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

Abstract

1 The prostanoid-IP receptor may be unique among G protein coupled receptors in that it is isoprenylated. In this study, we investigated the eects 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. 2 Both statins signi®cantly reduced IP receptor-mediated cyclic AMP generation and intracellular calcium ([Ca 2+ ] i ) mobilization in a time and concentration dependent manner but had no eect on signalling by the non-isoprenylated b 2 adrenergic receptor or by the human prostanoid-TP receptor isoforms. 3 Cerivastatin (IC 50 , 50 ± 90 nM) was signi®cantly more potent than lovastatin (IC 50 , 0.80 ± 4.2 mM) in inhibiting IP receptor signalling. 4 Whereas IC 50 values indicated that the hIP receptor was signi®cantly more sensitive than the mIP receptor to the statins, the extent of inhibition of cyclic AMP generation by the mIP receptor was signi®cantly greater than that of the hIP receptor to either statin, even at the highest concentrations used. 5 Pretreatment with either statin signi®cantly reduced IP receptor mediated desensitization of signalling by the h.TPa, but not by the h.TPb, receptor isoform. 6 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 aects of the statins not attributed solely to their lipid lowering properties.

Palmitoylation of the Human Prostacyclin Receptor

“…Deletion-and Site-directed Mutagenesis of Human Prostacyclin Receptor-The plasmids pBluescript-hIP and pHM-hIP have been previously described (20,26 , TGC to TAG). Deletion mutagenesis of hIP was performed using pBluescript-hIP as a PCR template, employing Expand High Fidelity Taq DNA polymerase and the oligonucleotide primers: 5Ј-G AGA AGC TTG ATG GCG GAT TCG TGC AGG-3Ј (sense primer; nucleotides ϩ1 to ϩ18 of hIP sequences are underlined) and 5Ј-ATA TGA ATT CTA GAC CCA GAG CTT GAG TCG C-3Ј (antisense primer; the sequences complimentary to nucleotides ϩ 902 to ϩ 920 of hIP are underlined, and the mutator in-frame stop codon is in bold type).…”

“…FURA2/AM was purchased from Calbiochem. [ 3 H] i P 3 (20 -40 Ci/mmol) and [ 3 H]cAMP (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Ci/mmol) were purchased from American Radiolabeled Chemicals Inc. [ 3 H]Iloprost (15.3Ci/mmol) was purchased from Amersham Biosciences. [9,10-3 H]Palmitic acid (60 Ci/mmol) was obtained from PerkinElmer Life Sciences.…”

“…Inhibition of isoprenylation of both the mouse and human (h) IP, either through site-directed mutagenesis of the critical acceptor Cys of the CAAX motif or through the use of the statin inhibitors of hydroxymethylglutaryl CoA reductase, established that although isoprenylation is not required for ligand binding by IP, it is absolutely required for receptor activation of adenylyl cyclase via G␣ s coupling and for efficient coupling to G␣ q /PLC (18,25,26). Moreover, it was also established that isoprenylation of the IP is required for efficient agonist-mediated receptor internalization (25).…”

“…Conversion of Cys 309 and Cys 311 of the hIP to Ser 309 and Ser 311 was performed using pHM-hIP C309S as a template and the oligonucleotide primers: 5Ј-GTC TGC AGC CTG AGC CTC GGG CCT G-3Ј (sense primer) and 5Ј-C AGG CCC GAG GCT CAG GCT (20,26). The plasmids pCMV-G␣ q , pCMV-G␣ s , and pHM:HaRas have been previously described (18,20,25,26).…”

“…Conversion of Cys 309 and Cys 311 of the hIP to Ser 309 and Ser 311 was performed using pHM-hIP C309S as a template and the oligonucleotide primers: 5Ј-GTC TGC AGC CTG AGC CTC GGG CCT G-3Ј (sense primer) and 5Ј-C AGG CCC GAG GCT CAG GCT (20,26). The plasmids pCMV-G␣ q , pCMV-G␣ s , and pHM:HaRas have been previously described (18,20,25,26). HEK 293 cells were transfected with pADVA (10 g/10-cm dish) and pCMV-or pHM-based vectors (25 g/10-cm dish) using the calcium phosphate/DNA co-precipitation procedure (27) (25 g/ 10-cm dish), respectively, using the calcium phosphate/DNA coprecipitation procedure (27).…”