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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02668

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CysLT₁ receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation

¹ Laboratory of Molecular Pharmacology, Section of Eicosanoid Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
² Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

View larger version (20K): [in a new window]   Fig. 1. Calcium responses to LTD4, ATP and UDP following LTD4-induced CysLT1 receptor activation. (A) Representative traces of the [Ca2+]i transient induced by 10 nM LTD4 before and after an initial challenge with the same concentration of LTD4. (B,C) Representative traces of the [Ca2+]i transient induced by 10 µM ATP (B) and 100 µM UDP (C) (2nd challenge) before and after an initial challenge with 10 nM LTD4. (D) Values shown representative means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of at least three independent experiments.

View larger version (27K): [in a new window]   Fig. 2. Calcium responses to LTD4 following ATP- and UDP-induced P2Y receptor activation. Representative traces of the [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with increasing concentrations of ATP (A) and UDP (B). (C) Values shown represent means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of at least three independent experiments. Statistical comparison of multiple groups were analyzed using one way ANOVA followed by Dunnett post hoc test (*P<0.05; **P<0.01, vs control).

View larger version (21K): [in a new window]   Fig. 3. Effect of GFX on CysLT1 receptor activation after LTD4, ATP and UDP challenge. Representative traces of the effect of increasing concentrations of GFX pretreatment (5 minutes) on [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with 10 µM ATP (A) and 100 µM UDP (B). (C) Values shown represent means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of at least three independent experiments.

View larger version (18K): [in a new window]   Fig. 4. Effect of GFX on CysLT1 receptor activation after PMA challenge, and effect of H89 on ATP- and UDP-induced CysLT1 desensitization. (A) Representative trace of the effect of increasing concentrations of GFX pretreatment (5 minutes) on the [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with 500 nM PMA (5 minutes). (B) Values shown represent means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of at least five independent experiments. (C) Effect of 10 µM H89 pretreatment (5 minutes) on the [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with 10 nM LTD4, 10 µM ATP or 100 µM UDP.

View larger version (34K): [in a new window]   Fig. 5. Rate of recovery of CysLT1 functionality after homologous or heterologous desensitization in dU937 cells and in the D5 clone, and effect of PTX or YM-254890 on extracellular nucleotide-induced heterologous desensitization. (A) Time course of recovery of CysLT1 functionality (10 nM LTD4, 2nd challenge) following homologous and heterologous desensitization in dU937 cells. Values shown represent means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of five independent experiments. (B) Time course of recovery of CysLT1 functionality (10 nM LTD4, 2nd challenge) following homologous desensitization in control dU937 cells and D5 clone. Values shown represent means of [Ca2+]i stimulation over basal (s/b) ± s.e.m. of at least three independent experiments. (C) Effect of 300 ng/ml PTX pretreatment (overnight) on the [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with 10 µM ATP or 100 µM UDP. Values shown represent the percentage response ± s.e.m. of three independent experiments. (D) Effect of 60 nM YM-254890 pretreatment (5 minutes) on the [Ca2+]i transient induced by 10 nM LTD4 (2nd challenge) after an initial challenge with 10 µM ATP or 100 µM UDP. Values shown represent the percentage response ± s.e.m. of three independent experiments.

View larger version (20K): [in a new window]   Fig. 6. Equilibrium binding and acid wash of [3H]LTD4 in intact dU937 cells. (A) Percentage of total [3H]LTD4 binding and displacement by unlabeled LTD4, ATP or UDP. (B) Effect of a 30 minute pretreatment with 0.1 µM LTD4, 100 µM ATP or 1 mM UDP on surface-specific [3H]LTD4 binding. (C) Acid wash: effect of the incubation at 0°C or 37°C (30 minutes) on the distribution of total [3H]LTD4 radioactivity (Tot); P, pellet; S, supernatant. Values shown represent means±s.e.m. of at least two independent experiments.

View larger version (29K): [in a new window]   Fig. 7. Confocal microscopy of the CysLT1 receptor trafficking in intact dU937 cells. Confocal laser scanning microscopy of the subcellular distribution of the CysLT1 receptors was performed using a specific antibody raised against the C-terminal tail. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 in basal conditions (control), and after 5 minutes were challenged with the indicated stimuli. Immunocytochemical staining of five representative dU937 cells after stimulation with vehicle (A), 10 nM LTD4 (B), 100 µM ATP (C), 1 mM UDP (D), 500 nM PMA (E) and 10 nM LTD4 after a 30 minute pretreatment with 10 µM GFX (F). The experiment shown is representative of at least two others performed.

View larger version (31K): [in a new window]   Fig. 8. CysLT1 receptor expression and heterologous desensitization in COS-7 cells. Confocal microscopy of the CysLT1 receptor trafficking in intact COS-7 cells. (A) COS-7 cells expressing HA-tagged CysLT1 receptor were immunoprecipitated with HA monoclonal antibody and visualized with an CysLT1 polyclonal antibody. (B) Equilibrium mixed-type binding curve of [3H]LTD4 in membranes from COS-7 cells transiently transfected with HA-tagged CysLT1 receptor. Binding of [3H]LTD4 is expressed as the ratio of bound ligand concentration to total ligand concentration (B/T, dimensionless) versus the logarithm of total ligand concentration (Log T). Two independent [3H]LTD4 mixed-type experiments were performed, each with triplicate determinations, and analyzed simultaneously. For the sake of clarity, only one representative curve is shown; the curve is computer generated. (Inset) The percentage of total [3H]LTD4 binding and displacement by unlabeled LTD4, ATP or UDP. (C) Representative traces of the [Ca2+]i transient induced by 30 µM ATP (2nd challenge) before and after an initial challenge with 1 µM LTD4. (D) Representative traces of the [Ca2+]i transient induced by 1 µM LTD4 (2nd challenge) before and after an initial challenge with 30 µM of ATP. (E) Confocal laser scanning microscopy of the subcellular distribution of the CysLT1 receptors performed using a specific antibody raised against the C-terminal tail. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 in basal conditions (control) and after a 5 minute challenge with the indicated stimuli. Immunocytochemical staining of COS-7 cells after stimulation with vehicle (left), 1 µM LTD4 (middle), 30 µM ATP (right). The experiment shown is representative of two others performed.

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