Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (⌬Cyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing ⌬Cyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of ⌬Cyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in ⌬Cyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in ⌬Cyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and ⌬Cyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase C␤3 in Wt and ⌬Cyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of ⌬Cyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase C␤3.Chemokines are a group of proteins that mediate directed migration and activation of leukocytes (1). These have been classified into two main families, CXC or CC-chemokines. Two newly identified proteins define additional groups of C and CX3C chemokines based on the position of conserved cysteines (2, 3). Both CC and CXC chemokines bind to seven transmembrane G-protein-coupled receptors which transduce signals through heterotrimeric G-proteins (4). Several recent studies showed that chemokines play a significant role in human immunodeficiency virus (HIV-1) 1 infection and that the chemokine receptors CCR5 and CXCR4 along with CD4 act as major co-receptors for the macrophage tropic and T-cell tropic HIV-1 strains entry, respectively, into target cells (5-10). While the C-C chemokines such as MIP1␣, MIP1␤, and regulated upon activation, normal T expressed and secreted can activate CCR5 the CXC chemokine SDF-1 is the ligand for CXCR4 (11-13). Recently, CD4 independent infection by HIV-2 was shown to be mediated by CXCR4 (14). HIV-1 envelope glycoproteins interact with chemokine receptors in a CD4-dependent and in one case...

Platelet-activating factor (PAF) stimulates a diverse array of cellular responses through receptors coupled to G proteins that activate phospholipase C (PLC). Truncation of the cytoplasmic tail of the receptor to remove phosphorylation sites (mutant PAF receptor, mPAFR) results in enhancement of PAF-stimulated responses. Here we demonstrate that PAF or phorbol 12-myristate 13-acetate (PMA) pretreatment inhibited wild type PAFR-induced PLC-mediated responses by ϳ90%, whereas these responses to the phosphorylation-deficient mPAFR were inhibited by ϳ50%, despite normal G protein coupling, suggesting a distal inhibitory locus. PAF and PMA, as well as a membrane permeable cyclic AMP analog, stimulated phosphorylation of PLC␤3. A protein kinase C (PKC) inhibitor blocked phosphorylation of PLC␤3 stimulated by PAF and PMA but not by cAMP. Activation of protein kinase A (PKA) by cAMP did not result in inhibition of Ca 2؉ mobilization stimulated by PAF. In contrast, cAMP did inhibit the response to formylpeptide chemoattractant receptor. These data suggest that homologous desensitization of PAF-mediated responses is regulated via phosphorylation at two levels in the signaling pathway, one at the receptor and the other at PLC␤3 mediated by PKC but not by PKA. Phosphorylation of PLC␤3 by PKA could explain the inhibition of formylpeptide chemoattractant receptor signaling by cAMP. As PAF and formylpeptide chemoattractant receptors activate PLC via different G proteins, phosphorylation of PLC␤3 by PKC and PKA could provide distinct regulatory control for classes of G proteincoupled receptors.

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein.Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cptcAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLC␤ 3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLC␤ 3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLC␤ 3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLC␤ 3 and appears to block the ability of G ␤␥ to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLC␤ 3 , but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.

Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet beta-adrenoceptors (betaARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, after betaAR stimulation or cAMP elevation. Basal NOS activity was 0.11+/-0.03 pmol L-citrulline/10(8) platelets. The betaAR agonist isoproterenol 1 micromol/L and the adenylyl cyclase activator forskolin 1 micromol/L each increased NOS activity, to 0.26+/-0.04 and 0.23+/-0.03 pmol L-citrulline/10(8) platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 micromol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca(2+). In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3+/-0.2% versus 2.6+/-0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through beta(2)ARs, as determined by coincubation with betaAR subtype-selective antagonists. We conclude that beta(2)ARs activate platelet NOS by increasing cAMP, and that this activation is Ca(2+)-independent. beta(2)ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of beta(2)ARs on adhesion but not aggregation.

In men with stable angina, an increase in plasma L-arginine/ADMA ratio after two weeks' oral supplementation with L-arginine is not associated with an improvement in endothelium-dependent vasodilatation, oxidative stress or exercise performance.

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Aims P256 is a divalent antibody which aggregates human platelets by interaction with glycoprotein (GP) IIb/IIIa receptors. We investigated the effect of tirofiban, an antagonist of the GP IIb/IIIa receptor, on P256-mediated platelet aggregation. Methods Responses to agonists were measured turbidometrically at 37°C in stirred citrated platelet-rich plasma from venous blood samples from healthy human volunteers. Inhibitory effects were determined by comparison with aggregation to the same concentration of agonist in a vehicle treated sample. Results Tirofiban inhibited a near maximally effective dose of P256 (10with an IC 50 of 9.3×10 −8 mol l −1 . Tirofiban (10 −7 mol l −1 ) inhibited responses to arachidonic acid, U46619 and P256 similarly, whereas aspirin (1.1×10−4 mol l −1 ) inhibited arachidonic acid more effectively than P256 ( P<0.007by anova).Conclusions Tirofiban potently and selectively inhibits P256-stimulated aggregation of human platelets.