Protein kinase A-dependent coupling of mouse prostacyclin receptors to Gi is cell-type dependent

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Abstract

The ability of the prostacyclin (IP) receptor agonist cicaprost to activate Gs-, Gq/11- and Gi-mediated cell signalling pathways has been examined in Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK 293) cells expressing the cloned human (hIP) or mouse (mIP) prostacyclin receptor, and compared with data from NG108-15 and SK-N-SH cells that endogenously express rat/mouse and human IP receptors, respectively. Cicaprost stimulated [3H]cyclic AMP production with EC50 values of 1.5–22 nM, and stimulated [3H]inositol phosphate production (EC50 values 49–457 nM) in all but the SK-N-SH cells. Cicaprost failed to inhibit forskolin-stimulated [3H]cyclic AMP production in any of these cell lines. Therefore, although both human and mouse IP receptors couple to Gs and Gq/11-mediated signalling pathways in a cell type-dependent manner, we could find no evidence for IP receptor coupling to Gi.

Introduction

The prostacyclin (IP) receptor is a G protein-coupled receptor belonging to the family of prostanoid receptors (Narumiya et al., 1999). IP receptors couple primarily to activation of adenylyl cyclase via Gs, and can also couple to activation of phospholipase C leading to phosphatidyl inositol turnover and mobilization of intracellular calcium ([Ca2+]i) (see Wise and Jones, 2000). Whether or not IP receptor coupling to Gq/11 is physiologically relevant remains uncertain, since to date, activation of this pathway in native cells has only been demonstrated using the non-specific IP receptor agonist iloprost in piglet cerebral microvascular smooth muscle cells (Parkinson et al., 2000) and in isolated rat dorsal root ganglion cells in vitro (Smith et al., 1998). Activation of multiple signalling pathways by IP receptor agonists (observed as increases in cyclic AMP, inositol trisphosphate or [Ca2+]i) has been reported in transformed cell lines such as BNu2cl3 mouse mast cells, Ob1771 mouse pre-adipocytes, human erythroleukaemia (HEL) cells and human megakaryoblastic leukaemia (MEG-01) cells (for references, see Wise and Jones, 1996).

A recent report has shown that the IP receptor endogenously expressed in mouse erythroleukaemia (MEL) cells and the cloned mouse (mIP) prostacyclin receptor stably overexpressed in human embryonic kidney 293 (HEK 293) cells can couple to Gi (Lawler et al., 2001). Furthermore, the mIP receptor in HEK 293 cells couples to both Gq/11 and Gi in a protein kinase A (PKA)-dependent manner (Lawler et al., 2001). In contrast, the cloned human (hIP) prostacyclin receptor stably overexpressed in HEK 293 cells couples independently to Gs and Gq/11 and does not couple to Gi (Miggin and Kinsella, 2002). We have previously demonstrated that IP receptor agonist-stimulated [3H]cyclic AMP production in Chinese hamster ovary (CHO) cells transiently expressing mIP receptors and in human neuroblastoma SK-N-SH cells was not affected by pertussis toxin treatment, suggesting a lack of coupling of both mIP and human IP receptors to Gi (Kam et al., 2001). Therefore, we have extended our studies here to examine the G protein coupling capacity of human and rodent IP receptors in a variety of cell lines stably or transiently expressing IP receptors. Our studies fail to support the hypothesis that mouse IP receptor switching from Gs to Gi is a universal phenomenon.

Section snippets

Cell culture

CHO cells were cultured in Ham's F-12 medium supplemented with 10% foetal bovine serum. HEK 293 cells were cultured in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% foetal bovine serum, and assays were performed in cell culture dishes treated with poly-d-lysine. Rat/mouse neuroblastoma-glioma (NG108-15) cells were cultured in DMEM medium supplemented with 6% foetal bovine serum, 0.1 mM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine and 2 mM l-glutamine. SK-N-SH

Coupling of IP receptors to Gs-mediated signalling pathways

Cicaprost stimulated [3H]cyclic AMP production in a concentration-dependent manner in CHO and HEK 293 cells transiently expressing hIP and mIP receptors, and in NG108-15 and SK-N-SH cells which endogenously express IP receptors (Table 1). The pEC50 values for cicaprost were similar for hIP and mIP receptors when transiently expressed in HEK 293 cells, but were significantly higher for hIP compared with mIP receptors transiently expressed in CHO cells (P<0.01). The affinity of human IP receptors

Discussion

When iloprost was first demonstrated to increase inositol phosphate production in CHO cells stably expressing the mIP receptor, it was also demonstrated that this process was pertussis toxin-insensitive and was not evoked by dibutyryl cyclic AMP (Namba et al., 1994). Furthermore, while pretreatment of these mIP receptor-expressing CHO cells with cholera toxin to degrade Gs resulted in a loss of iloprost-induced cyclic AMP production, it did not affect the inositol phosphate response. Together

Acknowledgments

This work was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (CA99/00.SC01).

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