D1 and D2 dopamine receptor-mediated inhibition of activated normal T cell proliferation is lost in jurkat T leukemic cells

Abstract

Dopamine is a catecholamine neurotransmitter, which plays an important role in the regulation of T cell functions. In activated T cells from normal volunteers, stimulation of D(1) and D(2) dopamine receptors inhibit cell proliferation and cytokine secretion. However, there is no report yet regarding the regulatory role of D(1) and D(2) dopamine receptors in abnormally proliferating T cells. The present study investigates the expression and effect of activation of these dopamine receptors in Jurkat cells, a leukemic T cell line showing uncontrolled proliferation. Like normal human T cells, in Jurkat cells, D(1) and D(2) dopamine receptors are also expressed; however, unlike activated normal T cells, stimulation of these dopamine receptors in Jurkat cells fails to inhibit their T cell receptor-induced proliferation. This alteration is due to failure of D(1) dopamine receptor-mediated activation of cyclic AMP signaling and a missense mutation at the third cytoplasmic loop of D(2) dopamine receptors affecting inhibition of phosphorylation of ZAP-70, an important downstream protein transducing signal from the T cell receptor. These results help to understand the biology of abnormal proliferation of T cells in pathophysiological conditions where dopamine plays an important role.

FIGURE 1.

Expression of D₁ and D₂ dopamine receptors in Normal and Jurkat cells. A, semiquantitative reverse transcription-polymerase chain reaction analysis of mRNA shows presence of D₁ and D₂ dopamine receptor expression in normal activated T cells and Jurkat cells. 15 S rRNA expression is the positive control. B, Western blot with specific antibodies against receptors also revealed similar expression pattern of D₁ and D₂ dopamine receptor proteins in normal and Jurkat T lymphocytes. Results are representative of six separate experiments.

FIGURE 2.

Effect of stimulation of D₁ and D₂ dopamine receptors on proliferation of activated normal T cells and Jurkat cells by [³H]thymidine incorporation assay. Treated cells were cultured for 72 h, and radiolabeled thymidine was added 18 h before termination of experiments. Incorporated radioactivity was measured as representative of cellular proliferation. Intracellular cAMP concentration also was measured from these treatment groups. A, stimulation of D₁ dopamine receptors by its specific agonist (SKF, SKF 82526) in CD3/CD28-stimulated normal T cells showed significant dose-dependent inhibition of proliferation with maximum inhibition found at a 1 μm concentration. In contrast, no significant inhibition of proliferation was observed in CD3/CD28-stimulated Jurkat cells following D₁ agonist treatment. B, CD3/CD28-stimulated normal T cells showed significant dose-dependent inhibition of proliferation following stimulation of D₂ DA receptors by a specific D₂ agonist (Q, quinpirole hydrochloride), and maximum inhibition was found at 2 μm concentration, but no significant inhibition of proliferation was observed in Jurkat cells following D₂ DA receptor agonist treatment. Results are mean ± S.E. of six separate experiments (*, p < 0.05). C, intracellular cAMP measured at different time points after D₁ agonist stimulation (1 μm) showed a significantly elevated level in normal activated T cells where the peak was reached at 5 h and then declined. But similar treatment failed to elevate intracellular cAMP pool of Jurkat cells. D, stimulation of D₂ dopamine receptors could not inhibit the cAMP concentration in CD3/CD28-activated Jurkat cells. Results are mean ± S.E. of six separate experiments (*, p < 0.05). Culture and treatment protocols are as described under “Experimental Procedures.”

FIGURE 3.

Effect of stimulation of D₂ dopamine receptors on ZAP-70 phosphorylation in activated normal T lymphocytes and Jurkat cells. A, phosphorylation of ZAP-70, an important signaling protein in T cell activation was abrogated following stimulation of D₂ dopamine receptors by specific agonist (Q, quinpirole hydrochloride; 2 μm) in CD3/CD28-activated normal T cells. However, similar D₂ agonist treatment failed to inhibit ZAP-70 phosphorylation in activated Jurkat cells. Protein loading was verified by reblotting the membrane by anti-ZAP-70, which showed that total ZAP-70 expression remained unchanged. Results are representative of six separate experiments. B, chromatogram of DNA from PCR product amplified from D₂ dopamine receptor of Jurkat cells shows a nonsynonymous mutation (C → G transversion). Base change to guanine at the indicated position of codon 311 results in substitution of amino acid cysteine and functional alteration of D₂ receptor activity in Jurkat cells.

FIGURE 4.

Inhibition of phosphodiesterase activity, together with stimulation of D₁ dopamine receptors in Jurkat cells, is associated with significant elevation in intracellular cAMP level and inhibition of cell proliferation. A, the D₁ DA receptor agonist (SKF, SKF 82526) induced dose-dependent inhibition of proliferation with 1 μm concentration, showing the maximum inhibition observed in CD3/CD28-stimulated Jurkat T cells when pretreated with phosphodiesterase inhibitor (Theo, theophylline; 1 mm), but not in CD3/CD28-stimulated Jurkat cells similarly treated with D₁ agonist alone. Results are based on [³H]thymidine incorporation assay. Results are mean ± S.E. of six separate experiments (*, p < 0.05). B, inhibition of phosphodiesterase activity by theophylline (1 mm) or stimulation with D₁ DA receptor-specific agonist (1 μm) separately was not sufficient to inhibit the CD3/CD28-induced Jurkat T cell proliferation, but stimulation of D₁ DA receptors with specific agonist (1 μm) together with inhibition of phosphodiesterase activity (theophylline; 1 mm) significantly inhibited proliferation of these cells as evident from [³H]thymidine incorporation assay. Inhibition of D₁ DA receptor activity by specific D₁ DA receptor antagonist (SCH, SCH 23390; 100 μm) abrogated this above-mentioned proliferation inhibition in Jurkat, suggesting the functional competence of D₁ DA receptors, and both inhibition of phosphodiesterase activity and D₁ receptor stimulation are required for effective inhibition of proliferation in Jurkat cells. C, significant increase in intracellular cAMP observed in CD3/CD28-stimulated Jurkat cells treated with phosphodiesterase inhibitor (theophylline; 1 mm) and D₁ DA receptor agonist (SKF 82526; 1 μm), when compared with only theophylline-treated (1 mm) or D₁ DA receptor agonist (SKF 82526; 1 μm)-treated groups which showed little or no increase of cAMP, respectively. Inhibition of D₁ DA receptor activity by D₁ DA receptor-specific antagonist (SCH 23390; 100 μm) and then treatment with theophylline (1 mm) and D₁ DA receptor agonist (SKF 82526; 1 μm) also showed only negligible increase in intracellular cAMP in Jurkat cells. Results are mean ± S.E. of six separate experiments (*, p < 0.05). Culture and treatment protocols are as described under “Experimental Procedures.”