations (up to 14 degrees C), and the halogen lights produced the most heat within the pulp chamber (up to 2 degrees C). Strain within the tooth was least with Heliomolar and greatest with Z100 Restorative and BISFIL II autocure resin. Clinical effects of strain relief were evident as white lines at the tooth-resin interface and cracks in enamel adjacent to the margins. This work implicates resin formulation, rather than light type or curing mode, as the important factor in polymerization problems. Lower light intensity and use of ramped and stepped curing modes did not provide significant lowering of shrinkage, modulus, or strain, and did not prevent enamel cracking adjacent to margins and formation of "white line" defects at the margins. Until materials with lower shrinkage and modulus are available, use of low-viscosity surface sealants as a final step in resin placement is suggested to seal defects.

Structure-function analysis of inhibitory adenosine receptor regulation.

Pharmacological and molecular cloning studies have revealed the presence of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3. Given that the A1 and A3ARs can both bind adenosine and couple productively to inhibitory G-proteins, the significance of the existence of multiple inhibitory AR subtypes remains obscure, although one possibility is that these receptors are regulated in a subtype-specific manner. In this review, we summarize our investigations into the mechanisms underlying the agonist-induced desensitization of inhibitory AR function. The results of this work demonstrate that while the A1AR desensitizes slowly over a time course of several hours, the A3AR desensitizes within minutes of agonist exposure. Molecular biological studies have begun to delineate the structural requirements responsible for these differences, and will provide a basis for future experiments designed to determine whether the ability of an inhibitory AR receptor subtype to 'turn-off' at a specific rate has implications for the physiological role of that receptor.

Induction of multiple effects on adenylyl cyclase regulation by chronic activation of the human A3 adenosine receptor.

The A3 adenosine receptor (A3AR) contributes to several cardiovascular effects of adenosine, including antihypertensive and cardioprotective effects. Although several studies have detailed the mechanisms underlying agonist-mediated desensitization of the rat A3AR, the regulation of the human A3AR, which displays only a 70% amino acid identity with the rat homologue, has not been addressed. Using a Chinese hamster ovary cell line stably expressing a recombinant human A3AR, we demonstrated that prolonged treatment with the AR agonist 5'-N-ethylcarboxamidoadenosine induces uncoupling of the A3AR from G proteins and functional desensitization. In addition to A3AR desensitization, a 1.5-2.5-fold increase was noted in the adenylyl cyclase (AC) activity achieved in the presence of GTP with or without forskolin. This sensitization of AC activity was not a consequence of the down-regulation of Gi proteins induced by NECA treatment and was not associated with sustained or transient increases in the expression of Gs. Time course experiments revealed that the onset of sensitization was half-maximal between 2 and 3 hr but was not due to the synthesis of new proteins because cycloheximide treatment failed to inhibit sensitization. The inability of the sensitization process to alter the AC activity obtained in the presence of manganese chloride suggests that prolonged A3AR activation increases the coupling efficiency between Gs and AC catalytic units. This phenomenon has implications for long term cellular adaptation to agonist because in agonist-treated cells, the extent to which a suboptimal concentration of forskolin could increase phosphorylation of the cAMP-responsive element binding protein was elevated compared with vehicle-treated controls.

Signalling enzymes: bursting with potential.

A newly identified form of phosphoinositide 3-kinase is regulated by G beta gamma subunits and is particularly abundant in phagocytic leukocytes. It is likely to be intimately involved in the process by which inflammatory signals regulate phagocyte activation and is a potential target for new anti-inflammatory drugs.

Identification of an A2a adenosine receptor domain specifically responsible for mediating short-term desensitization.

In an attempt to delineate the structural requirements necessary for agonist-induced desensitization, we have stably expressed wild-type and mutant A2a adenosine receptors (A2aARs) in Chinese hamster ovary cells and examined the effects of agonist pretreatment on adenylyl cyclase activity in subsequently isolated membranes. Deletion of 95 amino acids from the carboxyl-terminus of the A2aAR, thereby removing 10 potential phosphorylation sites, failed to alter radioligand binding, adenylyl cyclase activation, or functional desensitization parameters as compared with the wild-type receptor. However, simultaneous mutation of Thr 298 and Ser 305 to Ala residues attenuated the desensitization observed in response to short-term (30 min) agonist treatment while not blocking the ability to desensitize after long-term (24 h) agonist exposure. Individual mutation of these residues revealed that mutation of Thr 298 alone was sufficient to diminish both short-term desensitization and agonist-stimulated receptor phosphorylation. These data suggest that while the majority of the A2aAR carboxyl-terminus is dispensable with regard to observing functional desensitization, the integrity of Thr 298 is essential for observing agonist-stimulated receptor phosphorylation and short-term desensitization but not long-term desensitization of A2aAR function.

Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors. Analysis of a chimeric A1-A3 adenosine receptor.

The differing effects of short-term agonist exposure on the two inhibitory adenosine receptor (AR) subtypes have been examined using Chinese hamster ovary cells stably expressing the hemagglutinin epitope-tagged human A1AR and rat A3AR. Under conditions in which exposure of transfected cells to 5 microM (-)-(R)-N6-(phenylisopropyl)adenosine resulted in the functional desensitization and phosphorylation of the A3AR, neither property was exhibited by the A1AR. However, a stably expressed chimeric A1-A3AR, termed A1CT3AR, in which the C-terminal domain of the A1AR distal to its predicted palmitoylation site was replaced by the corresponding region of the A3AR, was able to undergo functional desensitization and agonist-stimulated phosphorylation in a manner similar to that exhibited by the A3AR. Moreover, purified G-protein-coupled receptor kinases 2, 3, and 5 were each capable of enhancing the agonist-dependent phosphorylation of the A3AR and A1CT3AR in vitro. Taken together, these data demonstrate that the C-terminal domain of the A3AR distal to its predicted palmitoylation site is responsible for this receptor's ability to undergo a rapid agonist-dependent desensitization and are consistent with a model in which phosphorylation of the A3AR within this domain by one or more G-protein-coupled receptor kinases initiates the desensitization process.

125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor.

The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.

Agonist-dependent phosphorylation and desensitization of the rat A3 adenosine receptor. Evidence for a G-protein-coupled receptor kinase-mediated mechanism.

A3 adenosine receptor (A3AR) activation contributes to both the cardioprotective and antihypertensive effects of adenosine. To date, no studies have examined the mechanisms by which this receptor undergoes rapid homologous desensitization. Therefore, a functional hemagglutinin epitope-tagged A3AR has been stably expressed in Chinese hamster ovary cells, and its regulation by the AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) has been studied. Cellular exposure to NECA induces rapid (t1/2 = approximately 1 min) A3AR phosphorylation on serine and threonine residues. This is associated with a functional desensitization and a 30-40% reduction in the number of high affinity agonist binding sites as determined by radioligand binding assays. Activation of second messenger-regulated kinases could not mimic the effect of NECA, suggesting a role for G-protein-coupled receptor kinases (GRKs). In vitro phosphorylation assays demonstrate that phosphorylation of agonist-occupied A3ARs is enhanced by GRK2 and that cellular pretreatment with NECA dramatically inhibits subsequent GRK2-mediated phosphorylation in vitro. Therefore, the A3AR is phosphorylated in situ by a kinase similar or identical to GRK2, and this may be involved in rapid functional desensitization of the A3AR.