ADP stimulation of inositol phosphates in hepatocytes: role of conversion to ATP and stimulation of P2Y2 receptors

Abstract

1 Accumulation of inositol (poly)phosphates (InsP(x)) has been studied in rat hepatocytes labelled with [(3)H]inositol. Stimulation with ADP resulted in a significant increase in total [(3)H]InsP(x), whereas 2-MeSADP had only a small effect and ADPbetaS was ineffective. UTP and ITP also stimulated substantial increases in [(3)H]InsP(x). 2 The dose-response curve to ADP was largely unaltered by the presence of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P). Similarly, inclusion of MRS 2179, a more selective P2Y(1) antagonist, had no effect on the dose-response curve to ADP. 3 The inclusion of hexokinase in the assay reduced, but did not abolish, the response to ADP. 4 HPLC analysis revealed that ADP in the medium was rapidly converted to AMP and ATP. The inclusion of hexokinase removed ATP, but exacerbated the decline in ADP concentration, leading to increased levels of AMP. 2-MeSADP was stable in the medium and ATP was largely unaffected. 5 The addition of the adenylate kinase inhibitor, diadenosine pentaphosphate (Ap(5)A) significantly reduced the ADP response. HPLC analysis conducted in parallel demonstrated that this treatment inhibited conversion of ADP to ATP and AMP. 6 Inclusion of the P1 antagonist CGS 15943 had no effect on the dose-response curve to ADP. 7 These observations indicate that hepatocytes respond to ADP with an increase in inositol (poly)phosphates following conversion to ATP. P2Y(1) activation in hepatocytes does not appear to be coupled to inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) production.

Figure 1

Effect of ADP, ATP, ADPβS and 2-MeSADP on inositol (poly)phosphate accumulation in rat hepatocytes. Total [³H]InsP_x accumulation was measured in [³H]inositol-labelled cells in response to incubation for 20 min with increasing concentrations of agonist. Results, shown as d.p.m. after subtraction of unstimulated controls, are from a single representative experiment, with stimulations performed in triplicate. Data pooled from three separate experiments are reported in the text.

Figure 2

Effect of ATP, UTP and ITP on inositol (poly)phosphate accumulation in rat hepatocytes. Total [³H]InsP_x accumulation was measured in [³H]inositol-labelled cells in response to incubation for 20 min with increasing concentrations of agonist. Results, shown as d.p.m. after subtraction of unstimulated controls, are from a single representative experiment, with stimulations performed in triplicate. Data pooled from three separate experiments are reported in the text.

Figure 3

Effects of the P2Y₁ antagonists, A3P5P and MRS 2179 on the inositol (poly)phosphate response to ADP. Dose–response curves to increasing concentrations of ADP were constructed in the presence and absence of 100 μM A3P5P (a) or 100 μM MRS 2179 (b). Results, shown as d.p.m. after subtraction of unstimulated controls, are from a single representative experiment, with stimulations performed in triplicate. Data pooled from three separate experiments are reported in the text.

Figure 4

Effect of hexokinase-treatment on the inositol (poly)phosphate response to ADP. Dose–response curves to increasing concentrations of untreated, and hexokinase-treated ADP. Hexokinase was also included in the assay (3 units well⁻¹) to counteract conversion of ADP to ATP during the 20 min stimulation period. Results, shown as d.p.m. after subtraction of unstimulated controls, are from a single representative experiment, with stimulations performed in triplicate. Data pooled from three separate experiments are reported in the text.

Figure 5

Effect of the adenylate kinase inhibitor, Ap₅A on the inositol (poly)phosphate response to ADP. Dose–response curves to increasing concentrations of ADP were constructed in the presence and absence of 300 μM Ap₅A. Results, shown as d.p.m. after subtraction of unstimulated controls, are from a single representative experiment, with stimulations performed in triplicate. Data pooled from three separate experiments are reported in the text.