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The effects of the statins lovastatin and cerivastatin on signalling by the prostanoid IP-receptor.

ABSTRACT: The prostanoid-IP receptor may be unique among G protein coupled receptors in that it is isoprenylated. In this study, we investigated the effects of the statins lovastatin and cerivastatin on signalling by the mouse (m) IP and the human (h) IP receptors, over-expressed in human embryonic kidney (HEK) 293 cells and by the hIP receptor, endogenously expressed in human erythroleukaemia cells. Both statins significantly reduced IP receptor-mediated cyclic AMP generation and intracellular calcium ([Ca(2+)](i)) mobilization in a time and concentration dependent manner but had no effect on signalling by the non-isoprenylated beta(2) adrenergic receptor or by the human prostanoid-TP receptor isoforms. Cerivastatin (IC(50), 50 - 90 nM) was significantly more potent than lovastatin (IC(50), 0.80 - 4.2 microM) in inhibiting IP receptor signalling. Whereas IC(50) values indicated that the hIP receptor was significantly more sensitive than the mIP receptor to the statins, the extent of inhibition of cyclic AMP generation by the mIP receptor was significantly greater than that of the hIP receptor to either statin, even at the highest concentrations used. Pretreatment with either statin significantly reduced IP receptor mediated desensitization of signalling by the h.TPalpha, but not by the h.TPbeta, receptor isoform. These data generated in whole cells point to the possibility that statin therapy may interfere with IP receptor signalling in vivo; such interference may be extenuated under conditions where circulating statin levels are elevated and may account, in part, for some of the pleiotropic affects of the statins not attributed solely to their lipid lowering properties.

SUBMITTER: Lawler OA

PROVIDER: S-EPMC1572747 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G(s)alpha. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the alpha subunits of its cognate G-protein G(s), G(i1), a G-protein which it fails to activate in co-expression studies, and a chimaeric G(i1)-G(s)6 (a form of G(i1) in which the C-terminal six amino acids were replaced with the equivalent sequence of G(s)) were stably expressed in HEK293 cells. These were detected by [(3)H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-G(s)alpha fusion protein, and both constructs were shown to interact with and activate endogenously expressed G(s)alpha. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-G(i1)alpha fusion. However, the fusion proteins containing either G(s)alpha or G(i1)-G(s)6alpha produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G(i1)-G(s)6alpha fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the G(s)alpha-containing fusion protein and a 9-fold improvement when using the fusion protein containing G(i1)-G(s)6alpha to detect G-protein activation compared with expression of the isolated receptor.

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The effects of the statins lovastatin and cerivastatin on signalling by the prostanoid IP-receptor.

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