Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells

Abstract

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.

Figure 1

Concentration-dependent inhibition by nucleotides of the forskolin-induced cyclic AMP accumulation in B10 cells. The maximum stimulated activity is set at 100%, defined in the presence of forskolin and IBMX alone (see Methods). The responses were measured on (a) a subclone capable of a maximum inhibition by adenine nucleotides of 60–70%, or on a second subclone (b) capable of up to 95% inhibition (see text).The data are represented as the mean±s.e.mean from (a) 5–15 independent determinations for each agonist, each performed in triplicate on subclone a, or (b) representative experiments, performed in triplicate on subclone b, with two of the nucleotides, illustrating the partial agonist activity of ATP. The curves are theoretical fits to a logistic equation.

Figure 2

Apparent antagonism by ATP of the 2-MeSADP-evoked decrease in 10 μM forskolin-stimulated cyclic AMP formation in B10 cells. The cyclic AMP accumulation shown is normalized to 100% as the value prior to 2-MeSADP addition.

Figure 3

The weak inhibition of the 10 μM forskolin-induced cyclic AMP formation, produced by A2P5P and A3P5P in B10 cells.

Figure 4

Concentration-dependent antagonism of the P2Y_T-receptor-selective antagonist PSFM-ATP (AR-C66096) (a) and BzATP (b) on the 2-MeSADP-mediated inhibition of cyclic AMP formation in B10 cells. cyclic AMP accumulation in the presence of 10 μM forskolin but in the absence of agonist and antagonist was normalized as 100%. From these and similar fitted curves (some removed for clarity from panel b) obtained at different concentrations the K_B values were computed.

Figure 5

Lack of effect of PSFM-ATP (AR-C66096) on the response to ADP of the P2Y₁ receptor in B10 cells. Intracellular Ca²⁺ mobilization measurements are shown. The lowest line shows that the antagonist alone gave no response.

Figure 6

The 2-MeSADP-induced inhibition of 10 μM forskolin-stimulated cyclic AMP formation in B10 cells with (upper curve) or without (lower plot) pertussis toxin (100 ng ml⁻¹) pre-treatment. Basal levels of cyclic AMP were unchanged by the PTX treatment. For further details, see Methods.

Figure 7

The rate of appearance (a) and metabolism (b) of ATP in the incubation medium of B10 cells. Confluent cultures were pre-washed and the medium replaced at zero time, with the conditions as in an adenylyl cyclase assay (without agonist), and the ATP content of the medium was monitored (see Methods). The stress due to the change of medium results in a release to give ∼100 nM ATP in the assay volume (a), but no further net release is seen. In another set of experiments (b), ATP was added initially to 1000 nM, and its disappearance with time was determined. Note that during the standard adenylyl cyclase assay period of 5 min, only a relatively small conversion of ATP to other products occurs, even in the absence (as here) of the ATP regeneration system.