Rapid resensitization of purinergic receptor function in human platelets.

BACKGROUND: Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. OBJECTIVES: To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. METHODS: These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. RESULTS: In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. CONCLUSIONS: This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

BACKGROUND: Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. OBJECTIVES: To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. RESULTS: Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. CONCLUSIONS: This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

An intensive field trial to assess hazards to birds and mammals from the use of methiocarb as a bird repellent on ripening cherries.

: A field trial was carried out in Kent, UK, in 1980, to assess the possible hazards to wildlife of methiocarb used as a spray to protect ripening cherries from damage by birds. A broad range of studies was undertaken on a single site subjected to a series of five applications. Ground deposition was measured by analysis of strips of chromatography paper and petri dishes placed between lines of trees. Airborne drift was measured on targets up to 11 m above the ground, and methiocarb residues were measured in samples of cherries. Birds were captured by intensive mist-netting throughout the trial, and breeding success was monitored in nest boxes and natural nest sites. Livetraps were set to catch small mammals in the orchard on five occasions during the trial. Systematic searches for possible casualties were made, resulting in the discovery of 21 birds found dead or incapacitated. Levels of activity of plasma, liver and brain esterases were measured in samples taken from several species, and liver slices from House Sparrows, Starlings and Thrushes were examined histologically for signs of cell damage. Samples of breast muscle tissue from Starlings and House Sparrows were analysed by GLC for residues of methiocarb and its principal breakdown product, methiocarb sulfoxide. Overall, the trial revealed that many birds and mammals were exposed to methiocarb, but its effects were largely transient and sublethal. Even a heavy repeated programme of spray applications did not cause any serious hazard to wildlife populations.

Species differences in avian serum B esterases revealed by chromatofocusing and possible relationships of esterase activity to pesticide toxicity.

Serum cholinesterase (BChE) and carboxylesterase (CbE) activities were investigated in ten species of birds. Multiple forms of serum BChE and CbE were also separated by chromatofocusing. Higher CbE activity and a wider range of CbE and BChE forms were present in the sera of omnivorous/herbivorous birds than carnivores. Omnivores/herbivores studied were the starling, house sparrow, tree sparrow, pigeon, partridge and magpie. Serum CbE activities of these species ranged from 0.46 to 2.93 mumol/min/mL with 2-6 forms separated by chromatofocusing. 0-6 forms of BChE were separated by the same method. The serum CbE activities of the little owl, tawny owl, barn owl and razorbill ranged from 0.19 to 0.58 mumoles/min/mL with 0-2 forms separated by chromatofocusing. No ChE forms were present within the pH gradient. These results may be significant in contributing to the understanding of the selective toxicity of organophosphorus and carbamate pesticides.

Distinction between 'A'-esterases and arylesterases. Implications for esterase classification.

'A'-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable 'A'-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both 'A'- and 'aryl'-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not 'A'-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications.