Pharmacological Characterization of Inositol 1,4,5-tris Phosphate Receptors in Human Platelet Membranes

Abstract

The phosphatidylinositol (PI) hydrolysis signaling system has been shown to be altered in platelets of depressed and schizophrenic subjects. Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)), an integral component of the PI signaling system, mobilizes Ca(2+) by activating Ins(1,4,5)P(3) receptors. To eventually investigate the role of Ins(1,4,5)P(3) receptors in depression and other mental disorders, we characterized [(3)H]Ins(1,4,5)P(3) binding sites in crude platelet membranes prepared from small amounts of blood obtained from healthy human control subjects. We found a single, saturable binding site for [(3)H]Ins(1,4,5)P(3) to crude platelet membranes, which is time dependent and modulated by pH, inositol phosphates, and heparin. Since cyclic adenosine monophosphate (cAMP) and Ca(2+) have been shown to be important modulators in Ins(1,4,5)P(3) receptors, in the present study we also determined the effects of various concentrations of CaCI(2) and forskolin on Ins(1,4,5)P(3) binding to platelet membranes. CaCI(2) modulated [(3)H]Ins(1,4,5)P(3) binding sites in a biphasic manner: at lower concentrations it inhibited [(3)H]Ins(1,4,5)P(3) binding, whereas at higher concentrations, it stimulated [(3)H]Ins(1,4,5)P(3) binding. On the other hand, forskolin inhibited [(3)H]Ins(1,4,5)P(3) binding. Our results thus suggest that the pharmacological characteristics of [(3)H]Ins(1,4,5)P(3) binding to crude platelet membranes are similar to that of Ins(1,4,5)P(3) receptors; and that both Ca(2+) and cAMP modulate [(3)H]Ins(1,4,5)P(3) binding in crude platelet membranes.

Figure 1

The effect of pH on [³H]Ins(1,4,5)P₃ binding to human platelet membranes. Binding at different pH values was performed as described in Section 2. The reaction mixture contained 20 nM [³H]Ins(1,4,5)P₃ and approximately 100 μg protein. Nonspecific binding was determined in the presence of 10 μM D-Ins(1,4,5)P₃. Incubations were carried out at 4°C for 10 minutes. Each point represents the mean value of two experiments performed in duplicate.

Figure 2

Time course of [³H]Ins(1,4,5)P₃ binding to human platelet membranes. Platelet membranes (100 μg/protein) were incubated at 4°C for different time intervals in the presence of 20 nM [³H]Ins(1,4,5)P₃. Nonspecific binding was estimated in the presence of 10 μM D-Ins(1,4,5)P₃. The incubations were rapidly terminated by vacuum filtration at different time points as shown in the figure. A single representative experiment is shown for duplicate determinations.

Figure 3

Saturation isotherm of [³H]Ins(1,4,5)P₃ binding to human platelet membranes. Each point is the mean of duplicate determination. Binding assays were carried out as described in Section 2. Nonspecific binding was determined in the presence of 10 μM D-Ins(1,4,5)P₃. A Scatchard plot of [³H]Ins(1,4,5)P₃ binding is shown in the inset. B = [³H]Ins(1,4,5)P₃ specifically-bound (fmol/mg protein), B/F = the ratio of specifically-bound to free ligand in fmol of Ins(1,4,5)P₃ (fmol/mg protein × nM). For this particular experiment, binding indices are B _max = 427.56 fmol/mg protein K _d = 21.73 nM; and the correlation coefficient (r) = 0.98.

Figure 4

Displacement curve for the inhibition of [³H]Ins(1,4,5)P₃ binding to human platelet membranes by inositol phosphates. Platelet membranes were incubated with 20 nM [³H]Ins(1,4,5)P₃ in the presence of increasing concentrations of D-Ins(1,4,5)P₃ (▲), D-Ins(2,4,5)P₃ (●), GPIP₂ (♦), and L-Ins(1,4,5)P₃ (■) at 4°C for 10 minutes. The data are the mean ± S.E.M. of three independent experiments, each run in duplicate. The IC50 values were calculated using the displacement curve (Table 1).

Figure 5

Inhibition of [³H]Ins(1,4,5)P₃ binding to human platelet membranes by heparin. Experimental conditions are similar to those described in Figure 4. The points represent the mean ± S.E.M. of three independent experiments, each run in duplicate.

Figure 6

Effects of CaCl₂ on [³H]Ins(1,4,5)P₃ binding to human platelet membranes in the presence (a) and absence (b) of EDTA. Platelet membranes were incubated with increasing concentrations of CaCl₂ in the presence of 20 nM [³H]Ins(1,4,5)P₃ in a buffer containing 50 mM Tris-HCl, pH 8.4; 1 mM EDTA; and 1 mM 2-mercaptoethanol at 4°C for 10 minutes. The data represent the inhibition or stimulation of [³H]Ins(1,4,5)P₃ binding to platelet membranes. The points represent the mean ± S.E.M. of three independent experiments, each run in duplicate.

Figure 7

Inhibitory effect of forskolin on [³H]Ins(1,4,5)P₃ binding to human platelet membranes. The experiments were performed in an assay medium (50 mM Tris HC1, pH 8.4; 1 mM EDTA; 1 mM 2-mercaptoethanol) containing 30 mM CaCl₂ 20 nM [³H]Ins(1,4,5)P₃, and increasing concentrations of forskolin at 4°C for 10 minutes. The data are the mean ± S.E.M. of three independent experiments, each carried out in duplicate.